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Mosquito-borne diseases remain a serious global public health problem. Growing resistance of disease vectors, such as Culex pipiens pallens, to traditional insecticides further exacerbates this problem. In this study, a series of novel thiophene-isoquinolinone hybrids were designed, synthesized, and evaluated as potential larvicides. Among the synthesized compounds, derivatives 5f, 6, and 7 demonstrated significant larvicidal activity against Culex pipiens pallens larvae with LC₅₀ values of 0.3, 0.1, and 1.85 μg/mL, respectively. Notably, all twelve thiophene-isoquinolinone derivatives demonstrated significantly higher toxicity than the reference organophosphate insecticide chlorpyrifos (LC₅₀ = 293.8 μg/mL), confirming the superior toxicity of these compounds. Interestingly, synthetic intermediate 1a (a thiophene semiester) exhibited the highest potency (LC₅₀ = 0.004 μg/mL), and although not yet fully optimized, its potency still exceeded that of all final derivatives. Mechanistic biological studies revealed robust neurotoxicity symptoms, suggesting impaired cholinergic function. Molecular docking and molecular dynamics simulations confirmed this observation, revealing strong specific interactions with acetylcholinesterase (AChE) and the nicotinic acetylcholine receptor (nAChR), suggesting a possible dual-action mechanism. Density functional theory (DFT) calculations further confirmed the favorable electronic properties and reactivity of the active compounds. The structural diversity and consistently high potency of this series of compounds may reduce the risk of cross-resistance and facilitate resistance management strategies through compound rotation or combination. Overall, these results indicate that thiophene-isoquinolinone hybrids are a promising option for the development of next-generation larvicides targeting neurophysiological pathways of insect vectors.
Mosquitoes are among the most effective vectors of infectious diseases, spreading a wide range of dangerous pathogens and posing a significant threat to global public health. Species such as Culex pipiens, Aedes aegypti, and Anopheles gambiae are particularly known for transmitting viruses, bacteria, and parasites, causing millions of infections and numerous deaths annually. For example, Culex pipiens is a major vector of arboviruses such as West Nile virus and St. Louis encephalitis virus, as well as parasitic diseases such as avian malaria. Recent research has also shown that Culex pipiens plays a significant role in the vector and transmission of harmful bacteria such as Bacillus cereus and Staphylococcus warwickii, which contaminate food and exacerbate public health problems. The high adaptability, survivability, and resistance of mosquitoes to control methods make them difficult to control and pose a persistent threat.
Chemical insecticides are a key tool in mosquito control, particularly during mosquito-borne disease outbreaks. Various classes of insecticides, including pyrethroids, organophosphates, and carbamates, are widely used to reduce mosquito populations and disease transmission. However, the widespread and long-term use of these chemicals has led to serious environmental and public health concerns, including ecosystem disruption, harmful effects on non-target species, and the rapid development of insecticide resistance in mosquito populations. 11,12,13,14 This resistance significantly reduces the effectiveness of many traditional insecticides, highlighting the urgent need for innovative chemical solutions with new mechanisms of action to effectively counter these evolving threats. 11,12,13,14 To address these serious challenges, researchers are turning to alternative strategies such as biocontrol, genetic engineering, and integrated vector management (IVM). These approaches demonstrate promise for sustainable, long-term mosquito control. However, during epidemics and emergencies, chemical methods remain crucial for rapid response.
Isoquinoline alkaloids are important nitrogen-containing heterocyclic compounds widely distributed in the plant kingdom, including families such as Amaryllidaceae, Rubiaceae, Magnoliaceae, Papaveraceae, Berberidaceae, and Menispermaceae.30 Previous studies have confirmed that isoquinoline alkaloids possess diverse biological activities and structural features, including insecticidal, antidiabetic, antitumor, antifungal, anti-inflammatory, antibacterial, antiparasitic, antioxidant, antiviral, and neuroprotective effects.
In this study, χ² values for all compounds were below the critical threshold, and p values were above 0.05. These results confirm the reliability of LC₅₀ estimates and demonstrate that probabilistic regression can effectively describe the observed dose-response relationship. Therefore, LC₅₀ values and toxicity indices (TIs) calculated based on the most active compound (1a) are highly reliable and suitable for comparing toxicological effects.
To evaluate the interactions of 12 newly synthesized thiophene-isoquinolinone derivatives and their precursor 1a with two key mosquito neuronal targets—acetylcholinesterase (AChE) and the nicotinic acetylcholine receptor (nAChR)—we conducted molecular docking modeling. These targets were selected based on neurotoxic symptoms observed in larval death assays, indicating impaired neuronal signaling. Furthermore, the structural similarity of these compounds to organophosphates and neonicotinoids further supports the preferred choice of these targets, as organophosphates and neonicotinoids exert their toxic effects by inhibiting AChE and activating nAChR, respectively.
Furthermore, several compounds (including 1a, 2, 5a, 5b, 5e, 5f, and 7) interact with SER280. SER280 residues are involved in shaping crystal structure conformations and are conserved in the redopated conformation of BT7. This diversity of interaction modes highlights the adaptability of these compounds in the active site, with SER280 and GLU359 potentially serving as adaptive anchor sites under docking conditions. The frequent interactions observed between synthetic derivatives and key residues such as GLU359 and SER280, which are components of the known SER-HIS-GLU catalytic triad in human acetylcholinesterase (AChE), further support the hypothesis that these compounds may exert potent inhibitory effects on AChE by binding to catalytically important sites. 29,61,64
Notably, compound 6 and its precursor 1a demonstrated the most potent activity against larvae in the bioassay, displaying the lowest LC₅₀ values among the compounds in the series. At the molecular level, compound 6 exhibits a critical interaction with chlorpyrifos at the GLU359 site, while compound 1a overlaps with re-doped BT7 via a hydrogen bond to SER280. Both GLU359 and SER280 are present in the original crystallographic binding conformation of BT7 and are components of the conserved catalytic triplet of acetylcholinesterase (SER–HIS–GLU), highlighting the functional significance of these interactions in maintaining the inhibitory activity of the compounds (Fig. 10).
The observed similarity in binding sites between BT7 derivatives (including native and reconstituted BT7) and chlorpyrifos, particularly at residues critical for catalytic activity, strongly suggests a common mechanism of inhibition between these compounds. Overall, these results confirm the significant potential of thiophene-isoquinolinone derivatives as highly potent acetylcholinesterase inhibitors due to their conserved and biologically relevant interactions.
A strong correlation between the molecular docking results and the larval bioassay results further confirms that acetylcholinesterase (AChE) and the nicotinic acetylcholine receptor (nAChR) are the primary neurotoxic targets of the synthesized thiophene-isoquinolinone derivatives. Although the docking results provide important information on receptor-ligand affinity, it should be recognized that binding energy alone is insufficient to fully explain insecticidal efficacy in vivo. Differences in LC₅₀ values between compounds with similar docking characteristics may be due to factors such as metabolic stability, absorption, bioavailability, and distribution in insects.⁶⁰ ,⁶⁴ However, the rational structural design, high receptor affinity simulated by computer simulation, and potent biological activity strongly support the view that AChE and nAChRs are the main mediators of the observed neurotoxicity.
In conclusion, the synthesized thiophene-isoquinolinone hybrids possess key structural and functional elements largely compatible with known neuroactive insecticides. Their ability to efficiently bind to acetylcholinesterase (AChE) and nicotinic acetylcholine receptors (nAChRs) via complementary interaction mechanisms highlights their potential as dual-target insecticides. This dual mechanism not only enhances insecticidal efficacy but also provides a promising strategy for overcoming existing resistance mechanisms, making these compounds promising candidates for the development of next-generation mosquito control agents.
Molecular dynamics (MD) simulations are used to validate and extend molecular docking results, providing a more realistic and time-dependent assessment of ligand-target interactions under physiologically realistic conditions. Although molecular docking can provide valuable preliminary information on potential binding positions and affinities, it is a static model and cannot account for receptor flexibility, solvent dynamics, or temporal fluctuations in molecular interactions. Therefore, MD simulations are an important complementary method for assessing complex stability, interaction robustness, and conformational changes in ligands and proteins over time. 60,62,71
Based on their superior binding properties to acetylcholinesterase (AChE) compared to the nicotinic acetylcholine receptor (nAChR), we selected the parent molecule 1a (with the lowest LC₅₀ value) and the most active thiophene-isoquinoline compound 6 for molecular dynamics (MD) simulations. The goal was to evaluate whether their binding conformation in the AChE active site remained stable over 100 ns of simulation and to compare their binding behavior with that of chlorpyrifos and the rebound cocrystallized AChE inhibitor BT7.
Molecular dynamics simulations included root mean square deviation (RMSD) to assess overall complex stability; root mean square deviation of fluctuations (RMSF) to study residue flexibility; and ligand-acceptor interaction analysis to determine the stability of hydrogen bonds, hydrophobic contacts, and ionic interactions (Supplementary Data). Although the RMSD and RMSF values for all ligands remained within a stable range, indicating no significant conformational changes in the AChE-ligand complex (Figure 12), these parameters alone are insufficient to fully explain the differences in binding mass between the compounds.
Post time: Dec-15-2025