In vitro evaluation of 1,3,4-oxadiazolethiones for insecticidal activity in agricultural pest cell lines

Avaliação in vitro de 1,3,4-oxadiazoltionas para atividade inseticida em linhagens celulares de pragas agrícolas

Abstract

The paper was dedicated to the investigation of the insecticidal activities of recently prepared 1,3,4-oxadiazole compounds and their in vitro efficacy was determined with the help of cell lines taken out of Helicoverpa zea, Trichoplusia ni and Lymantria dispar. The MTT assay yielded results that showed that the compounds synthesized had insecticidal activities between 88.4 -97.2 - 100 µM that were similar to those of the commercial insecticide Imidacloprid 2F of 96.4 -98.3 - 100 µM. In silico testing showed that the newly prepared compounds bind to a variety of amino acid residues either with several interaction modes at the same time such as π-π stacking, hydrogen bonding, π-π anion interaction, and π-π alkyl interaction, specifically against acetylcholinesterase (AChE) of the insect species used. Since they are relatively harmless to the environment, these 1,3,4-oxadiazole derivatives have a potential as alternative agents in sustainable pests control in agriculture, which could be beneficial compared to the use of conventional insecticides. The results highlight the effectiveness of such compounds as effective agents against insect cell lines in agriculture. However, in order to completely prove their practical implementation, there is a need to conduct additional in vivo research and field tests are suggested to verify their effectiveness in real-world situations.

Keywords:
Helicoverpa zea; Trichoplusia ni; Lymantria dispar; oxadiazole; AChE; insecticidal activity

Resumo

O artigo foi dedicado à investigação das atividades inseticidas de compostos de 1,3,4-oxadiazole recentemente preparados, e sua eficácia in vitro foi determinada com a ajuda de linhagens celulares derivadas de Helicoverpa zea, Trichoplusia ni e Lymantria dispar. O ensaio de MTT produziu resultados que mostraram que os compostos sintetizados apresentaram atividades inseticidas entre 88,4–97,2–100 µM, semelhantes às do inseticida comercial Imidacloprid 2F (96,4–98,3–100 µM). Os testes in silico demonstraram que os compostos recém-preparados se ligam a diversos resíduos de aminoácidos através de vários modos de interação simultâneos, como empilhamento π-π, ligação de hidrogênio, interação π-ânion e interação π-alquil, especificamente contra a acetilcolinesterase (AChE) das espécies de insetos utilizadas. Por serem relativamente inofensivos ao meio ambiente, esses derivados de 1,3,4-oxadiazole têm potencial como agentes alternativos no controle sustentável de pragas na agricultura, podendo ser benéficos em comparação com o uso de inseticidas convencionais. Os resultados destacam a eficácia desses compostos como agentes efetivos contra linhagens celulares de insetos na agricultura. No entanto, para comprovar completamente sua aplicabilidade prática, é necessário realizar pesquisas adicionais in vivo, e testes de campo são recomendados para verificar sua eficácia em condições reais.

Palavras-chave:
Helicoverpa zea; Trichoplusia ni; Lymantria dispar; oxadiazole; AChE; atividade inseticida

1. Introduction

The pest insects are posing a major menace of agricultural sustainability in the world, leading to massive losses in crops, compromised food security and annual economic losses worth billions of dollars. The growing resistance to traditional insecticides is increasing the urgency of new environmentally harmless approaches to the elimination of pests (Quandahor et al., 2024; Folake et al., 2023; Simon-Delso et al., 2015; Kollmeyer et al., 1999). Insect pests can cause losses in yield of up to 40% in some areas, which is a significant economic cost of billions of dollars a year in both the developed and developing agricultural systems of the United States, Europe, Asia and Africa (Simon-Delso et al., 2015; Mdeni et al., 2022). Consequently, it is crucial that effective and innovative pest -management approaches, including the production of new insecticidal compounds and breeding of pest-resistant crop cultivars, be developed to ensure agricultural sustainability, strengthening food security and maintaining ecosystem integrity in the face of changing environmental and biological pressures (Quandahor et al., 2024; Folake et al., 2023).

The use of insecticides is generally viewed as a convenient and effective mode of control over pests, thus, supporting its use and increasing demand. Insecticides are sub-types of pesticides that are specifically used to kill, repel or inhibit the growth and development of insects at different stages of life (Quandahor et al., 2024; Folake et al., 2023; Simon-Delso et al., 2015; Erika et al., 2023; Mdeni et al., 2022). Even though there are still conventional insecticides that are highly effective, their use is highly questionable due to their toxicity to non-target beneficial insects and the growing resistance development in the pest population (Simon-Delso et al., 2015). Insecticides have diverse modes of action, and they include interference with hormonal control, neural transmission, and cellular damage (Simon-Delso et al., 2015; Araújo et al., 2023). However, before the field implementation toxicological evaluations should be carried out to determine that such agents do not cause residues that are harmful or cause long term effects that are harmful to the human health and environment (Kollmeyer et al., 1999; Vidau et al., 2009).

The compounds of synthetic insecticides play a critical role in protecting farm crops against pest attacks and consequently lead to high production. These substances consist of a unique category of chemicals designed to be useful in handling insect pests, which impart their action at various physiological tiers, including interference with the insect nervous system, reproduction, and interference with metabolic functions (Jeschke et al., 2014; Jeanguenat, 2012; Luczynski and Kudelko, 2022).

The insecticides could be divided into various groups such as organophosphates, carbamates, pyrethroids, neonicotinoids and organochlorines and each of them interacting with different physiological systems of insects could be chosen based on particular pest management needs. An example of this is the organophosphates which block acetylcholinesterase and hence disrupt the insect nervous system leading to paralysis and death. Examples of such representatives are malathion (C₁₀H₁₉O₆PS₂) and diazinon (C₁₂H₂₁N₂O₃PS) (Erika et al., 2023). Synthetic analogs of natural pyrethrins, known as pyrethroids, act by blocking sodium channels in the insect nervous system; examples of this group include permethrin (C₂₁H₂₀Cl₂O₃) and deltamethrin (C₂₂H₁₉Br₂NO₃), both of which are highly potent insecticides and act rapidly (Davies et al., 2007). Carbamates like carbaryl (C₁₂H₁₁NO₂) and methomyl (C₅H₁₀N₂O₂S) (Mdeni et al., 2022) also have an inhibitory effect on neuronal activity, although they are less persistent and tend to be less toxic (Folake et al., 2023; Simon-Delso et al., 2015; Erika et al., 2023; Davies et al., 2007; Mdeni et al., 2022; National Toxicology Program, 2019; Kollmeyer et al., 1999).

Neonicotinoids are a group of neuroactive insecticides, which share a similar chemical structure to that of nicotine, in that they stimulate acetylcholine receptors. Examples of representative compounds are imidacloprid (C₉H₁₀ClN₅O₂) and thiamethoxam (C₈H₁₀ClN₅O₃S); these agents are widely used under commercial names like Admire, Advantage, Bayer Advanced, Decis, Condifor, Arrivo, Gaucho, Marathon, Merit, Premier, Premise, Provado (Nurmatov and Ochilov, 2013; Plumlee, 2004) and Imidacloprid 2F (Quali-Pro, USA) (Yentel, 2011). Despite its effectiveness, the long-term use of neonicotinoids is linked to harmful ecological effects, the most significant being the impact on social insects (Yentel, 2011; Kollmeyer et al., 1999; National Toxicology Program, 2019; Pesticide Information Profiles, 2012). They are highly toxic to aquatic biota including shrimps and aquatic insect species especially. Imidacloprid is particularly dangerous to honeybees (Reuters, 2018).

One example of a synthetic insecticide that disrupts γ-aminobutyric acid (GABA) receptors is phenylpyrazoles (such as fipronil) (Vidau et al., 2009). Butenolides are newer classes with a similar mode of action as neonicotinoids: they act like acetylcholine receptors, but add a unique pharmacophore. These wide spectrum systemic insecticides are mostly sprayed on crops, seeds and soil as protection against pests (Jeschke et al., 2014; Hesselbach and Scheiner, 2018). Diamides specifically target insect ryanodine receptors (RyRs) and thus affect the calcium homeostasis, resulting in the onset of paralysis, feeding cessation, and death. The first insecticide to be registered in this category was flubendiamide (Ebbinghaus-Kintscher et al., 2006; Jeanguenat, 2012; Du and Hu 2023).

On the other hand, 1,3,4-oxadiazole analogues are seen as an alternative to be pursued, due to their less harmful ecological toxicity and new mechanistic profiles (Ziyaev et al., 2017). Oxadiazoles: five-membered heterocyclic substances, with one oxygen atom and two nitrogen atoms, based on furan (Ziyaev et al., 2025). Their wide spectrum of biological action makes them useful in agriculture, where they are used as herbicides, insecticides, and plant-protective agents against bacterial, viral and fungal pathogens (Luczynski and Kudelko, 2022; Ziyaev et al., 2023, 2017).

The extensive use of an already restricted range of insecticides has triggered the development of resistance in pests, which has led to the need to continually synthesize new compounds with insect-specific mechanisms of action and altered molecular structures to maintain high and stable crop productivity (Araújo et al., 2023). Although 1,3,4-oxadiazole derivatives have been the subject of intensive pharmacological research, little has been done to study their insecticidal potential, especially in vitro activity against agricultural pests (Glomb and Świątek, 2021; Akram et al., 2018).

In our current study, we discussed the biological activity of new S-alkyl and S-acyl analogs of 1,3,4-oxadiazoles that had earlier been synthesized by our laboratory (Ismailova et al., 2019; Okmanov et al., 2023; Ziyaev et al., 2017). Herein, this is the first time that the in vitro activity of these compounds are tested on insect cell lines. To overcome this scarsity of data on their insecticidal potential, the derivatives were assessed in vitro in relation to cell lines of Helicoverpa zea, Trichoplusia ni, and Lymantria dispar.

Helicoverpa zea (corn earworm) and Trichoplusia ni (cabbage looper) are important agricultural pests, which are in the family, Noctuidae. H. zea causes harm to corn and other crops, but T. ni larvae consume over 160 plant species, majorly, cruciferous vegetables including cabbage and broccoli (Lambert et al., 1996; Light et al., 1993; Capinera., 2001; United States Agricultural Research Service, 2017). Lymantria disperse (gypsy moth) is an extremely destructive forest pest that belongs to the family Erebidae and is predisposed to feeding on the trees of the genus Decidua, such as oak, birch, and poplar, to name a few (Kimber, 2024; Akram et al., 2022; Boukouvala et al., 2022).

2. Materials and Methods

2.1. Synthesis of S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate 4, S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate 5 and 2-[(2-chloro-6-fluorobenzyl)sulfanil]-5-[4-(dimethylamino)phenyl]-1,3,4-oxadiazole 6

Novel derivatives of 1,3,4-oxadiazole were synthesized as detailed in our prior research publication (Ziyaev et al., 2017; Ismailova et al., 2019; Okmanov et al., 2023). Figures 1 and 2 depict the synthesis of the novel 1,3,4-oxadiazole derivatives.

Figure 1
Synthesis scheme of S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate 4 and S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate 5.

Figure 2
Synthesis Scheme of 2-[(2-chloro-6-fluorobenzyl)sulfanyl]-5-[4-(dimethylamino)phenyl]-1,3,4-oxadiazole 6.

The structures of the synthesized compounds were confirmed by IR, ¹H and ¹³C NMR spectra (Ziyaev et al., 2023, 2017), as well as by X-ray structural analysis (XRD) (Okmanov et al., 2023).

2.2. Cell line

Three insect cell lines, namely, Helicoverpa zea, Trichoplusia ni and Lymantria dispar were used in in vitro screening of insecticidal activity. The collection of the Laboratory of Molecular Genetics of the Institute of Chemistry of Plant Substances kept these lines. Cells were grown in the Grace Insect Medium (Gibco, USA), which was supplemented with 1.0% antimycotic-antibiotic solution (Lonza, Belgium), 2 mM glutamine (Himedia, India), and 10% fetal bovine serum (FBS) (Gibco, USA) (Drugmand et al., 2011).

2.3. Viable cell density was determined

The Trypan Blue exclusion method was used to determine cell viability and density

(Strober, 2015). Cells were combined with 0.4% of Trypan Blue solution in a 1:1 ratio and left to incubate at room temperature over a period of 5 minutes. Viable (unstained) and non-viability (blue-stained) cells were counted using Neubauer hemocytometer (Marienfeld, Catalog No. 0640010) using a 10x objective. Cell density, OD₆₀₀, and protocols of long-term storage and recovery followed standard protocols as described in the Gibco Insect Cell Culture User Guide (USA).

2.4. Bioassay in vitro against Helicoverpa zea, Lymantria dispar and Trichoplusia ni

In a bid to measure the insecticidal in vitro efficacy, the above insect cell lines were cultured and exposed to the MTT colorimetric assay to determine viability and estimate insecticidal impacts. Imidacloprid 2F (Quali-Pro, USA) was used as the positive control and Insect Medium of Grace (Gibco, USA) was used as the negative control.

To determine the assay, the cells were seeded in a 96-well plate (Costar, USA) at 2×10³ cells per well. Test compounds or Imidacloprid 2F (active ingredient: 21.8 to 100 mM/mL) were added to each well at a concentration of 10-100 µM/mL. The medium was changed after 24 h incubation with fresh Grace medium with 0.05 mg/ml - MTT. After another 4 h incubation, the MTT solution was removed, and 100 µL dimethyl sulfoxide (DMSO) was added in order to dissolve the formazan crystals. An EnSpire 2600 spectrophotometer (PerkinElmer, USA) was used to read the absorbance at 620 nm. Averages of the inhibition were obtained using three replicates (Turaeva et al., 2024).

2.5. Simulation of molecular docking

Extensive molecular docking investigations were conducted to determine the binding reactions of new 1,3,4-oxadiazole analogs with the target enzyme acetylcholinesterase (AChE). Four computational platforms were used, which include Discovery Studio 2019, SwissDock, CB-Dock2, and Neurosnap. The crystal structure of AChE (PDB ID: 6XYS; (RCSB PDB Protein Data Bank, 2025) was ready through the addition of the missing hydrogen atoms, the assignment of correct protonation states, and the energy minimization. The preparation of ligands was done by building molecular structures, explicit hydrogen addition, partial charges assignment, generation of multiple conformations, and optimization of the ligands. The calculation of dockings was carried out separately in each platform to investigate the binding modes and verify predictions. Consensus binding modes involving results were analyzed by ranking by binding scores and the key molecular interactions were examined. This multi-platform strategy was intended to be used to provide dependable, sturdy predictions of molecular recognition processes.

2.6. Statistical analysis

The results obtained were statistically processed in OriginPro 8.6 (Origin Lab Corp, USA). The results of the experiment were presented in the form of mean values (obtained through the calculation of three replicates) and the standard deviation. The statistical significance of the results was considered as p≤0.05.

3. Results

The first step of the research involved the determination of the best cultivation and maintenance conditions of insect cell lines. The cell lines of Hi5, Hz-AM1 and IPLB-LdEp were cultured in 25 mL, filter-free culture flasks at 26-27°C in incubator systems. The cells were kept in serum-free Express Five SFM medium, but the cells of Hz-AM1 and IPLB-LdEp were kept in Grace medium with 10% fetal bovine serum (FBS). During the experiment, the signs of cellular health, such as density, viability, and the rate of proliferation, were measured, and the obtained results are discussed in the following section.

Considering the unique biological attributes of the Trichoplusia ni (Hi5) line compared to Hz-AM1 and IPLB-LdEp, the experimental protocol was modified to that effect. The propagation of Hi5 cells took place in the Express Five SFM medium at 27°C in 25 cm² culture flasks. Cell count, viability and optical density at 600nm (OD₆₀₀) were determined at 0, 24, 48, 72 and 96 h using Trypan Blue exclusion and the results are tabulated in Table 1.

Table 1
Growth kinetics of Trichoplusia ni (Hi5) cells in Express Five SFM Medium over 96 hours.

Nº	Time (hours)	Viable cell density (×106 cells/mL)	Optical density (OD600)	Cell viability (%)
1	0	0.5	0.05	98 ±0.12
2	24	1.2	0.13	96 ±0.12
3	48	2.8	0.30	95 ±0.12
4	72	4.2	0.48	90 ±0.12
5	96	4.8	0.56	87 ±0.12

Kinetics of growth of the cells of Trichoplusia ni (High Five) were tested in the simple culture flasks at 26-27°C in Express Five SFM medium during a 96 hour time span. The viable cell density at the point of 0.5×10⁶ cells/m decreased to 4.8×10⁶ cells/ml at 96 h and OD₆₀₀ rose from 0.05 to 0.56, while viability decreased from 98% to 87%. The most significant exponential growth rate occurred between 24 and 72 h; after 72 h, the rate of growth slowed down, which was likely to be caused by the lack of oxygen during nonagitation. High Five cells are therefore seen to be quite convenient to be used in baculovirus research, due to their high biomass production and fast growth.

Since the Hz-AM1 and LdEp cells differ in their properties compared to the High Five cells, the experimental procedures that followed were modified. The cultivation of the cells was in Grace Insect Medium with 10% FBS at 27°C in 25 cm² flasks. Trypan Blue exclusion was used to measure quantitatively the number of cells, their viability and density (OD₆₀₀) at 24 hours (0, 24, 48, 72, 96 h). Table 2 below shows the resulting data.

Table 2
Growth kinetics of Helicoverpa zea (Hz-AM1) and Lymantria dispar (LdEp) cells in Grace’s Insect Medium over 96 hours.

Time (hours)	Viable cell density (×106 cells/mL)	Optical density (OD600)	Cell viability (%)
0 (Hz-AM1)	0.22	0.06	97 ±0.11
24	0.36	0.11	95 ±0.11
48	1.00	0.30	93 ±0.11
72	2.50	0.75	89 ±0.11
96	3.48	1.05	86 ±0.12
0 (LdEp)	0.5	0.05	98 ±0.11
24	1.1	0.11	96 ±0.11
48	2.4	0.24	94 ±0.11
72	3.6	0.36	91 ±0.12
96	4.2	0.45	88 ±0.12

The kinetics of growth of cells of Helicoverpa zea in a 96-hour period were outlined into three phases. The lag phase (0-24 h) showed a small increase of cell density 0.22×10⁶ to 0.36×10⁶ cells/mL, a parallel increase in optical density of 600 nm (OD₆₀₀) of 0.06 to 0.11 and viability decreasing from 97% to 95%. During the exponential phase (24-72 h) the cell density reached 2.50×10⁶ cell/mL, OD₆₀₀ was 0.75, the specific growth rate was 0.039 h^-1, and the viability was 89%. The stationary phase (72-96 h) was defined by the stabilization of the density at 3.48×10⁶ cells/mL, OD₆₀₀ attaining 1.05 and further decrease in viability to 86%, which indicated a correlation with the nutrient loss with time. This contributes to the fact that the growth rate of Helicoverpa zea cells in the medium of Grace at 27°C is slower than that of Hi5 cells, but the latter has a higher OD₆₀₀ value, which can be explained by the different growth dynamics in various media and volumes of the culture.

Further, the kinetics of the growth of IPLB-LdEp (Lymantaria dispar) cells were also measured under the same conditions. Viable cell density grew 0.5×10⁶ to 4.2×10⁶ cells ml between 0 and 96 h OD₆₀₀ increased to 0.45, and viability decreased from 98% to 88%. The phase of exponential proliferation was more intense between 24 and 72 h, and after that the growth rate slowed down. As such, the IPLB-LdEp cells had a moderate growth rate, which makes them the right choice in baculovirus research.

Following the above observations, further experiments were carried out to streamline the nutrient medium, pH, subculturing times, cryopreservation conditions and post recovery viability evaluation of the insect cell lines. These experiments were carried out at least three times under different conditions and media; the results in Table 3 were found as being the best conditions.

Table 3
Optimal culture conditions and cryopreservation media for Hz-AM1, Hi5, and LdEp cell lines.

Cell line	Optimal culture medium	Optimal pH	Doubling time (hours)	Optimal cryopreservation medium	Post-thaw viability (%)
Hz-AM1	Grace’s + 10% FBS, 4 g/L glucose, 2 mM glutamine	6.0	17.2	80% Grace’s + 10% DMSO + 10% FBS	91 ±0.12
Hi 5	Express Five SFM, 4 g/L glucose, 4 mM glutamine	6.5	13.5	80% Express Five SFM + 10% DMSO + 10% FBS	93 ±0.12
LdEp	Grace’s + 10% FBS, 4 g/L glucose, 4 mM glutamine	6.0	18.0	80% Grace’s + 10% DMSO + 10% FBS	90 ±0.12

In the case of Hz-AM1 cells cultured in Grace’s medium with + 10% FBS cells in 4 g/L glucose, 2 mM glutamine, pH 6.0, the cell doubling time was determined as 17.2 h. A vitrified solution of 80% Grace’s medium, 10% dimethyl sulfoxide and 10% fetal bovine serum were used in the cryopreservation and after thawing, a cell viability of 91% was noted. Under the same chosen freezing medium, High Five (Hi5) insect cells growing on Express Five SFM medium (4 g/L glucose, 4 mM glutamine, pH 6.5) had a doubling time of 13.5 h and a viability of 93%. In the case of IPLB-LdEp cells, the nutrient and freezing conditions of the Hz-AM1 cells were used, and the cells obtained a pH of 6.0, a doubling time of 18.0 h, and a recovery viability of 90% after freezing.

The mixture of 80% nutrient media, 10% DMSO, and 10% FBS turned out to be the best cryoprotectant in all the studied cell lines. Storage and freezing were done in liquid nitrogen. These results define the best conditions to culture and freeze baculovirus-insect cell systems.

The synthesized derivatives S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate, S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate, and 2-[(2-chloro-6-fluorobenzyl)sulfanil]-5-[4-(dimethylamino)phenyl]-1,3,4-oxadiazole were evaluated for their in vitro insecticidal activities against insect cell lines of Trichoplusia ni (Cabbage Looper Moth), Helicoverpa zea (Corn Earworm Moth), and Lymantria dispar (Gypsy Moth).

The insecticidal effect was measured through the use of MTT assay and initial screening was done at 10-100 μM/mL. Imidacloprid 2F (21.8%) was used as a commercial standard insecticide (see Table 4).

Table 4
In vitro insecticidal activity of 1,3,4-Oxadiazolethione derivatives against Helicoverpa zea, Trichoplusia ni, and Lymantria dispar cell lines. The experiments were repeated three times.

Test compound	Concentration, µM/ml	Inhibition of test organisms, %
		Helicopvera zea	Trichoplusiani	Lymantria dispar
Control: Imidacloprid 2F 21.8%	10	82.1 ±0.4	79.7 ±0.3	80.2 ±0.2
	100	98.2 ±0.3	98.3 ±0.2	96.4 ±0.2
S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate	10	86.5 ±0.2	77.8 ±0.4	83.6 ±0.2
	100	97.2 ±0.4	93.7 ±0.2	91.6 ±0.3
S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate	10	80.5 ±0.4	79.3 ±0.3	80.2 ±0.6
	100	89.4 ±0.2	90.9 ±0.4	88.4 ±0.5
2-[(2-chloro-6-fluorobenzyl)sulfanyl]-5-[4-(dimethylamino)phenyl]-1,3,4-oxadiazole	10	84.1 ±0.3	82.3 ±0.7	88.8 ±0.4
	100	96.8 ±0.4	94.1 ±0.3	91.7 ±0.4

The compounds assessed as shown in Table 4 exhibited insecticidal effects on lepidopteran insects, Helicoverpa zea, Trichoplusia ni and Lymantria dispar. S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)-O-isobutylcarbonothioate, at a concentration of 100 μM/mL, inhibited proliferation of H. zea cells by 97.2% ±0.4, T. -ni cells by 93.7% ±0.2 and L. dispar cells by 91.6% ±0.3. S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl)O-isobutylcarbonothioate inhibited the growth of cells by 89.4% ±0.2, 90.9% ±0.4, and 88.4% ±0.5, respectively, while 2-[(2-chloro-6-fluorobenzyl)-sulfanil]-5-[4-(dimethylamino)-phenyl]-1,3,4-oxadiazole inhibited cell growth by 96,8% ±0.4, 94,1% ±0.3, and 91,7% ±0.4, respectively.

Helicoverpa zea, Trichoplusia ni and Lymantria dispar. had higher percentages of inhibition of 98.2% ±0.3, 98.3% ±0.2, and 96.4% ±0.2 respectively with reference insecticide “Imidacloprid 2F” (21.8% imidacloprid). These results verify the exceptional insecticidal activity of the produced syntheses and indicate their applicability to the agricultural pest management programmes.

The insecticidal activity of the synthesized compounds was explained by the mechanistic nature of the insecticidal effect using the Discovery Studio 2019, SwissDock, CB-Dock 2 and Neurosnap platforms with the aid of extensive in silico investigations. Another key molecular target of insecticidal effect of 1,3,4‑oxadiazole derivatives is acetylcholinesterase (AChE), an important neuronal signal transduction enzyme (Begum et al., 2023; Shi et al., 2022). Based on this, computational docking experiments were conducted to assess the inhibitory activity of the new compounds on AChE of three economically important lepidopteran pests, namely, Helicoverpa zea, Trichoplusia ni, and Lymantria dispar (Figures 3-5).

Figure 3
In silico analyses of receptor-ligand interaction of S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate and 6XYS.

Figure 5
In silico analyses of receptor-ligand interaction of 2-[(2-chloro-6-fluorobenzyl)sulfanyl]-5-[4-(dimethylamino)phenyl]-1,3,4-oxadiazole and 6XYS.

Molecular docking is a core computational approach to the description of the interaction space between small-molecule ligands and target protein structures. Based on mechanistic understanding of previous research (Song et al., 2022), the current study examined the molecular recognition patterns of new 1,3,4-oxadiazolethiones derivatives in complex with acetylcholinesterase (AChE). Crystal structure of carboxylesterase (CES; PDB ID: 6XYS) was used as a structural template of the docking simulations of the synthesized compounds. The derivatives were tested in these simulations to evaluate their binding conformations, inter-molecular interactions and relative affinities.

Computational analysis showed that there are a number of significant interactions that lead to stabilization of the compound in the enzyme active site (Figure 3). Carbon-hydrogen bonds between the C17/C18 methyl groups and Trp321 were found whereas a hydrogen bond between the oxygen atom at 14 and Tyr73 was observed. The sulfur atom at the 12-position was interacting with Tyr324 through π-sulfur interactions and the 1,3,4-oxadiazole moiety was interacting with Asp375 through π-anion interactions. Moreover, the 2-chlorophenyl moiety was π-π stacked with Tyr374. Together, the above complementary interactions increase binding affinity and provide useful structure-activity relationships relevant to the rational design of compounds with high biological activity.

The outcomes of a molecular docking analysis are shown in Figure 4, and they reveal that the current affinity of the compound to its target site is based on several simultaneous binding interactions. The indole moiety of residue Trp321 is involved in 2 aromatic contacts: a parallel π–π stacking contact between the 3-pyridyl fragment and a T-shaped π-π contact between the 1,3,4-oxadiazole ring system. The phenolic ring of Tyr73 participates in a π-donor hydrogen bonding interaction with the electron-deficient 1,3,4-oxadiazole heterocycle. Additionally, the aromatic residues Tyr374, Tyr370, Tyr71, and Phe371 collectively engage in π-alkyl interactions with the carbon atom at the 16-position, creating a hydrophobic binding environment that further stabilizes the ligand-receptor complex.

Figure 4
In silico analyses of receptor-ligand interaction of S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate and 6XYS.

This binding interaction examination as shown in Figure 5 indicates a complex web of intermolecular interactions that hold the ligand in place in the active site. The 2-chloro-6-fluoro benzyl group forms strong π-anion interactions with the negatively charged carboxylate groups of Asp482 and Glu485. At the same time, phenyl ring system is additionally involved in other π-anion interactions with Glu485 forming a dual interaction pattern, which increases the stability of binding. The aromatic phenyl ring also undergoes hydrophobic π-alkyl interactions with the aliphatic side chains of Ile82 and Ile161 to add to the binding affinity in terms of favorable van der Waals contacts. Remarkably, the Ile82 also forms a π-σ contact with the heterocycle 1,3,4-oxadiazole and, thus, illustrates the ability of the compound to interact with the residue in a variety of binding modes. Other stabilizing contacts include a traditional hydrogen bond between the fluorine atom of the 2-cloro-fluorobenzyl ring and the indole NH group of Trp472. In addition, Asp160 is also linked to the methyl group of the 4-(dimethylamino)-phenyl moiety, which gives further binding sites in the binding pocket.

4. Discussion

In this study, synthesized 1,3,4-oxadiazole derivatives, namely S-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate, S-(5-(3-pyridyl)-1,3,4-oxadiazol-2-yl) O-isobutylcarbonothioate, and 2-[(2-chloro-6-fluorobenzyl)sulfanil]-5-[4-(dimethylamino)phenyl]-1,3,4-oxadiazole compounds, demonstrated high insecticidal activity in vitro against Helicoverpa zea, Trichoplusia ni, and Lymantria dispar cell lines. According to the literature, compounds containing the 1,3,4-oxadiazole heterocycle, and specifically 3H-1,3,4-oxadiazol-2-thione derivatives, exhibit diverse types of biological activity (Glomb and Świątek, 2021; Erika et al., 2023; Liu et al., 2022). Because of the presence of the thioamide (NH–C=S) group, 5 -substituted 3H-1,3,4-oxadiazol-2-thiones have significant practical importance, because they allow the derivatives to be synthesized by two nucleophilic reactive sites within this group, both exocyclic sulfur atom and endocyclic nitrogen atom in the oxadiazole ring, depending on the conditions of the experiment. It is noteworthy that the S-alkyl(acyl)-derivatives of the 5-aryl-1,3,4-oxadiazol-2-thione exhibit significantly high biological activity.

The molecular docking findings in Figures 3-5 indicate that, the newly synthesized 1,3,4-oxadiazole derivatives have a high binding affinity with the acetylcholinesterase (AChE) active sites in all three target lepidopteran species. These computerized projections outline that the new compounds have strong possibilities of the inhibition of AChE, thus justifying their future application as effective insecticides against pests of agricultural significance. The overall binding-interaction study shows that there are several complementary patterns of molecular-recognition which in combination result in the high binding affinity of the compounds and also a possible level of selectivity.

The differences between the intrinsic variation in the molecular mechanisms of action of oxadiazole derivatives and conventional neonicotinoids can be attributed to the environments safety profile of the oxadiazole derivatives. In contrast, the synthesized oxadiazole compounds are reversible, non-covalent inhibitors of acetylcholinesterase (AChE) in contrast with neonicotinoids that are non-covalent irreversible agonists of nicotinic acetylcholine receptors (Begum et al., 2023). This mechanistic separation is what drives their weakened environmental perseverance and watered down toxicity on non-target organisms.

The molecular docking studies have shown that oxadiazole analogs mainly bind to the peripheral anionic site (PAS) and anionic subsite of AChE through reversible interactions, including π interaction, π anion interactions, and hydrogen bonding (Begum et al., 2023; Pflégr et al., 2022). This binding mode is in sharp contrast to organophosphate insecticides forming a covalent adduct with the catalytic serine residue of the enzyme, thus, causing irreversible inhibition and triggering increased mammalian toxicity (Pflégr et al., 2022). The reversible oxadiazole activation promotes endogenous enzymatic reactivation and countermeasures the possibility of protracted cholinergic toxicity in non-target organisms.

Structural differences between insect and vertebrate acetylcholinesterases can be attributed to the selective toxicity of oxadiazole derivatives on insects compared to mammalian homologues (Wang et al., 2024; Adedeji et al., 2020). The insects usually encode two different AChE isoforms (AChE1, AChE2), in which AChE1 is the majority of enzyme responsible in the degradation of acetylcholine at synaptic junctions (Adedeji et al., 2020). The binding pocket architecture particularly the PAS region which selectively binds the oxadiazole entities exhibits strong amino-acidic and conformational differences between the insect and mammalian AChE variants (Begum et al., 2023; Wang et al., 2024).

The computational analysis showed that there are certain amino acid residues (Trp321, Tyr73, Tyr324, Asp375, Tyr374) which aid in high-affinity binding of the oxadiazole derivatives. The residues and spatial configuration of the insect and mammalian acetylcholinesterase (AChE) are different and as such offer a molecular solution to species selectivity. The allosteric binding mode adopted by oxadiazoles is reversible and hence they can be inhibited using these structural differences hence may selectively inhibit insect AChE and leave mammalian enzymes uninhibited (Begum et al., 2023; Pflégr et al., 2022).

The recent in vitro cytotoxicity experiments have added up useful information regarding the safety profile of oxadiazole derivatives on human cell systems. According to Begum et al. (Begum et al., 2023), a group of 1,3,4-oxadiazole derivatives were reported to exhibit AChE inhibitory action with an IC₅₀ of 41.87 ±0.67 to 1580.25 ±0.70 μM which is in the range of micromolar potency with regard to the target enzyme. Notably, different studies by Serag et al. (2024) demonstrated selective cytotoxicity, with oxadiazole analogs demonstrating strong antiproliferative effects on cancer cell lines (IC₅₀ 1.82-5.55 μM) and significantly lower toxicity on normal human fibroblasts (WI-38 cells, IC₅₀ = 41.17 μM).

Further cytotoxicity determinations by Tariq et al. (Tariq et al., 2022) determined 1,3,4-oxadiazol-2-thiol targets against AChE concomitant with reasonable indices of selectivity of normal human cell lines. This evidence indicates that the fact that insecticidal activity can be readily obtained with the corresponding oxadiazole derivatives does not exclude the observation that the same compounds can be used to attain an acceptable level of safety when exposing it to human beings.

The environmental benefits of oxadiazole derivatives are especially obvious in comparison to such neonicotinoids as imidacloprid. Neonicotinoids have been well documented to induce acute toxicity on beneficial insects, especially honeybees, and have been reported to have LD₅₀ of 4-17 ng/bee on imidacloprid (Li et al., 2024; Fu et al., 2018). These are compounds with great systemic action and longevity in plant tissues causing chronic pollinators to be exposed to contaminated nectar and pollen (Li et al., 2024).

On the contrary, the reversible binding mode of oxadiazole derivatives implies decreased bioaccumulation propensity and enhanced metabolic clearance in non-target organisms (Pflégr et al., 2022; Sousa et al., 2020). Molecular docking findings show that part of the binding interactions are mainly due to the non-covalent forces that are easily broken by physiological conditions and that may shorten the enzyme inhibition in insects of use. Although the toxicity of oxadiazoles in bees is not fully studied as yet, the mechanistic differences indicate that the toxicity is more favourable to the environment as opposed to its persistent neonicotinoid counterparts (Sousa et al., 2020; Zoroddu et al., 2022).

Neonicotinoids have also been linked with development of resistance in different pest populations, which is in the form of increased metabolism detoxification and target-site mutation (Fu et al., 2018; Bass et al., 2014). Resistance-management strategies the multimodal, reversible, multi-point binding model of oxadiazole derivatives has the potential to be useful.

The molecular docking studies have indicated that the synthesized compounds interact with multiple amino acid residues at once with different types of interaction (π-π stacking, hydrogen bonding, π-anion interactions, π-alkyl interactions). This multi-modal binding pattern enhances the probability that simultaneous mutations in many locations would be necessary to obtain resistance, which would slow the pace of the development of resistance, relative to compounds with a single-point binding-ability (Pflégr et al., 2022; Pesticide Information Profiles, 2012; Sparks and Nauen, 2015).

The obtained experimental evidence proves that the produced oxadiazole derivatives showed much more successful inhibition of target pest cells, which is the performance that is similar to that of the control by means of imidacloprid. Such efficacy, together with the enhanced safety profile, makes oxadiazole derivatives promising candidates to be used in sustainable pest management activity.

5. Conclusion

The 1,3,4-oxadiazole derivatives synthesized during this study were found to exhibit a high insecticidal efficacy on cellular proliferation of Helicoverpa zea, Trichoplusia ni and Lymantria dispar in vitro with a reduction of up to 97.2%. The data can be compared to those produced by the commercially available imidacloprid-based pesticide Imidacloprid 2F, thus highlighting the profile of oxadiazole as a safe pesticide in terms of its environmental application in the control of agricultural pests. Molecular docking studies indicated that the novel compounds have been able to bind to several amino-acid residues at the same time in a range of binding modalities: π-π stacking, hydrogen bonding, π-anion interactions, π-alkyl interactions, which selectively bind the insect acetylcholinesterase (AChE). These constituents, due to their relatively limited environmental toxicity, come out as viable alternatives to neonicotinoids in the context of a sustainable agricultural system. Future in vivo studies will determine the efficacy of these oxadiazole analogs in the long-term in agricultural environments.

Acknowledgements

Financial support from Agency of Innovative Development of the Republic of Uzbekistan (Project No. F-FA-2021-360) is gratefully acknowledged.

Data Availability Statement

The entire dataset supporting the results of this study was published in the article itself.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  27 Feb 2026
• Date of issue
  2026

History

• Received
  31 Oct 2025
• Accepted
  29 Dec 2025