Revista Brasileira de Entomologia Revista Brasileira de Entomologia
Rev Bras Entomol 2018;62:112-8 - Vol. 62 Num.2 DOI: 10.1016/j.rbe.2018.01.004
Medical and Veterinary Entomology
Synthesis of new α-amino nitriles with insecticidal action on Aedes aegypti (Diptera: Culicidae)
Andrés G. Ruedaa, Aurora L. Carreño Oteroa, Jonny E. Duqueb,, , Vladimir V. Kouznetsova,,
a Universidad Industrial de Santander, Escuela de Química, Laboratorio de Química Orgánica y Biomolecular, Santander, Colombia
b Universidad Industrial de Santander, Escuela de Medicina, Centro de investigaciones en enfermedades tropicales, Santander, Colombia
Received 30 August 2017, Accepted 26 January 2018
Abstract

Aedes aegypti is the principal vector of arboviral pathogens that may cause diseases as dengue fever, chikungunya and zika. The harmful environmental effects of commercial pesticides coalesced with the development of insecticide-resistant populations encourage the discovery and generation of new alternative products as a tool to reduce the incidence of vector-borne diseases. In this work, through the classic three component Strecker reaction of commercial benzaldehydes, cyclic secondary amines and KCN, a new series of nine α-amino nitriles, girgensohnine analogs, has been synthetized and screened for larvicide and adulticide properties against A. aegypti, one of the dominant vectors of dengue, chikungunya and zika in tropical and subtropical areas all over the world. Molecules 3 and 4 were identified as potential larvicidal agents with LC50 values of 50.55 and 69.59ppm, respectively. Molecule 3 showed 100% of mortality after 2h of treatment when a concentration of 30ppm in adulticidal assays was evaluated. Additionally, in order to elucidate the mode of action of these molecules, their acetylcholinesterase (AChE) inhibitory properties were evaluated using the Ellman assay. It was found that the molecules possess a weak AChE inhibitory activity with IC50 values between 148.80 and 259.40μM, indicating that AChE could not be a principal target for insecticide activity.

Keywords
Arthropod-borne diseases, Girgensohnine analogs, Strecker reaction, Insecticidal activity, Vector control
Introduction

The arboviruses (arthropod-borne viruses) such as dengue (DENV), chikungunya (CHIKV) and zika (ZIKV) viruses are important causes of human diseases that affect public health in tropical and subtropical areas all over the world (Weaver and Reisen, 2010). According to the National Health Institute of Colombia, in 2017 approximately 23,000 cases of dengue, 1000 cases of Chikungunya and 1900 cases of Zika virus have been reported. The increase of the number of cases has positioned these diseases among the most worrisome for public health in different Latin America countries (Instituto Nacional de Salud INS, 2017).

Aedes (Stegomyia) aegypti (Linnaeus 1762) and Aedes (Stegomyia) albopictus (Skuse 1895) female mosquitoes are hematophagous insects that transmit pathogens to humans which may or may not cause diseases as the described above. The main symptoms of these virus infections are acute fever and polyarthralgia. Dengue infection is a serious disease and it is estimated that approximately 96 million cases are annually reported worldwide (Guzman and Harris, 2015; Simmons et al., 2012). CHIKV virus have an increasingly important impact in humankind morbidity, with potentially life-threatening and a painful arthritis. According to Staples and Fischer other symptoms are headache, myalgia, conjunctivitis, vomiting, and maculopapular rash (Staples and Fischer, 2014). ZIKV virus, like CHIKV virus, is an emerging arbovirus (Musso and Gubler, 2016; Weaver et al., 2016), which is widespread in neotropical regions with recent epidemics outbreaks in Africa, Asia, Europe and recently in America (Hayes, 2009; Ioos et al., 2014; Rodriguez-Morales, 2015; Zanluca et al., 2015). Zika infection is usually asymptomatic but in some cases rash, conjunctivitis and not very high fever can be observed. However, symptomatic ZIKV virus infection has been associated to Guillain-Barré syndrome (Cao-Lormeau et al., 2016) and neonatal microcephaly (Rasmussen et al., 2016).

Currently, there are no specific treatments, effective vaccines, or preventive drugs for these infectious diseases (Rashad et al., 2014; Shan et al., 2016; Stevens et al., 2009). Treatments are only palliative and include rest, hydration, analgesics, and antipyretics. Therefore, these virus infections are best prevented by avoiding the vector bites. Vector control methods involve strategies as the Integrated Vector Management promoted by WHO (2012). These activities include the interruption of human-vector contact, environmental management, biological and chemical controls and self-initiative for individual and household protection. The use of repellents, insecticide-treated mosquito nets, aerosol insecticides, mosquito coils and even clothing for minimizing skin exposure and air conditioning to reduce mosquito bite are some of the most popular household strategies used (Kawada et al., 2014; Prajapati et al., 2005). These efforts have been shown not to be sufficient due mainly to the wide range of artificial and natural larval habitats that A. aegypti can use for its breeding. Clean water collected in man-made container in rural areas without public water service or for storage in drought periods, provide favorable conditions for the production of a large number of mosquito larvae and adults (Grisales et al., 2013).

Because different reasons, as those explained above, make impossible the elimination of mosquito breeding sites completely, the most common vector control alternative in stages of larvae and adults is the use of synthetic insecticides (larvicides and adulticides). Among them, organophosphorus larvicides (temephos), organophosphorus insecticides (malathion and fenitrothion), carbamates (carbaryl, propoxur) and pyrethroids (permethrin and deltamethrin) (Fig. 1), also toxic to mammals and harmful to the environment, are usually employed against A. aegypti mosquitoes and larvae (Braga and Valle, 2007).

Fig. 1.
(0.21MB).

Structures of insecticide agents and molecules studied in this work.

However, resistance to insecticides, documented for more than 500 species of arthropods is one of the main problems in vector control, this phenomenon has been described to all these pesticides (Hemingway et al., 2004; Russell et al., 2004; Smith et al., 2016) and reported in several countries such as Colombia, Brazil and Malaysia (Aguirre-obando et al., 2015; Bona et al., 2016; Low et al., 2015). The increase in doses and frequencies of application are some of the issues to control, leading to preserve the effectiveness of commercial and new pesticides, this is why it is necessary to take into account that resistance is a multifactorial problem that involves environmental, operational and genetic factors (Guedes et al., 2017; Sparks and Nauen, 2015). For these reasons, it is particularly significant to know which are the modes of action of the insecticides and those changes that result in the resistance to the applied product (Lima et al., 2011). Furthermore, the use of new compounds against A. aegypti could inhibit various detoxifying enzymes at the same time, leading to not being easily identified by these systems and decreasing the risk of resistance (Carreño Otero et al., 2018). It is well known that action mode of organophosphates and carbamates is through acetylcholinesterase (AChE) inhibition whose decreased sensitivity is attributed to insecticide resistance (Pang, 2014), while pyrethroids are potent disrupters of voltage-sensitive sodium channels (Shafer et al., 2005). This selectivity of insecticides in the action mode is one of the reasons to produce new molecules that affect different targets in the insect.

On the other hand, it was proved that some plant secondary metabolites possess larvicidal and adulticidal activity against A. aegypti mosquitoes (Liu et al., 2016) and thus could serve as suitable prototypes for designing new bioactive molecules. We recently reported that alkaloid girgensohnine, a N-cianomethyl piperidine metabolite present in shrub Girgensohnia oppositiflora (Amaranthaceae) (Nahrstedt et al., 1993), exhibited moderate in vitro anti-AChE properties (IC50=93μM), while its closer analog, 2-(3,4-dimethoxyphenyl)-2-(piperidin-1-yl) acetonitrile (α-amino nitrile A) (Fig. 1) showed reasonable in vitro AChE inhibition activity (IC50=45μM) exhibiting in vivo larvicidal activity (LC50=88ppm) on A. aegypti larvae (Carreño et al., 2014; Vargas and Kouznetsov, 2013).

Considering these previous results and the importance of control methods for increasing incidence of vector-borne diseases, this present work aimed to synthetize new series of α-amino nitriles B using three component Strecker reaction, assessing first their activity on A. aegypti larvae, then screening their activity on adults, and evaluating their anti-AChE properties. All this is in order to contribute to the development of new molecules with larvicides and adulticides properties against A. aegypti and to elucidate the mode of action of the obtained α-amino nitrile derivatives.

Materials and methodsIn silico evaluation of proposed compounds

Before the synthesis of the girgensohnine analogs, a theoretical study was performed to predict their bioavailability properties, known as Absorption, Distribution, Metabolism, Excretion and Toxicity properties (ADME-Tox) (Lipinski et al., 2001). The Lipinsky's rules evaluate the physicochemical properties of the proposed structures such as molecular weight, partition coefficient (LogP), solubility, polar surface area (TPSA) and the number of rotatable bonds. For this study, Molinspiration and Osiris online calculation resources were used. Osiris predicts the toxicity of proposed molecules by comparing their structural fragments with those found in toxic compounds reported.

Equipment and purification of compounds

The melting points (uncorrected) were determined on a Fisher-Johns melting point apparatus (Flores-Conde et al., 2012). Infrared (FT-IR) spectra were recorded on a Lumex Infralum FT-02 spectrometer, νmax in cm−1 (Ertürk et al., 2012). Bands are characterized according to the functional group. 1H-NMR spectra were obtained with a Bruker AM-400 spectrometer (400MHz). Data were reported as follows: chemical shift, integration, multiplicity (s=singlet, d=doublet, t=triplet, dd=doublet of doublets, dt=doublet of triplets, ddd=doublet of doublet of doublets, td=triplet of doublets, qd=quartet of doublets, pd=pentet of doublets, m=multiplet), coupling constants (Hz) and proton assignation. 13C-NMR spectra were obtained with a Bruker AM-400 (100MHz) spectrometer with complete proton decoupling. Chemical shifts were reported in ppm (δ) relative to the solvent peak (CDCl3, 7.24ppm for 1H and 77.23ppm for 13C). An Amazon X Bruker Datonics mass spectrophotometer with electrospray nebulization (ESI-MS) was used for MS identification. Elemental analyses were performed on a PerkinElmer 2400 Series II analyzer with theoretical values of ±0.4. The work-up for the reactions, extraction, and purification procedures in column chromatography were carried out using reactants and reagent grade solvents (purchased from Merck, Sigma–Aldrich and J.T. Baker). Thin-layer chromatography (TLC) was performed using Silufol UV254 precoated plates (0.25mm). UV light of 254nm was used to observe components and iodine vapor was used for revealing. Column chromatography was performed using neutral aluminum oxide column as solid support (Al2O3 neutral active 90, 70–230 Mesh, Merck) using as eluents solvent mixtures of petroleum ether e and ethyl acetate.

General procedure for preparation of the title compounds

Using a 50mL round-bottom flask, benzaldehydes 1 (10mmol) and secondary amines 2 (13mmol) were dissolved with a magnetic stirrer in acetonitrile (10mL) and kept stirring for 30min at room temperature. Subsequently, KCN (0.35g, 15mmol) and 0.80g of SSA catalyst were added to the flask. Then, the resulting suspension was stirred for 18h (TLC control) and filtered. The resulting filtrate was concentrated using a rotary evaporator and the obtained mass was purified with alumina column chromatography (Al2O3) eluting with different concentrations of petroleum ether: ethyl acetate to obtain pure α-amino nitrile compounds 311 (Table 1).

Table 1.

Properties and yields of synthesized α-amino nitriles 311.

Comp.  Yield (%)  Mp (°C)  MW (g/mol) ≤500  LogP ≤5  TPSA (Å) ≤90  H-bond donor ≤5  H-bond acceptor ≤10 
3  66  78–79  230.31  2.674  36.264 
4  88  95–96  244.33  2.915  36.264 
5  82  87–88  274.36  2.505  45.498 
6  81  88–89  258.32  2.794  45.498 
7  66  57–58  244.33  3.004  36.264 
8  91  94–95  274.36  2.594  45.498 
9  68  80–81  258.32  2.838  45.498 
10  63  Oil  216.28  2.169  36.264 
11  57  79–80  232.28  1.612  45.498 

Mp, melting point; MW, molecular weight (g/mol); LogP, n-octanol–water partition coefficient; TPSA, topological polar surface area; NER, number of rotatable bonds; H-bond donor, hydrogen bond donors (expressed as the sum of OHs and NHs); H-bond acceptor, hydrogen bond acceptors (expressed as the sum of Ns and Os).

In vivo evaluation of insecticidal action on A. aegypti larvae and adults

Larvae between third and fourth instar of Rockefeller strain were used, these were reared in CINTROP laboratory, keeping in plastic containers with a temperature of 25°C±5°C, wet conditions of 80±5% and a photoperiod of 12:12h. Reaching the third instar, larvae were chosen for the assay beginning with exploratory dosage of three concentrations between 50 and 300ppm of synthesized molecule dissolved in dimethylsulfoxyde (DMSO) at 1%. For this assay, fifteen larvae were added in each plastic recipient with 50mL of water using three replications for each concentration and a negative control with DMSO, mortality was registered after 24 and 48h. For those molecules with highest mortality value, multiple dosage assays were applied, using six concentrations assigned in an asymmetric way and below the dosage when most of the larvae were death in exploratory dosage assay. In these dosages, ten larvae were added in each recipient with 100mL of water and mortality was registered after 24 and 48h. Four replicates, a negative control with DMSO and a positive control with the insecticide Temephos were used in this assay (Hemingway, 2005).

For those molecules with the highest values of mortality in multiple dosage larvae assay, adulticidal assays were developed using the CDC bottles protocol of Brogdon and Mcallister. Three replicates for each synthesized molecule concentration (30, 300 and 1000ppm) and a negative control with acetone were tested. The sides and bottom of the bottles were impregnated with three concentrations of synthesized molecule dissolved in acetone and four replicates for each concentration were made. Solvent used was allowed to dry overnight before the test. Ten adults were added to each bottle and each bottle was read for 2h started testing each 15min and a final check within 24h (Brogdon et al., 2005).

In vitro AChE inhibition assay

For AChE inhibition evaluation, a modified protocol of Ellman colorimetric assay with commercial Sigma–Aldrich®Electrophorus electricus AChE was used. This method determines the production of thiocholine caused by hydrolysis of acetylthiocholine, followed by its reaction with 5,5-dithiobis-2-nitrobenzoate ion (DTNB), confirming the development of reaction with a yellow color (Ellman et al., 1961).

Statistical analysis

Evaluation on A. aegypti larvae: LC50, LC98 and confidence limits values were calculated with Probit analysis (Ashford and Sowden, 1970). For the evaluation on A. aegypti adults: Data was tabulated and subjected to a normality test. When the data in each concentration were normal, an ANOVA test was carried out. If the distribution was not normal, non-parametric tests were conducted. The results were analyzed by Kruskal–Wallis and Lilliefors method using Statistica v11 software. Only data with p<0.05 were considered significant.

Results and discussionIn silico evaluation of the proposed compounds

Prior to synthetizing desired molecules, their physico-chemical parameters were evaluated, employing the Lipinski's rule (Lipinski, 2001). All products reported molecular weights below 500g/mol (216.28–274.36g/mol), a partition coefficient below 5 (LogP 1.612–3.004), hydrogen bond donors (expressed as the sum of OHs and NHs) less than 5 hydrogen bond acceptors (expressed as the sum of Ns and Os) lees than 10 and a polar surface area below 90Å (36.264–45.498Å). The obtained results showed that the proposed α-amino nitriles have pharmacokinetic profiles, and fulfill all parameters established. As shown in Supporting information (Table S1); no risk of toxicity was predicted for all compounds. With these results, synthesis of all proposed compounds was performed.

Obtention of title compounds

One of most common and simplest methods for the preparation of α-amino nitriles is the direct three component reaction of aldehyde, amine and potassium cyanide known as Strecker reaction (Wang et al., 2011; Strecker, 1850; Shafran et al., 1989; Otto and Opatz, 2014). As α-amino nitriles are important for synthesizing useful α-amino acids in both laboratory and industrial scale, Strecker-type reactions have been studied during the last few years looking mainly for new and better catalyst systems and cyanide sources. Looking for literature survey in Strecker synthesis, we could note that methods reported for this reaction often require not only the use of expensive reagents, and hard work-up as high catalyst loadings, high temperatures and pressures, but also involve long reaction times and tedious post-reaction procedures (Brahmachari, 2016). Moreover, all these reactions are carried out for small-scale α-amino nitrile preparation. Taking into consideration the latter comment and that the reported in vivo insecticidal assays need a large amount of substances, our attention addressed to a simple protocol, which involves the use of classical inorganic cyanide source KCN in the presence of sulfuric acid supported on silica gel (SSA, SiO2–O–SO3H), as a robust procedure proceeding under mild reaction conditions. In order to obtain the desired 311 α-amino nitriles (series B), commercial aldehyde components 1 (p-anisaldehyde, piperonal and 3,4-dimethoxybenzaldehyde) and amine components 2 (piperidine, pyrrolidine, morpholine, 2-methylpiperidine and 4-methylpiperidine) were chosen. Thus, a mixture of benzaldehydes 1, secondary amines 2, and KCN was stirred in the presence of SSA in acetonitrile at room temperature for 18h (Scheme 1).

Scheme 1.
(0.2MB).

Preparation of new α-amino nitriles 311 by modified Strecker reaction.

These mild reaction conditions allowed the large-scale preparation of the needed products as stable white crystal solids purified by flash column chromatography on alumina, using petroleum ether/ethyl acetate as eluent (Table 1). Structure of the obtained compounds 311 was confirmed by common spectral methods. The obtained α-amino nitriles showed in the IR spectra the characteristic CN bands appearing in the region of 2190–2222cm−1. Their structures were also confirmed with their 1H-, 13C-NMR and bidimensional experiments (COSY, HSQC), and supported by the mass spectrometric data (Supporting information, General Methods). Compounds 49 are diastereoisomeric mixtures, which resulted to be inseparable by conventional column chromatography, and were proceed to be tested as an enantiomeric mixture. All the α-amino nitriles obtained are moderate lipophilic compounds (1.61<LogP<3.00) with favorable values of TPSA (36.26–45.49) for agrochemical substances (Table 1). Having pure nine α-amino nitriles 311 with suitable pharmacokinetic profiles, we began their screening on A. aegypti mosquitoes and larvae.

In vivo evaluation of insecticidal activity on A. aegypti larvae and adults

Following by published protocols (Carreño et al., 2014), larvae between third and fourth instar of A. aegypti were exposed to different concentrations of the obtained girgensohnine analogs 311 (range of 300–50ppm). These diagnostic tests looked for the higher mortality rates. Larvae mortality produced by comp. 311, dissolved in DMSO at 1%, was determined after 24 and 48h, using fifteen larvae in each triplicated experiment. Analyzing results obtained (Table 2), it could be concluded that: (1) all compounds tested were active at the highest concentration (300ppm) registering between 93 and 100% of larvae mortality, (2) no mortality was observed in control evaluation, (3) only three α-amino nitriles (comp. 3, 4 and 7) conversed high larvicidal activity (100% of larvae mortality) at concentration of 120ppm, (4) at a concentration of 70ppm compound 4 still killed all fifteen larvae, and (5) in contrast of compounds 3 and 10 that reported high mortality at the lowest dose, all molecules reported low mortality at a concentration of 50ppm). Having these preliminary biological data, it could discuss on α-amino nitrile structure – larvicidal activity relationship. Noteworthy, all three active comp. 3, 4 and 7 possess para-methoxy group on aryl ring and piperidine or methyl-piperidine skeleton.

Table 2.

Larvicidal activity of compounds (Comp.) 311 on A. aegypti larvae after 24 and 48h.

Comp.  Concentrations, ppmControl (DMSO)  Time of exposure 
  300  120  70  50  L/D   
  L/Da  L/D  L/D  L/D     
3  0/15  0/15  7/8  8/7  15/0   
4  0/15  0/15  0/15  14/1  15/0   
5  0/15  11/4  13/2  14/1  15/0   
6  0/15  14/1  12/3  13/2  15/0  24
7  0/15  0/15  10/5  12/3  15/0   
8  0/15  7/8  14/1  15/0  15/0   
9  0/15  5/10  14/1  14/1  15/0   
10  1/14  8/7  ntb  10/5  15/0   
11  0/15  5/10  nt  13/2  15/0   
3  0/15  0/15  6/9  7/8  15/0   
4  0/15  0/15  0/15  12/3  15/0   
5  0/15  10/5  12/3  12/3  15/0   
6  0/15  12/3  11/4  13/2  15/0  48
7  0/15  0/15  10/5  12/3  15/0   
8  0/15  4/11  13/2  14/1  15/0   
9  0/15  5/10  15/0  14/1  15/0   
10  1/14  7/8  nt  10/5  15/0   
11  0/15  4/11  nt  14/1  15/0   
a

.L/D, live/death.

b

Not tested.

Incorporation of another methoxy substituent or dioxymethylen fragment into aryl ring of α-amino nitrile derivatives (comp. 5, 6, 8 and 9) decreased insecticide activity. α-Amino nitriles containing p-methoxyphenyl moiety (comp. 10 and 11) were inactive, thus chemical nature of cyclic amine is also important factor (piperidine skeleton vs pyrrolidine and morpholine rings).

Thus, the multiple dosage assays were applied for molecules 3 and 4, which showed the highest larvicidal activity, calculating their lethal concentrations (LC50 and LC98 values) with Probit analysis (Table 3). The chi-square values were significant at p<0.05 level. Possessing respective LC50 values of 50.55 and 69.59ppm, these two compounds resulted to be better than reported early α-amino nitrile A (Fig. 1) and could be considered as suitable models for developing new agents against A. aegypti larvae (Dias and Moraes, 2013).

Table 3.

LC50 values for compounds (Comp.) 3, 4 and temephos®.

Comp.  34Temephos 
  24h (confidence limits)  48h (confidence limits)  24h (confidence limits)  48h (confidence limits)  24h (confidence limits) 
LC50a (ppm)  50.55 (48.13–52.98)  48.53 (46.18–50.94)  69.59 (66.01–73.32)  64.51 (61.32–67.79)  0.0021 (0.0019–0.0022) 
LC98 (ppm)  86.39 (79.02–97.77)  82.18 (74.21–95.81)  139.54 (125.51–160.76)  122.07 (111.09–138.24)  0.0057 (0.0046–0.0080) 
χ2b  3.77  2.63  4.50  3.78  8.01 
AIc  8.82±0.82  8.98±0.99  6.79±0.55  7.42±0.58  9.21±0.56 
a

LC, lethal concentration.

b

Chi-square.

c

Slope standard deviation.

It should be commented that larvae mortality and their morphologic changes (darkness of the body and decreasing of their size) were observed 2h after starting the bioassay. Although their LC50 values are higher than those reported for commercial insecticides (temephos, LC50=0.0059ppm) (Harris et al., 2010), these α-amino nitriles showed in the in silico analysis good pharmacokinetic properties and low toxicity compared to temephos (Supporting information, Table S1). Evaluation of larvicidal activity using products extracted from plants have been studied by several authors. Low LC50 values such as those found for Croton zehntneri (LC50=26.2ppm) and Croton nepetaefolius (LC50=66.4ppm) essential oils have been reported (Pacelli et al., 2013), furthermore, Seo et al. (2015) evaluated the constituents of Apiaceae essential oils against Aedes albopictus, revealing for carvacrol a larvicidal activity of 80% at a concentration of 50ppm and an IC50 of 57ppm, showing a correlation between larvae mortality and acetylcholinesterase activity for this constituent of ajowan plant (Trachyspermum ammi), in spite of this activity, carvacrol is found in ajowan plant just in 0.55%. Although the active compounds found in these works showed larvicidal activity in a concentration range comparable to those previously reported for essential oils, it should be noted that these synthetic compounds can be produced in considerable quantities with high purity and that also their in silico analysis indicates a low toxicity in mammals.

Molecules 3 and 4, with the highest mortality values in larvae assay, were evaluated according to Brogdon and Mcallister method (Brogdon et al., 2005). Results showed that 2-(4-methoxyphenyl)-2-(piperidin-1-yl) acetonitrile 3 had the highest adulticidal activity with 100% of mortality at 300ppm. In contrast, 2-(4-methoxyphenyl)-2-(4-methylpiperidin-1-yl) acetonitrile (4) presented more than 50% of mortality at the same concentration (Fig. 2).

Fig. 2.
(0.09MB).

Mortality percentage on A. aegypti adults of α-amino nitriles 3,4 after 2h started the assay.

Calculated probability values in Kruskal–Wallis test confirmed significant differences between the concentrations used in this experiment with probability values below 0.05 at the concentrations of 300ppm and 1000ppm. After that, AChE inhibition properties of the obtained α-amino nitriles 311 were evaluated looking for some relationship between enzymatic and larvicidal activities.

In vitro AChE inhibition assay

Evaluation of enzyme inhibitory capacity of these compounds was performed by Ellman assay (Ellman et al., 1961), in which acetylcholinesterase from Electrophorus electricus (EC 3.1.1.7, Type VI-S) was used (Carreño et al., 2014). According to these results (Supporting information, Table S2), the synthesized compounds possess weak anti-AChE activity with IC50 values between 36.31 and 60.25ppm (148.80–259.40μM). It could be noted that 2-(4-methoxyphenyl)-2-(4-methylpiperidin-1-yl) acetonitrile 4 resulted to be the more active compound, followed by 2-(4-methoxyphenyl)-2-(2-methylpiperidin-1-yl)acetonitrile 7. It was also observed that pyrrolidine or morpholine rings (comp. 10 and 11) did not contribute in AChE inhibition activity. Taking into consideration that agrochemical Propoxur, insecticide and acetylcholinesterase inhibitor, exhibited IC50 0.0150ppm and comparing results of enzyme and insecticide activities, it was observed that more active comp. 3 against A. aegypti larvae (LC50 50.55ppm) showed IC50 60.25μg/mL, while another active comp. 4 (LC50 69.59ppm) exhibited an IC50 value of 36.31ppm that means there is not relationship between AChE inhibition activity and larvicidal activity of the tested compounds.

Conclusion

New α-amino nitriles analogs of alkaloid girgensohnine were designed and synthetized. These compounds were obtained in good yields under mild conditions as stable white powders with defined melting points. Prior to synthetic and enzymatic studies ADME parameters of these molecules were calculated revealing their acceptable pharmacokinetic profiles showing in silico favorable physicochemical properties and a low risk of toxicity for humans. Their insecticide action tested on mosquito larvae reported mortality at concentrations below 120ppm, highlighting compounds 3 and 4 with LC50 values of 50.55 and 69.59ppm, respectively. Insecticide action test on mosquitoes showed a mortality of 100% for compound 3 and a 53.33% of mortality for compound 4 when a concentration of 30ppm was evaluated. Additionally, it was found that these molecules possess weak anti-AChE activity with values of IC50=36.31–60.25ppm (148.80–259.40μM) indicating that AChE enzyme could not be a principal target responsible for their insecticide activity and that the evaluated molecules would present another mechanism or mode of action on insects. With these biological results, α-amino nitriles 3 and 4 could be considered as suitable models for developing new agents against A. aegypti larvae as insecticide candidates. Further investigations on detailed biochemical mechanism of action, as detoxifying enzyme evaluations, are now under way in our laboratories and their results will be published elsewhere.

Conflicts of interest

The authors declare no conflict of interest concerning this work.

Acknowledgements

This work was supported by the Research program: “Financial support from Departamento Administrativo de Ciencia, Tecnología e Innovación de Colombia, COLCIENCIAS and Patrimonio Autónomo, Fondo Nacional de Financiamiento para la Ciencia, Francisco José de Caldas”, contracts code 110265740528 (grant 624-2014). A.L.C.O. thanks COLCIENCIAS for the fellowship No. 567/2012 to carry out doctoral studies.

Appendix A
Supplementary data

The following are the supplementary data to this article:

mmc1.docx

References
Aguirre-obando et al., 2015
O.A. Aguirre-obando,A.C.D. Bona,J.E.L. Duque,M.A. Navarro-silva
Insecticide resistance and genetic variability in natural populations of Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Colombia
Zoologia, 32 (2015), pp. 14-22
Insecticide
Ashford and Sowden, 1970
J. Ashford,R.R. Sowden
Multi-variate probit analysis
Biometrics, 26 (1970), pp. 535-546
Bona et al., 2016
A.C.D. Bona,R.F. Chitolina,M.L. Fermino,L. de Castro Poncio,A. Weiss,J.B.P. Lima,N. Paldi,E.S. Bernardes,J. Henen,E. Maori
Larval application of sodium channel homologous dsRNA restores pyrethroid insecticide susceptibility in a resistant adult mosquito population
Parasit. Vectors, 9 (2016), pp. 397-410 http://dx.doi.org/10.1186/s13071-016-1634-y
Braga and Valle, 2007
I.A. Braga,D. Valle
Aedes aegypti: inseticidas, mecanismos de ação e resistência
Epidemiol. Serv. Saúde, 16 (2007), pp. 279-293
Brahmachari, 2016
G. Brahmachari
Design of organic transformations at ambient conditions: our sincere efforts to the cause of green chemistry practice
Chem. Rec., 16 (2016), pp. 98-123 http://dx.doi.org/10.1002/tcr.201500229
Brogdon et al., 2005
W.G. Brogdon,J. Rojanapremsuk,S. Suvannadabba,W. Pandii,J.W. Jones,R. Sithiprasasna,P. Provincial,P. Health,V. Biology,M. Sciences
Bottle and biochemical assays on temephos
Southeast Asian J. Trop. Med. Public Heal., 36 (2005), pp. 417-425
Cao-Lormeau et al., 2016
V.-M. Cao-Lormeau,A. Blake,S. Mons,S. Lastère,C. Roche,J. Vanhomwegen,T. Dub,L. Baudouin,A. Teissier,P. Larre,A.-L. Vial,C. Decam,V. Choumet,S.K. Halstead,H.J. Willison,L. Musset,J.-C. Manuguerra,P. Despres,E. Fournier,H.-P. Mallet,D. Musso,A. Fontanet,J. Neil,F. Ghawché
Guillain-Barré syndrome outbreak associated with Zika virus infection in French Polynesia: a case–control study
Carreño et al., 2014
A.L. Carreño,L.Y. Vargas Méndez,L.J.E. Duque,V.V. Kouznetsov
Design, synthesis, acetylcholinesterase inhibition and larvicidal activity of girgensohnine analogs on Aedes aegypti, vector of dengue fever
Eur. J. Med. Chem., 78 (2014), pp. 392-400 http://dx.doi.org/10.1016/j.ejmech.2014.03.067
Carreño Otero et al., 2018
A.L. Carreño Otero,A.M. Palacio-Cortés,M.A. Navarro-Silva,V.V. Kouznetsov,L J.E. Duque
Behavior of detoxifying enzymes of Aedes aegypti exposed to girgensohnine alkaloid analog and Cymbopogon flexuosus essential oil
Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol., 204 (2018), pp. 14-25
Dias and Moraes, 2013
C.N. Dias,D.F.C. Moraes
Essential oils and their compounds as Aedes aegypti L (Diptera: Culicidae) larvicides: review
Parasitol. Res., 113 (2013), pp. 565-592 http://dx.doi.org/10.1007/s00436-013-3687-6
Ellman et al., 1961
G. Ellman,D. Courtney,A. Valentino,M. Featherstone
A new and rapid colorimetric determination of acetylcholinesterase activity
Biochem. Pharmacol., 7 (1961), pp. 88-95
Ertürk et al., 2012
E. Ertürk,M.A. Tezeren,T. Atalar,T. Tilki
Regioselective ring-opening of epoxides with ortho-lithioanisoles catalyzed by BF 3-OEt 2
Tetrahedron, 68 (2012), pp. 6463-6471
Flores-Conde et al., 2012
M.I. Flores-Conde,L. Reyes,R. Herrera,H. Rios,M.A. Vazquez,R. Miranda,J. Tamariz,F. Delgado
Highly regio- and stereoselective Diels–Alder cycloadditions via two-step and multicomponent reactions promoted by infrared irradiation under solvent-free conditions
Int. J. Mol. Sci., 13 (2012), pp. 2590-2617 http://dx.doi.org/10.3390/ijms13032590
Grisales et al., 2013
N. Grisales,R. Poupardin,S. Gomez,I. Fonseca-Gonzalez,H. Ranson,A. Lenhart
Temephos resistance in Aedes aegypti in Colombia compromises dengue vector control
PLoS Negl. Trop. Dis., 7 (2013), pp. e2438 http://dx.doi.org/10.1371/journal.pntd.0002438
Guedes et al., 2017
R.N.C. Guedes,S.S. Walse,J.E. Throne
Sublethal exposure, insecticide resistance, and community stress
Curr. Opin. Insect Sci., 21 (2017), pp. 47-53 http://dx.doi.org/10.1016/j.cois.2017.04.010
Guzman and Harris, 2015
M.G. Guzman,E. Harris
Dengue
Harris et al., 2010
A.F. Harris,S. Rajatileka,H. Ranson
Pyrethroid resistance in Aedes aegypti from Grand Cayman
Am. J. Trop. Med. Hyg., 83 (2010), pp. 277-284 http://dx.doi.org/10.4269/ajtmh.2010.09-0623
Hayes, 2009
E.B. Hayes
Zika virus outside Africa
Emerg. Infect. Dis., 15 (2009), pp. 1347-1350 http://dx.doi.org/10.3201/eid1509.090442
Hemingway, 2005
J. Hemingway
Guidelines for laboratory and field testing of mosquito larvicides
World Health Organization, (2005),
Hemingway et al., 2004
J. Hemingway,N.J. Hawkes,L. McCarroll,H. Ranson
The molecular basis of insecticide resistance in mosquitoes
Insect Biochem. Mol. Biol., 34 (2004), pp. 653-665 http://dx.doi.org/10.1016/j.ibmb.2004.03.018
Instituto Nacional de Salud INS, 2017
Instituto Nacional de Salud INS
Boletín epidemiológico semana 43 de 2017
(2017)
Ioos et al., 2014
S. Ioos,H.P. Mallet,I. Leparc Goffart,V. Gauthier,T. Cardoso,M. Herida
Current Zika virus epidemiology and recent epidemics
Med. Mal. Infect., 44 (2014), pp. 302-307 http://dx.doi.org/10.1016/j.medmal.2014.04.008
Kawada et al., 2014
H. Kawada,K. Ohashi,G.O. Dida,G. Sonye,S.M. Njenga,C. Mwandawiro,N. Minakawa
Preventive effect of permethrin-impregnated long-lasting insecticidal nets on the blood feeding of three major pyrethroid-resistant malaria vectors in western Kenya
Paras. Vectors, 7 (2014), pp. 1-9
Lima et al., 2011
E.P. Lima,M.H.S. Paiva,A.P. de Araújo,E.V.G. da Silva,U.M. da Silva,L.N. de Oliveira,A.E.G. Santana,C.N. Barbosa,C.C. de Paiva Neto,M.O.F. Goulart,C.S. Wilding,C.F.J. Ayres,M.A.V. de Melo Santos
Insecticide resistance in Aedes aegypti populations from Ceará
Brazil. Parasit. Vectors, 4 (2011), pp. 5 http://dx.doi.org/10.1186/1756-3305-4-5
Lipinski, 2001
C.A. Lipinski
Drug-like properties and the causes of poor solubility and poor permeability
J. Pharmacol. Toxicol. Methods, 44 (2001), pp. 235-249
Lipinski et al., 2001
C.A. Lipinski,F. Lombardo,B.W. Dominy,P.J. Feeney
Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings
Adv. Drug Deliv. Rev., 46 (2001), pp. 3-26
Liu et al., 2016
X.C. Liu,D. Lai,Q.Z. Liu,L. Zhou,Q. Liu,Z.L. Liu
Bioactivities of a new pyrrolidine alkaloid from the root barks of Orixa japonica
Molecules, 21 (2016), pp. 1-8
Low et al., 2015
V.L. Low,W.Y. Vinnie-Siow,Y.A.L. Lim,T.K. Tan,C.S. Leong,C.D. Chen,A.A. Azidah,M. Sofian-Azirun
First molecular genotyping of A302S mutation in the gamma aminobutyric acid (GABA) receptor in Aedes albopictus from Malaysia
Trop. Biomed., 32 (2015), pp. 554-556
Musso and Gubler, 2016
D. Musso,D.J. Gubler
Zika virus
Clin. Microbiol. Rev., 29 (2016), pp. 488-524
Nahrstedt et al., 1993
A. Nahrstedt,M. Lechtenberg,A. Brinker,D.S. Seigler,R. Hegnauer
4-Hidroximandelonitrile glucosides, dhurrin in Sucleya suckleyana and taxiphillin in Girgensohnia oppositiflora (chenopodiaceae)
Phytochemistry, 33 (1993), pp. 847-850
Otto and Opatz, 2014
N. Otto,T. Opatz
Heterocycles from α-Aminonitriles
Chem. – Eur. J., 20 (2014), pp. 13065-13077
Pacelli et al., 2013
G. Pacelli,G.P. De Lima,T.M. De Souza,G.D.P. Freire,D.F. Farias,A.P. Cunha,N. Maria,P. Silva,S.M. De Morais
Further insecticidal activities of essential oils from Lippia sidoides and Croton species against Aedes aegypti L.
Parasitol. Res., 112 (2013), pp. 1953-1958 http://dx.doi.org/10.1007/s00436-013-3351-1
Pang, 2014
Y.P. Pang
Insect acetylcholinesterase as a target for effective and environmentally safe insecticides
Advances in Insect Physiology, 1st ed., Elsevier Inc., (2014) http://dx.doi.org/10.1016/B978-0-12-417010-0.00006-9
Prajapati et al., 2005
V. Prajapati,A.K. Tripathi,K.K. Aggarwal,S.P.S. Khanuja
Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus
Bioresour. Technol., 96 (2005), pp. 1749-1757 http://dx.doi.org/10.1016/j.biortech.2005.01.007
Rashad et al., 2014
A.A. Rashad,S. Mahalingam,P.A. Keller
Chikungunya virus: emerging targets and new opportunities for medicinal chemistry
J. Med. Chem., 57 (2014), pp. 1147-1166 http://dx.doi.org/10.1021/jm400460d
Rasmussen et al., 2016
S.A. Rasmussen,D.J. Jamieson,M.A. Honein,L.R. Petersen
Zika virus and birth defects — reviewing the evidence for causality
N. Engl. J. Med., 374 (2016), pp. 1-7 http://dx.doi.org/10.1056/NEJMp1514202
Rodriguez-Morales, 2015
A.J. Rodriguez-Morales
Zika: the new arbovirus threat for Latin America
J. Infect. Dev. Ctries, 9 (2015), pp. 684-685
Russell et al., 2004
R.J. Russell,C. Claudianos,P.M. Campbell,I. Horne,T.D. Sutherland,J.G. Oakeshott
Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides
Pestic. Biochem. Physiol., 79 (2004), pp. 84-93
Seo et al., 2015
S.M. Seo,C.S. Jung,J. Kang,H.R. Lee,S.W. Kim,J. Hyun,I.K. Park
Larvicidal and acetylcholinesterase inhibitory activities of apiaceae plant essential oils and their constituents against Aedes albopictus and formulation development
J. Agric. Food Chem., 63 (2015), pp. 9977-9986 http://dx.doi.org/10.1021/acs.jafc.5b03586
Shafer et al., 2005
T.J. Shafer,D.A. Meyer,K.M. Crofton
Developmental neurotoxicity of pyrethroid insecticides: critical review and future research needs
Environ. Health Perspect., 113 (2005), pp. 123-136
Shafran et al., 1989
Y.M. Shafran,V.A. Bakulev,V.S. Mokrushin
Synthesis and properties of a-aminonitriles
Russ. Chem. Rev., 58 (1989), pp. 148-162
Shan et al., 2016
C. Shan,X. Xie,A.D.T. Barrett,M.A. Garcia-Blanco,R.B. Tesh,P.F.D.C. Vasconcelos,N. Vasilakis,S.C. Weaver,P.Y. Shi
Zika virus: diagnosis therapeutics, and vaccine
ACS Infect. Dis., 2 (2016), pp. 170-172 http://dx.doi.org/10.1021/acsinfecdis.6b00030
Simmons et al., 2012
Simmons
Current concepts, Dengue
N. Engl. J. Med., 366 (2012), pp. 1423-1432 http://dx.doi.org/10.1056/NEJMra1110265
Smith et al., 2016
L.B. Smith,S. Kasai,J.G. Scott
Pyrethroid resistance in Aedes aegypti and Aedes albopictus: important mosquito vectors of human diseases
Pestic. Biochem. Physiol., 133 (2016), pp. 1-59 http://dx.doi.org/10.1016/j.pestbp.2016.03.005
Sparks and Nauen, 2015
T.C. Sparks,R. Nauen
IRAC: mode of action classification and insecticide resistance management
Pestic. Biochem. Physiol., 121 (2015), pp. 122-128 http://dx.doi.org/10.1016/j.pestbp.2014.11.014
Staples and Fischer, 2014
J.E. Staples,M. Fischer
Chikungunya virus in the Americas — what a vectorborne pathogen can do
N. Engl. J. Med., 371 (2014), pp. 887-889 http://dx.doi.org/10.1056/NEJMp1407698
Stevens et al., 2009
A.J. Stevens,M.E. Gahan,S. Mahalingam,P.A. Keller
The medicinal chemistry of dengue fever
J. Med. Chem., 52 (2009), pp. 7911-7926 http://dx.doi.org/10.1021/jm900652e
Strecker, 1850
A. Strecker
Ueber die künstliche Bildung der Milchsäure und einen neuen, dem Glycocoll homologen Körper
Eur. J. Org. Chem., 75 (1850), pp. 27-45
Vargas and Kouznetsov, 2013
L.Y. Vargas,V.V. Kouznetsov
First girgensohnine analogs prepared through InCl3-catalyzed strecker reaction and their bioprospection
Curr. Org. Synth., 10 (2013), pp. 969-973
Wang et al., 2011
J. Wang,X. Liu,X. Feng
Asymmetric strecker reactions
Chem. Rev., 111 (2011), pp. 6947-6983 http://dx.doi.org/10.1021/cr200057t
Weaver et al., 2016
S.C. Weaver,F. Costa,M.A. Garcia-Blanco,A.I. Ko,G.S. Ribeiro,G. Saade,P.Y. Shi,N. Vasilakis
Zika virus: history, emergence, biology, and prospects for control
Weaver and Reisen, 2010
S.C. Weaver,W.K. Reisen
Present and future arboviral threats
Antiviral Res., 85 (2010), pp. 328-345 http://dx.doi.org/10.1016/j.antiviral.2009.10.008
WHO, 2012
World Health Organisation
Report of a WHO Technical Working Group Meeting on Dengue Prevention and Control, Meeting Report,
Zanluca et al., 2015
C. Zanluca,V.C.A. De Melo,A.L.P. Mosimann,G.I.V. Dos Santos,C.N.D. dos Santos,K. Luz
First report of autochthonous transmission of Zika virus in Brazil
Mem. Inst. Oswaldo Cruz, 110 (2015), pp. 569-572 http://dx.doi.org/10.1590/0074-02760150192
Corresponding authors. (Vladimir V. Kouznetsov kouznet@uis.edu.co)
Copyright © 2018. Sociedade Brasileira de Entomologia
Rev Bras Entomol 2018;62:112-8 - Vol. 62 Num.2 DOI: 10.1016/j.rbe.2018.01.004