Synthesis, molecular docking studies, and larvicidal activity evaluation of new fluorinated neonicotinoids against Anopheles darlingi larvae

Article abstract of DOI:10.1371/journal.pone.0227811

Anopheles darlingi is the main vector of malaria in Brazil, characterized by a high level of anthropophilia and endophagy. Imidacloprid, thiacloprid, and acetamiprid are the most widespread insecticides of the neonicotinoid group. However, they produce adverse effects on the non-target insects. Flupyradifurone has been marketed as an alternative to non-fluorinated neonicotinoids. Neonicotinoids containing trifluoroacethyl substituent reveal increased insecticidal activity due to higher hydrophobicity and metabolic stability. We synthesized novel neonicotinoid insecticides containing fluorinated acceptor groups and their interactions were estimated with the nicotinic acetylcholine receptor (nAChR) binding site by molecular docking studies, to evaluate their larvicidal activity against A. darlingi, and to assess their outdoor photodegradation behavior. New neonicotinoid analogues were prepared and characterized by NMR and mass-spectrometry. The synthesized molecules were modelled by time-dependent density functional theory and analyzed, their interaction with nAChR was investigated by molecular docking. Their insecticide activity was tested on Anopheles larvae collected in suburban area of Manaus, Brazil. Four new fluorinated neonicotinoid analogs were prepared and tested against 3^rd instars larvae of A. darlingi showing high larvicidal activity. Docking studies reveal binding modes of the synthesized compounds and suggest that their insecticidal potency is governed by specific interactions with the receptor binding site and enhanced lipophilicity. 2-Chloro-5-(2-trifluoromethyl-pyrrolidin-1-ylmethyl)pyridine 5 showed fast degradation in water maintaining high larvicidal activity. All obtained substances possessed high larvicidal activity in low concentrations in 48 hours of exposure, compared to commercial flupyradifurone. Such activity is connected to a unique binding pattern of the synthesized compounds to insect's nAChR and to their enhanced bioavailability owing to introduction of fluorinated amino-moieties. Therefore, the compounds in question have a high potential for application as control agents for insects transmitting tropical diseases, and they will be less persistent in the environment.

Full text of DOI:10.1371/journal.pone.0227811

RESEARCH ARTICLE
Synthesis, molecular docking studies, and
larvicidal activity evaluation of new fluorinated
neonicotinoids against Anopheles darlingi larvae
Rochelly da Silva Mesquita1, Andrii Kyrylchuk 2, Iryna Grafova3, Denys Kliukovskyi2,
ID
2
Andriy Bezdudnyy , Alexander Rozhenko , Wanderli Pedro Tadei , Markku Leskela ,
2
4
3
¨
3
Andriy Grafov
*
ID
1 Department of Chemistry, Federal University of Amazonas, Manaus, Amazonas, Brazil, 2 Institute of
Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine, 3 Department of Chemistry,
University of Helsinki, Helsinki, Finland, 4 Malaria and Dengue Laboratory, National Institute for Amazonian
Research, Manaus, Amazonas, Brazil
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
* andriy.grafov@helsinki.fi
Abstract
Anopheles darlingi is the main vector of malaria in Brazil, characterized by a high level of
anthropophilia and endophagy. Imidacloprid, thiacloprid, and acetamiprid are the most wide-
spread insecticides of the neonicotinoid group. However, they produce adverse effects on
the non-target insects. Flupyradifurone has been marketed as an alternative to non-fluori-
nated neonicotinoids. Neonicotinoids containing trifluoroacethyl substituent reveal
increased insecticidal activity due to higher hydrophobicity and metabolic stability.
We synthesized novel neonicotinoid insecticides containing fluorinated acceptor groups
and their interactions were estimated with the nicotinic acetylcholine receptor (nAChR) bind-
ing site by molecular docking studies, to evaluate their larvicidal activity against A. darlingi,
and to assess their outdoor photodegradation behavior. New neonicotinoid analogues were
prepared and characterized by NMR and mass-spectrometry. The synthesized molecules
were modelled by time-dependent density functional theory and analyzed, their interaction
with nAChR was investigated by molecular docking. Their insecticide activity was tested on
Anopheles larvae collected in suburban area of Manaus, Brazil. Four new fluorinated neoni-
cotinoid analogs were prepared and tested against 3rd instars larvae of A. darlingi showing
high larvicidal activity. Docking studies reveal binding modes of the synthesized compounds
and suggest that their insecticidal potency is governed by specific interactions with the
receptor binding site and enhanced lipophilicity. 2-Chloro-5-(2-trifluoromethyl-pyrrolidin-1-
ylmethyl)pyridine 5 showed fast degradation in water maintaining high larvicidal activity. All
obtained substances possessed high larvicidal activity in low concentrations in 48 hours of
exposure, compared to commercial flupyradifurone. Such activity is connected to a unique
binding pattern of the synthesized compounds to insect’s nAChR and to their enhanced bio-
availability owing to introduction of fluorinated amino-moieties. Therefore, the compounds in
question have a high potential for application as control agents for insects transmitting tropi-
cal diseases, and they will be less persistent in the environment.
OPEN ACCESS
Citation: da Silva Mesquita R, Kyrylchuk A, Grafova
I, Kliukovskyi D, Bezdudnyy A, Rozhenko A, et al.
(2020) Synthesis, molecular docking studies, and
larvicidal activity evaluation of new fluorinated
neonicotinoids against Anopheles darlingi larvae.
PLoS ONE 15(2): e0227811. https://doi.org/
10.1371/journal.pone.0227811
Editor: Ahmed Ibrahim Hasaballah, Al-Azhar
University, EGYPT
Received: May 6, 2019
Accepted: December 30, 2019
Published: February 5, 2020
Copyright: © 2020 da Silva Mesquita et al. This is
an open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors gratefully acknowledge
financial support of the present research by: the
European project Horizon2020-MSCA-RISE-2016-
734759, acronym VAHVISTUS; Magnus Ehnrooth
Foundation and Finnish Center of Excellence in ALD
(Finland); the Adaptation of the Amazonian Aquatic
Biota Project (ADAPTA) (Brazil); Malaria Network
Project (Rede Malaria) financially supported by the
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New fluorinated neonicotinoids against Anopheles darlingi larvae
National Council for Scientific Development - CNPq
(Brazil); and the Foundation for Research Support
in the State of Amazonas - FAPEAM (Brazil). The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Introduction
Malaria is a parasitic and endemic disease in several regions of the world with wide distribu-
tion mainly in tropical and subtropical areas. Currently, more than 3 billion people are at risk
of contracting the disease and about 148 to 304 million of new cases are recorded annually [1].
Anopheles darlingi Root 1926 is the main vector of malaria in Brazil. On one hand, the spe-
cies is characterized by a high level of anthropophily and endophagy in relation to other mos-
quito species of the Amazon region. On the other hand, A. darlingi is the malaria transmitting
species the most favored by environmental changes resulting from human activities such as
occupation of urban and peri-urban spaces in an uncontrolled manner; the construction of
hydroelectric power plants, irrigation projects, and fishponds; the exploitation of fossil fuels,
minerals, and natural gas; forest fires; deforestation; and road construction [2–5].
Mosquito vector control measures are the most commonly used approach to reduce the
number of malaria cases. In relation to entomological data, the above measures include such
traditional actions to combat vector-borne diseases as space spraying, indoor residual spray-
ing, long-lasting insecticidal nets, larval source management, biolarvicide application, active
search, and appropriate case treatment [6,7].
Competing interests: The authors have declared
that no competing interests exist.
Abbreviations: FPF, Flupyradifurone; nAChR,
Nicotinic acetylcholine receptor; DT50, Degradation
half-time; TD-DFT, Time dependent density
functional theory; QSAR, quantitative structure-
activity relationship; QM/MM, quantum mechanical
molecular dynamics simulations; NMR, Nuclear
magnetic resonance; TLC, thin layer
chromatography; TMS, Tetramethyl silane; BP-86/
TZVP, Functional composed of the Becke 1988
exchange functional and the Perdew 86 correlation
functional / Triple zeta for valence electrons plus
polarization function; DMSO, dimethylsufoxide;
LC50, Median lethal dose; UV, ultraviolet; ESP,
electrostatic potential surface; AChBP,
Management of larval breeding sites plays an important role in the control of malaria. Espe-
cially in those specific sites, where the vectors tend to reproduce, which may have characteris-
tics of permanent or semi-permanent breeding sites. The WHO suggests the use of larvicides
in those breeding sites located in urban areas, to protect the human population [5,8–10].
Larvicide control actions represent an effective tool for vector control, reducing the trans-
mission of malaria in specific areas. This procedure is particularly important in the Amazonian
region, where different breeding sites of malaria vectors are widely distributed [9].
According to the WHO recommendations, only the use of pyrethroid insecticides is indi-
cated for the actions on adult mosquitoes in current vector control program [8,9]. Recently,
resistance of the vector to these insecticides were reported in some areas, making research tar-
geted at new classes of insecticides highly relevant [8,11].
acetylcholine binding protein; TRP, tryptophan;
TYR, tyrosine; MET, methionine.
The above dependence on the only one class of insecticides can be potentially overcome by
implementation of neonicotinoids, a promising class of insecticides that exhibit excellent effi-
ciency and low risk to humans and the environment. They are extensively used throughout the
world for crop protection, particularly against sucking insect pests, accounting for one-fourth
of the total world insecticide market [12]. Imidacloprid, thiacloprid, and acetamiprid are the
most widespread insecticides of the neonicotinoid group. Toxicity of neonicotinoids to mam-
mals is usually much lower than for many insect species. One can find reports on adverse
effects of several widely adopted neonicotinoids on the non-target insects (especially honey-
bees) [13,14]. Prolonged periods of exposure of aquatic insects to neonicotinoids even though
at low levels, has given rise to serious risks, since those insects are as sensitive to that class of
insecticides, as the bees. Recently, imidacloprid toxic residues have been detected in water sur-
faces and are associated with the reduction of aquatic insect populations [15,16].
By that reason, the imidacloprid, thiamethoxam, and clothianidin were banned for use on
flowering crops in the EU for the two-year term in 2013 [17,18]. After thorough risk assess-
ment, the three pesticides were completely banned in the EU for outdoor use on crops since
2018 [19–21]. At the same time, it was established that several neonicotinoids (e.g. acetami-
prid) represent low risk to honeybees [22,23], thus any further restrictions of those substances
is neither scientifically nor legally appropriate [24]. Negative effects were mostly caused by
improper and excessive use of the insecticides, or laboratory-based experiments that employed
greater concentrations of the neonicotinoids than those found in nectar and pollen of the
treated plants [17].
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Fig 1. Flupyradifurone.
https://doi.org/10.1371/journal.pone.0227811.g001
Flupyradifurone (FPF, Fig 1) is a new butenolide insecticide chemically similar to neonico-
tinoids, despite it has been marketed as an alternative to them for commercial reasons [25].
Both FPF and neonicotinoids have the same mode of action being a nicotinic acetylcholine
receptor (nAChR) antagonists. The FPF possess low toxicity to non-target insects and mam-
mals, and has recently been registered for use in Canada and the United States [26]. Like neo-
nicotinoids, the FPF is highly water soluble and persistent in the environment [27]. However,
it is practically non-toxic to young adult bees when in acute contact [28].
Agricultural and public health (vector control) importance of neonicotinoids have raised
the need of their further development, taking into a consideration their mechanism of action.
The nAChR is a biochemical target for neonicotinoids, it plays a central role in the mediation
of fast excitatory synaptic transmission in the insect central nervous system [12]. Structural
differences of nAChRs receptor subunits between vertebrate and in the invertebrate species
[29] provides relatively low risk of neonicotinoids to mammals [29]. Interactions of the active
compound with the nAChR may be efficiently investigated using the whole gamut of in silico
methods, from inexpensive quantitative structure-activity relationships (QSAR) to computer
docking and hybrid quantum and molecular mechanics (QM/MM) approaches [18,30–32].
In this study, we perform modeling of nAChR-ligand interactions via molecular docking.
By analyzing various binding conformations and quick estimation of binding affinity for each
of them, molecular docking allows finding of the best binding mode for virtually any potential
ligand. This way we can rank compounds according to the strength of their interaction with
the receptor and find specific intermolecular interactions that are important for the activity of
a potential insecticide [33].
Trifluoromethyl and perfluoroalkyl groups can dramatically affect functional properties of
organic molecules, thereby increasing a possibility of their application as agrochemicals, medi-
cines or building blocks to create new organic materials [34–36].
Rationale for the introduction of fluorine atoms is based on the following reasons:
• Metabolic stability is one of the key factors in determining the bioavailability of a compound.
A frequently employed strategy to circumvent this problem is to block the reactive site by a
fluorine atom introduction. There are many examples illustrating an increase of a molecule
metabolic stability by replacement of an oxidizable C-H group with a C-F one [37–40].
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New fluorinated neonicotinoids against Anopheles darlingi larvae
• Fluorine can change basicity of a compound. Presence of highly basic groups may limit bio-
availability of a molecule. Introduction of the fluorine atom or fluorinated group in the
vicinity of the basic moiety reduces the basicity. Thus, better membrane permeation and
improved bioavailability may be expected for the fluorinated compound [41].
• Moreover, fluorine substituents are introduced to increase receptor-binding affinity of a
compound. For example, most of the neurokinin1 receptor antagonist drugs contain a 3,5-di
(trifluoromethyl)phenyl group to increase the binding affinity [42].
Earlier, it was shown that neonicotinoids containing trifluoroacethyl substituent reveal
increased insecticidal activity due to higher hydrophobicity and metabolic stability [43–45].
Besides that, the presence of an acceptor substituent (such as a nitro- or a cyano-group) in a
terminal moiety is one of the most important factors determining the insecticide activity [29].
Some pesticides have an obvious drawback, because of their possible persistence in the envi-
ronment. In addition to being environmental contaminants, persistent insecticides develop a
resistance to them much more frequently than non-persistent ones. Sunlight photodegrada-
tion is one of the most destructive pathways for pesticides after their release into the environ-
ment [46]. However, the biocidal activity should be preserved before the photodegradation
occurs [47]. Several studies have been devoted to the investigation of the photolysis of the neo-
nicotinoids [48–50]. Half-life of the FPF in water under sunlight was estimated to be as high as
2.5 days [51]. Therefore, reduced photostability becomes an important requirement in the
design of new neonicotinoid insecticides.
During last few years we have developed synthetic approaches to generate novel low-molec-
ular weight fluorinated amines and investigated their properties [52–54]. Substances of that
kind have never been used as neonicotinoid building blocks. Therefore, the aim of this work
was to synthesize new neonicotinoid insecticides containing fluorinated acceptor groups to
estimate the interactions of the synthesized compounds with the nAChR binding site by
molecular docking studies, to evaluate their larvicidal activity against A. darlingi, and to assess
their outdoor photodegradation behavior.
Materials and methods
3-trifluoromethylaniline was purchased from Acros Organics, all other reagents were pur-
chased from Sigma-Aldrich and used as supplied. The solvents (Aldrich) were purified by stan-
dard procedures used in synthetic organic chemistry.
Spectroscopy
1H NMR spectra were recorded on a Varian VXR-300 and Mercury 300 spectrometers, 19
F
NMR spectra were obtained on Gemini 200 Varian instrument at 188 MHz. UV- spectra were
recorded on an UV-Vis Evolution 220 spectrophotometer (Thermo Fischer Scientific) in the
range of 200–500 nm at a bandwidth of 2nm and integration time of 1s.
General synthetic procedures
a) synthesis of fluorosubstituted anilines. 3,5- Bis-trifluoromethylaniline was obtained
following the two-step procedure with a total yield of 56% [55,56]. First, the 5-nitroisophthalic
acid was fluorinated with SF4 taken in a 1:5 molar ratio at 80–901C for 16 hours in a presence
of 10% of HF as a catalyst. The 3,5-bis(trifluoromethyl)nitrobenzene obtained was then
reduced with hydrogen in a methanol solution over 10% Pd/C catalyst. The product was sepa-
rated by distillation and identified spectroscopically. The spectra correspond to those
described in [56].
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New fluorinated neonicotinoids against Anopheles darlingi larvae
4-(pentafluoroethoxy)aniline. 4-(pentafluoroethoxy)nitrobenzene was obtained according
to the previously described procedure [57]. The fluorination product was then reduced with
hydrogen in a methanol solution over 10% Pd/C catalyst, separated by distillation, and identi-
fied spectroscopically. The spectra correspond to those described in [58]. The total yield after
two steps is 71%.
2-(trifluoromethyl)pyrrolidine was obtained according to our previously described proce-
dure [52].
b) synthesis of neonicotinoids. New neonicotinoids were synthesized as shown in the Fig
2 according to the protocol http://dx.doi.org/10.17504/protocols.io.9h5h386.
A mixture of 2-chloro-5-(chloromethyl)pyridine 1 (0.324 g, 1.9 mmol), the corresponding
substituted aniline 2a-c or 4 (2.0 mmol), and anhydrous potassium carbonate (0.828 g, 6.0
mmol) in 5 ml of anhydrous acetonitrile was vigorously stirred under reflux (Fig 2). Progress
of the reaction was monitored by thin layer chromatography (TLC). After the conversion was
complete, the reaction mixture was separated from insoluble inorganic salts. The latter were
thoroughly washed with dichloromethane. The combined organic solutions were evaporated
under reduced pressure and the residue was purified by preparative thin layer chromatography
(TLC) on SiO2 (EtOAc/hexane 1:2).
Fig 2. Synthesis of new neonicotinoids containing fluorinated acceptor groups.
https://doi.org/10.1371/journal.pone.0227811.g002
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New fluorinated neonicotinoids against Anopheles darlingi larvae
N-[(6-chloropyridin-3-yl)methyl]-4-(pentafluoroethoxy)aniline (3a). Yield 68%. Light-yel-
low viscous oil.1H NMR (300 MHz, CDCl3, TMS), δ: 3.93 (br. s, 1H, NH); 4.26 (s, 2H, CH2);
6.49 (d, 2H, C3’H, C5’H); 6.79 (d, 2H, C2’H, C6’H); 7.23 (d, 1H, C3H); 7.58 (m, 1H, C2H); 8.31
(s, 1H, C5H). 19F NMR (188 MHz, CDCl3, CCl3F), δ: -86.46 (s, 3F, CF3); -88.41 (s, 2F, CF2).
ME (EI) m/z 353 (M+1, 100%).
N-[(6-chloropyridin-3-yl)methyl]-3-(trifluoromethyl)aniline (3b). Yield 61%. Light-yellow
viscous oil.1H NMR (300 MHz, CDCl3, TMS), δ: 4.15 (br. s, 1H, NH); 4.29 (s, 2H, CH2); 6.64
(d, 1H, C6’H); 6.74 (s, 1H, C5’H); 6.89 (d, 1H, C4H); 7.16 (d, 1H, C2H); 7.22 (d, 1H, C3H); 7.57
(m, 1H, C2H); 8.23 (s, 1H, C5H). 19F NMR (188 MHz, CDCl3, CCl3F), δ: -62.09 (s). ME (EI)
m/z 287 (M+1, 100%).
N-[(6-chloropyridin-3-yl)methyl]-3,5-bis(trifluoromethyl)aniline (3c). Yield 73%. Yellow-
ish viscous oil. 1H NMR (300 MHz, CDCl3, TMS), δ: 3.59 (br. s, 1H, NH); 4.34 (s, 2H, CH2);
6.89 (s, 2H, C2’H, C5’H); 7.13 (s, 1H, C4H); 7.26 (d, 1H, C3H); 7.58 (m, 1H, C2H); 8.32 (s, 1H,
C5H). 19F NMR (188 MHz, CDCl3, CCl3F), δ, m. d.: -62.55 (s). ME (EI) m/z 355 (M+1, 100%).
2-Chloro-5-(2-trifluoromethyl-pyrrolidin-1-ylmethyl)pyridine (5). Yield 83%. Light-yellow
viscous oil. 1H NMR (300 MHz, CDCl3, TMS), δ: 1.82 (m, 2H, CH2CH2CH2 pyrrolidine), 2.01
(m, 2H, CH2CHCF3), 2.36 (m, 1H, CHCF3), 2.93 (m, 1H, CHHN pyrrolidine), 3.28 (m, 1H,
CHHN pyrrolidine), 3.88 (ABX, 2JHH = 153.4 Hz, 4JHH = 13.6 Hz, 2H, ArCH2N), 7.29 (d, 1H,
H-5 pyridine), 7.69 (dd, 1H, H-4 pyridine), 8.29 (d, 1H, H-3 pyridine). 19F NMR (188 MHz,
CDCl3, CCl3F), -77.03 (s broad, 6F, CF3); ME (EI) m/z 265 (M+1, 100%).
Calculation details
Electronic population analysis was conducted within the Gaussian-09 set of programs [59]
using BP-86/TZVP approximation [60–62]. Multiwfn program was used for electrostatic
potential (ESP) surfaces generation [63,64]. The TD-DFT calculations were carried out at the
BP86/6-311G�� approximation level using the Gaussian-03 program package. Molecular dock-
ing was carried out using AutoDock Vina package [65] and analyzed in AutoDockTools4
[66,67], VMD [68], and BIOVIA Discovery Studio 4.5 [69] programs were used for ESP sur-
faces and docking results visualization.
Ethical considerations
The larvae used in this study were collected in suburban areas of the city of Manaus, Amazo-
nas, Brazil. We declare that no permission was required for these locations. In addition, the
collecting of anopheline larvae did not involve endangered or protected species.
Test organism
Anopheles larvae were collected in suburban areas of the city of Manaus, Amazonas, Brazil and
transported to insectary of the Laboratory of Malaria and Dengue at the National Institute of
Amazonian Research (INPA). The mosquito larvae were reared according to the protocols
described in [70]. Species of the larvae were identified after the realization of the bioassays.
All 7’920 larvae used in the bioassays were identified at the species level according to their
morphological characteristics following the taxonomic keys of [71–73]. The specimens were
placed in Petri dishes containing a few drops of larval fixative prepared according to the proto-
col adopted at the Laboratory of Malaria and Dengue of the INPA (http://dx.doi.org/10.17504/
protocols.io.9qrh5v6) and observed under an optical microscope with magnifications of 100×
to 400×. The identification of species was made based on the external morphological aspects,
such as spiracular apparatus, head, antenna, apex, abdominal segments, palmate hairs, set of
prothoracic hairs, thorax, dorsal view, and ventral integument appearance.
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New fluorinated neonicotinoids against Anopheles darlingi larvae
The colony was maintained at a temperature of 26 2˚C, a relative humidity around 70–
80%, a photoperiod of 12:12 hours [70], and fed with a Tetramin1 fish food.
Larvicidal activity
The larvicidal activity tests for new substances were performed on the 3rd instar Anopheles lar-
vae according to a methodology recommended by the WHO [74,75] https://dx.doi.org/10.
17504/protocols.io.9kch4sw. All the experiments were carried out at the temperature of 26
2˚C, the relative humidity of 70–80% and the photoperiod of 12:12 hours light/dark. For each
substance, a stock solution in dimethylsufoxide (DMSO) was prepared with a concentration of
2000 μg/mL followed by a serial dilution to obtain 3.0, 5.0, 10.0, 20.0, and 30.0 μg/mL for 3a;
2.5, 4.5, 9.5, 14.0, and 19.0 μg/mL for 3b; 2.5, 4.5, 9.0, 14.0, and 18.0 μg/mL for 3c; 6.0, 8.0,
10.0, 12.0, and 14.0 μg/mL for 5; and 2.0, 3.0, 4.0, 5.0, and 6.0 μg/mL for FPF.
The experiment was performed in five replicates of each dosage and two controls: one of
them containing 1.0% aqueous solution of DMSO (negative control) and the other one con-
taining flupyradifurone (Sigma Aldrich1) as a positive control. Twenty larvae of the 3rd instar
were transferred into each dish of replicas and controls containing 50 mL of distilled water,
food, and an aliquot of the stock solution in an appropriate amount for different concentra-
tions. The larval mortalities were observed and registered at 48 hour intervals of exposure.
Mortality rate of 10% and a confidence interval of 95% were set as the limits. The tests were
repeated 3 times. The larvae were considered dead, when they were immobile and unable to
reach the water surface.
Statistical analysis
In order to obtain LC50, the mortality data were treated by a statistical software Polo plus [76],
with 95% confidence interval and values of p < 0.05 were considered statistically significant.
In the case of �10% mortality in the control group, the larval mortality was corrected using
Abbott’s formula [77].
Photodegradation test
40 mg/L solution of 5 was prepared using a deionized milli-Q water. The experiment was per-
formed in Manaus (AM, Brazil; -3.096240ºS, -59.986194˚W; under an average daily solar irra-
diation of 4.92 kWh/m2, http://www.cresesb.cepel.br/index.php#data). The test solution was
placed into a quartz cuvette and tightly stopped there. The cuvette was places under a direct
sunlight, maintaining the temperature at 35˚C. Quantity of the compound 5 in the solution
was monitored by UV-spectrophotometry, using Thermo Scientific Evolution 220- UV-vis
spectrophotometer; Software INSIGHT: 1.3.10; Firmware 3.0.0.109; Scan Speed 120,00 nm/
min; data Interval 1,00 nm; Integration Time 0,500 sec; Bandwidth 2 nm, following the absorp-
tion maximum at 268 nm. The spectra were recorded at different time intervals of 1h - 4h, and
1–4 days from 09:00 until 16:00 (i.e. 7 h each day) and analyzed according to [78].
Results
New fluorinated neonicotinoids 3a-c and 5 (Fig 3) were synthesized from 2-chloro-(5-chloro-
methyl)pyridine 1 and the corresponding fluorinated aromatic or alicyclic amines as shown in
the Fig 2. The starting amines 2a-c or 4 were refluxed with chloropyridine 1 in acetonitrile in
the presence of potassium carbonate. Light-yellow viscous oils of the final compounds were
obtained after their purification by preparative TLC.
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Fig 3. New neonicotinoids containing fluorinated acceptor groups.
https://doi.org/10.1371/journal.pone.0227811.g003
In order to estimate structure-properties relationship for the substances in question, we
performed DFT calculations of the molecules and generated their electrostatic potential sur-
faces (ESP) (Fig 4).
The ESP maps visualize electrostatic potential energy, i.e. energy of interaction between the
molecule and an imaginary positively charged (+1) ion. Color scale in the right side of the fig-
ure shows a correspondence between the surface color and the interaction energy. Positive val-
ues (red) indicate higher interaction energy and therefore electron-poor positively charged
part of the molecule. Negative values (blue) display opposite characteristics. Prevalence of the
red and blue colors in the ESP maps evidence a polar character of the molecule, and appropri-
ately colored regions point at regions of the structure that will more likely interact with posi-
tively (blue parts of the map) or negatively (red parts) charged groups of the receptor active
site. Intermediate values of interaction energy (green) indicate weakly polar hydrophobic parts
of the molecule.
Possible binding models of the synthesized neonicotinoids with nAChR were examined by
docking studies with AutoDock Vina [65] and shown in the Fig 5. Since it is known that
amino acids forming binding pockets are conserved in an AChBPs (acetylcholine binding pro-
teins), a crystal structure of a Lymnaea stagnalis AChBP co-crystallized with imidacloprid
(PDB ID: 2zju) [79] was used as the receptor template.
The calculations also enabled to estimate the protein-ligand binding affinities and partition
coefficients P shown in the Table 1.
Fig 4. ESP surfaces for Flupyradifurone and new neonicotinoids 3a-c and 5.
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Fig 5. Binding modes of compounds 3a-c and 5 to L. stagnalis AChBP.
https://doi.org/10.1371/journal.pone.0227811.g005
Approximately 7’920 Anopheles larvae were collected in suburban areas of the city of
Manaus (AM), Brazil. After the tests were performed successfully, all larvae were identified to
verify the percentage of the species present. A. darlingi was the predominant species represent-
ing 77.38% of the larvae used in the tests. The remaining percentage was distributed among
Table 1. Estimated binding affinities and calculated logP values for the compounds 3a-c, 5, and FPF.
Compound
Predicted binding affinity, kJ/mol
logP
4.42
3.65
4.53
2.94
1.84
3a
3b
3c
-33.472
-35.146
-34.727
-30.125
-29.288
5
FPF
P = partition coefficient
https://doi.org/10.1371/journal.pone.0227811.t001
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Table 2. LC50 of new substances and FPF tested against 3rd instars larvae of A. darlingi at the interval of 48 hours.
Substances
LC50 μg/mL
0.57
Regression equation
y = (0.26+5) + 1.10�log x
y = (0.01+5) + 1.50�log x
y = (0.06+5) + 1.35�log x
y = (7.33+5) - 5.08�log x
y = (3.02+5) - 0.22�log x
χ2
DF
3
3a
3b
3c
0.02
4.70
2.83
1.46
3.30
0.97
3
0.90
3
5
4.93
3
FPF
1.18
3
LC50: Median Lethal Concentration, χ2: Neill’s lack-of-fit test, DF: degrees of freedom
https://doi.org/10.1371/journal.pone.0227811.t002
the following species: A. nuneztovari (16.36%), A. triannulatus (5.08%), A. albitarsis (0.80%),
A. oswaldoi (0.20%), A. evansae (0.11%), A. matogrossensis (0.03%), and A. nimbus (0.01%).
The larvicidal activity of all synthesized compounds were evaluated, showing high mortality
of the 3rd instar larvae of A. darlingi (Table 2). The highest larvae mortality rates; i.e. the lowest
LC50 values were observed for 3a, 3c and 3b with the LC50 values of 0.57, 0.90 and 0.97 μg/mL
respectively, which are lower than that of the commercially available FPF standard (LC50
=
1.18 μg/mL). Within the series under investigation, the highest LC50 value of 4.93 μg/mL was
found for the compound 5. No mortality was observed in the negative control.
The dose-response graphs (Fig 6) show scatter plots of the experimental data on percentage
of mortality of A. darlingi larvae vs concentration and the trendlines for each of the com-
pounds under investigation and the FPF. The coefficients of determination R2 show perfect fit
of the data points by the regression trendlines. According to the graph, all tested compounds
showed stable very high larvae mortality rates (more than 70%) starting from the lowest con-
scentrations applied. Thus, the data show high efficiency of the new substances against larvae
of the main vector of malaria in the Amazonian region. The sample of commercial FPF insec-
ticide was responsible for the highest larvae mortality. This implies that FPF was the com-
pound showing the highest stability of action in the 48-hour interval, because it maintains its
high larvicidal efficacy in the first doses tested (Fig 6). The compound 5 revealed a similar
behavior, being the most efficient compound within the series under investigation. The com-
pound 5 achieved 100% mortality rates at doses nearly 2 times lower than the compounds 3a-
c. By that reason, it was chosen for a photodegradation test under real outdoor conditions in
the city of Manaus (AM, Brazil).
The photodegradation was monitored by UV spectra. The UV spectra of 5 in aqueous solu-
tion showed absorption band maxima of 210 nm and 267 nm, which correspond well to the
values predicted by TD-DFT calculations (Fig 7A). The outdoor photodegradation results are
shown in the Fig 7B. As observed in the Fig 7, the compound starts to degrade shortly after 2
hours of exposure. The half-life of the compounds 5 was assessed from 0 h to 4 days of direct
exposure to sunlight. The photodegradation half-life time for 5 (DT50 = 11.9 h) was derived
based on UV/VIS spectral data according to the US Environmental Protection Agency stan-
dard operating procedure [78]. Comparison of residual deviances of the first- and second-
order kinetic models shows that a single first-order rate model can be considered appropriate.
Discussion
Various habitat forms, favorable environmental conditions for the development of mosqui-
toes, and their distribution in the environment, are the main factors that favor the diversity of
species of the genus Anopheles in the Amazon region. The anophelines collected in the study
region include A. darlingi, A. nuneztovari, A. triannulatus, A. albitarsis, A. oswaldoi, A. evan-
sae, A. matogrossensis, and A. nimbus; all had previously been reported in the literature [2,3].
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Fig 6. Mortality of A.darlingi larvae (%) vs concentration of the compounds 3a, 3b, 3c, 5, and flupyradifurone in the intervals of 48 hours. Trendlines are shown as
the corresponding dash lines, R2 values are reported near each line.
https://doi.org/10.1371/journal.pone.0227811.g006
However, this diversity may vary according to the hydrological cycle, a phenomenon that
influences the seasonality of species; also, a variety of different breeding sites, the intensifica-
tion of human/vector contact, and the recorded cases of malaria have to be taken into account
[5,80]. The dominance of certain anopheline species has also been related to their major ability
to adapt to various environmental transformations, especially in the ecosystems altered by
anthropic actions and environmental changes, such as increased exposure to sunlight and
absence of frequently growing aquatic plants. The above factors affect the development of the
mosquito larvae and cause a direct impact on the presence of different species of the same
genus [80,81]. This study showed that A. darlingi was the dominant species in the collection
area. It is considered the main malaria vector in the region, being more susceptible to infection
by Plasmodium spp. parasites [80,81].
In the present study, we synthesized a series of new fluorinated neonicotinoid analogs start-
ing from some novel amines containing -CF3 and -OC2F5 groups. Introduction of fluorine
into a biologically active molecule increases its biological activity by affecting several
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Fig 7. UV spectra. (a) spectra of the compound 5 and FPF. (b) Dependence of the absorption (at λmax = 268 nm) from the duration of irradiation.
https://doi.org/10.1371/journal.pone.0227811.g007
parameters, e.g. binding to a target receptor. We suppose the presence of fluorinated amino-
moieties will improve hydrophobic interactions with the receptor site.
Introduction of fluorine into a biologically active molecule increases its biological activity
by affecting several parameters, e.g. binding to a target receptor. Thus, we synthesized a series
of new fluorinated neonicotinoid analogs starting from some novel amines containing -CF3
and -OC2F5 groups. We suppose the presence of fluorinated amino-moieties will improve
hydrophobic interactions with the receptor site.
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New fluorinated neonicotinoids against Anopheles darlingi larvae
In order to find structural features that may contribute to activity of the synthesized com-
pounds compared to the FPF, the calculations of the electrostatic potential surfaces and molec-
ular docking were carried out. Comparison of the molecular surface maps (Fig 4) shows a wide
diversity of configurations for the compounds under investigation. The molecules of 3a-b are
bulkier than that of the FPF, a commercially available insecticide. Earlier, it was shown that
bulky neonicotinoid analogs often have insecticidal potencies comparable to or even higher
than popular commercial insecticides [31,43,82–84]. Such bulky substances can engage more
regions of the ligand-binding domain of the nAChRs than conventional ones, or even dock to
entirely different locations of those receptors [85,86].
Comparison of the ESP maps for the compounds 3a-c and 5 with that of FPF (Fig 4) reveals
two main distinctive features of the synthesized neonicotinoid analogues. First, the electronic
density distribution and shape of the molecular surfaces vary substantially within the series
and considerably differ from those of the FPF. Second, all the substances 3a-c and 5 have
enlarged weakly polar (green) lipophilic areas, capable to hydrophobic interactions with the
receptor active site.
Binding modes of the synthesized neonicotinoids 3a-c and 5 to nAChR are shown in the
Fig 5. General feature of fluoroalkyl-phenyl substituted compounds 3a-c is represented by ori-
entation of the fluorinated groups and phenyl moiety to the hydrophobic region of the recep-
tor binding pocket consisting of five aromatic amino acid residues–TRP53, TRP143, TYR164,
TYR185 and TYR192. They contribute to the π-stacking interactions with phenyl rings at the
distances of ca. 3.6 Å (that is within a usual range for parallel displaced π-stacking mode) [87]
and to hydrophobic interactions with fluorinated groups. The stacking seems to have a large
contribution to binding affinities, since the molecule of pyrrolydine derivative 5 is turned
over, when compared to 3a-c ones. Thus, the pyridine moiety can participate in stacking inter-
action with TYR185 phenyl ring. Three out of four compounds under discussion also take part
in S-π interactions with MET114 sulfur atom at a distance of ca. 3.5 Å. The compound 3a falls
out of the row, evidently, because of the large volume of the -OC2F5 group. However, it is ori-
ented similarly to the 3b and 3c allowing the hydrophobic interactions between the fluorine
atoms of the neonicotinoid and aromatic amino acid residues.
Besides the non-covalent interactions described above, a hydrogen bonding between the
NH-moiety of the neonicotinoid and a hydroxyl group of the TYR192 is present for 3c and 3b
compounds. That bonding enhances stability of the receptor-ligand complex that is immedi-
ately seen in the results of molecular docking (compounds 3a,b have the highest affinities).
Measured larvicidal activities confirm the docking results, both compounds have low LC50
values.
According to the docking calculation results, the best predicted binding affinities do not
vary significantly, being in the range of -30 � -35 kJ/mol (Table 1). The LC50 values deter-
mined generally decrease along with a growing interaction strength. Fluorine substitution
greatly enhances the lipophilicity of the amines employed for the synthesis. A common mea-
sure of lipophilicity of a compound is the partition coefficient P, i.e. ratio of its equilibrium
concentrations in two immiscible liquids, usually octanol and water. Calculated logP values for
the compounds under investigation and the FPF are shown in the Table 1. Comparison of the
logP values for the compound 3b having one CF3-group and the 3c one with two CF3-groups
shows that fluorine substitution has a substantial influence on the lipophilicity of the com-
pound. Lethal concentrations of the substances 3a-c, 5 decrease with the increase of the calcu-
lated logP. The above results lead to an additional explanation of high larvicidal activity of the
neonicotinoids in question. In addition to the binding strength to the insect’s nAChR, the
enhanced activity of compounds 3a-c, and 5 could also result from their high bioavailability
due to facile blood-brain barrier crossing [29].
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Fig 8. Comparison of the binding modes of FPF (green) and 5 (pink).
https://doi.org/10.1371/journal.pone.0227811.g008
Position of the compound 5 at the receptor binding site is also very similar to that of flupyr-
adifurone (Fig 8), which indicates similar receptor-ligand interactions of these compounds.
CF3-group in 5 is a substitution of electronegative lactone pharmacophore of the FPF, while
conformational mobility of 5 allows it to achieve simultaneous hydrogen bonding, aromatic
interactions, and halogen bond.
Very high larvicidal activity of the new substances was confirmed in the test using A. dar-
lingi larvae. The compound 3a showed the best larvicidal potential (LC50 = 0.57 μg/mL)
among the compounds under investigation, considering higher mortality of the larvae during
the 48 h interval, even when compared to the FPF positive control LC50 = 1.18 μg/mL.
Since the compounds under investigation promoted a high mortality in contact with the
larvae, they may be considered as potential larvicides. However, more specific tests are neces-
sary to evaluate the mechanism of action and to classify them as possible insecticides to control
the vectors of malaria in the Amazonian region.
Larvae of Anopheles mosquitos prefer to live in clean, unpolluted water. Despite a high
insecticidal potential of neonicotinoids for vector control applications outdoors, quite a little is
known about direct sunlight photodegradation in water of neonicotinoid insecticides contain-
ing fluorinated acceptor groups.
Since the habitat of Anopheles larvae is water, the eventual use of the compounds under real
outdoor conditions may be modelled in an aqueous solution under intense natural sunlight
and elevated environmental temperature, typical for the region. In this work we studied exper-
imentally a photodegradation of 5 under real outdoor conditions (sunlight, environment, tem-
perature) (Fig 7B) and compared those data to similar data for the FPF [25,88]. A reported
DT50 value for the FPF (13.8 h) [88] is slightly higher than for the compound 5 (11.9 h).
Hence, the 2-chloro-5-(2-trifluoromethyl-pyrrolidin-1-ylmethyl)pyridine would be less persis-
tent in the environment.
Rather often, continuous and indiscriminate use of persistent in the environment insecti-
cides in inadequate concentrations has led to the development of insect resistance to agents
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New fluorinated neonicotinoids against Anopheles darlingi larvae
currently used in vector control. In the areas of Amazon region with high incidence of malaria,
the application of insecticides is restricted to round the house places and some mosquito
breeding site places. The former method is not very effective, since the houses are scattered in
rural areas, and thus, the impact is minimal and should be monitored regularly. The latter is
only permitted on a non-continuous base, when malaria outbreak happens and it is not possi-
ble to permanently eliminate the breeding site [9,89]. In such a way, environmental contami-
nation may be avoided.
The fluorinated neonicotinoids under investigation showed high efficiency and rapid
photodegradation; hence, they may be regarded as a viable option for insect vector control in
places, where the resistance was observed. Another important safety factor is related to higher
selectivity of neonicotinoids to the mosquito nAChRs [12,29] with respect to the mammalian
ones.
The above would require further testing of the proposed compounds for their action on
non-target organisms and persistence in the environment to ensure a more selective spectrum
of action.
Conclusion
Our results showed that the A. darlingi larvae were highly susceptible to new neonicotinoid
analogs 3a-c and 5 containing fluorinated acceptor groups. The substances 3a-c revealed
higher larvicidal activity at low concentrations in 48 hours of exposure, when compared to flu-
pyradifurone. The reason of such activity may be connected to the unique binding pattern of
the synthesized compounds to insect’s nAChR and to their enhanced bioavailability owing to
introduction of fluorinated amino-moieties. The larvicidal activity tests showed the LC50 val-
ues that are in a good agreement with the theoretically predicted binding of the active com-
pound to the nAChR. The photodegradation of 5 was studied under real outdoor conditions
(sunlight, environment, temperature) and compared to the FPF data. The compound 5
degrades rapidly in water under direct sunlight, the DT50 value found was slightly lower than
that of the FPF. Thus, in view of all the data reported in this study, the compound 5 has a
lower persistence time in water without reducing its efficacy as a larvicide. However, further
testing is required to evaluate those and other factors in real field conditions.
Acknowledgments
RSM and WPT gratefully acknowledge the assistance of Dr R.C.S.Nunomura from the Depart-
ment of Chemistry of the Federal University of Amazonas (UFAM, Manaus, Brazil) for the
UV-vis measurements and Mr A.C. de Oliveira from the Department of Pharmacy, UFAM for
his help in statistical data treatment.
Author Contributions
Conceptualization: Alexander Rozhenko, Markku Leskela¨, Andriy Grafov.
Data curation: Andrii Kyrylchuk, Alexander Rozhenko, Wanderli Pedro Tadei, Markku Les-
kela¨, Andriy Grafov.
Investigation: Rochelly da Silva Mesquita, Iryna Grafova, Denys Kliukovskyi, Andriy Bezdud-
nyy, Wanderli Pedro Tadei, Markku Leskela¨.
Methodology: Rochelly da Silva Mesquita, Andrii Kyrylchuk, Iryna Grafova, Denys Kliukovs-
kyi, Andriy Bezdudnyy, Alexander Rozhenko, Wanderli Pedro Tadei.
Supervision: Andriy Grafov.
PLOS ONE | https://doi.org/10.1371/journal.pone.0227811 February 5, 2020
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New fluorinated neonicotinoids against Anopheles darlingi larvae
Visualization: Andrii Kyrylchuk.
Writing – original draft: Rochelly da Silva Mesquita, Andrii Kyrylchuk, Iryna Grafova, Denys
Kliukovskyi, Andriy Bezdudnyy, Alexander Rozhenko, Wanderli Pedro Tadei, Andriy
Grafov.
Writing – review & editing: Rochelly da Silva Mesquita, Andrii Kyrylchuk, Iryna Grafova,
Andriy Bezdudnyy, Markku Leskela¨, Andriy Grafov.
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