Novel synthesis of some heterocycles and their molluscicidal activity against the destructive land snail, Theba pisana

Background Terrestrial snails, specifically Theba pisana , represent significant agricultural pests in the Mediterranean region, particularly in Egypt. They are gastropods that cause substantial damage to a variety of vegetation, rendering them important agricultural pests that result in economic losses. In response, we have developed unique and non‑ toxic molluscicides. The study assessed nine novel heterocycles compared with methomyl as a reference compound, to study their molluscicidal effects on T. pisana . These heterocycles include 2‑pyrazolines, 2‑oxocyclohex‑3‑ene, and 3‑cyano‑2‑ethoxypyridine. Results The obtained data revealed that the majority of the produced chemicals were remarkably effective against T. pisana snails, exhibiting different degrees of toxicity seven days post‑treatment. Methomyl exhibited the highest toxicity, followed by prop‑2‑en‑1‑one and 1‑thiocarbamoyl‑2‑pyrazoline derivatives, with LC 50 values of 44.14, 59.54, and 72.00 ppm, respectively. Conclusions These findings will inform the potential of these synthetic compounds as one of the sources for mollus‑ cicide development and their integration into pest management strategies.


Background
The land snails are creatures known as gastropods that seriously harm a variety of vegetation, making them important agricultural pests.They target buds, roots, leaves, flowers, and tree trunks, resulting in significant harm to growing plants [1].These pests harm plants at different stages of growth, resulting in decreased yields [2] and posing a danger to sustainable agriculture [3].Land snails cause economic loss because of their eating habits and their contamination of crops with their bodies, excrement, or slime.This not only reduces product quality but also causes major financial losses [4].Furthermore, land snails act as intermediary hosts, indirectly spreading infections to humans and domestic animals through infected plants and fruits [5,6].
Land snails have grown to be a major agricultural pest in several Egyptian regions, with reported attacks on diverse plant components [7][8][9][10][11].Of these pests, the terrestrial snail species Theba pisana, is one of the most common and dangerous land snails in the area known as the Mediterranean, especially in Egypt, due to its small size, climbing inclination, ability to cause plant damage, and high reproductive rate [12].

Chemistry
The building blocks for the production of different heterocycles are acetyl pyrazolopyridine 1 [37] and pyrazole-4-carboxaldehyde, which reacted by Claisen-Schmidt condensation to produce 1-propen-2-one derivative 2 (Scheme 1).Different 2-pyrazoline derivatives 3-5 can be obtained through cycloaddition of 2 using hydrazine hydrate 80% at different reaction conditions.If the cycloaddition occurred in ethanol, unsubstituted 2-pyrazoline 3 was formed.Conversely, the use of formic or Spectral data and elemental analysis confirmed the structures of products 3-5.For instance, the 1 H-NMR spectra of 3 exhibited three doublets of doublets caused by the two methylene protons in the 2-pyrazoline ring.The two methylene protons form an ABC structure, with H A , H B , and H C appearing as double doublets at δ = 3.11-3.16,3.40-3.46,and 6.06-6.09ppm, with J AB = 18 Hz, J AC = 12 Hz, and J BC = 5.5 Hz, respectively.The NH proton of 3 appeared as a signal at δ = 8.43 ppm.Similarly, the 13 C-NMR exhibited two signals at δ = 44.6 and 57.0 ppm referring to pyrazoline-CH 2 , and pyrazoline-CH.
The spectral data indicated the structures of 1-phenyl-2-pyrazoline 6 and 1-thiocarbamoyl-2-pyrazoline 7, and their infrared spectra confirmed the lack of carbonyl bond absorption bands.In addition, compound 7 showed new absorption bands from the amino group at v = 3431 and 3230 cm −1 , and a new signal from the C = S group at δ = 176.5 ppm, according to the 13 C-NMR spectra.
For additional reactions of 1-propen-2-one 2 functionality, it undergoes treatment with ethyl acetoacetate or malononitrile to produce ethyl 2-oxocyclohexene carboxylate 8 or 3-cyano-2-ethoxypyridine derivative 9, separately (Scheme 4).FT-IR spectra of 8 exhibited the appearance of two C = O at υ = 1665 with 1712 cm −1 .Moreover, the 1 H-NMR spectra confirmed the existence of the ethyl group, a signal (triplet) at δ = 1.20-1.27ppm for CH 3 , and a signal (quartet) at δ = 4.58-4.63ppm due to CH 2 .The cyclohexene ring protons gave three signals at δ = 3.05-3.20due to cyclohexene-CH 2 , 3.65-3.78due to two protons of cyclohexene-CH, and at 6.50 due to the other proton of cyclohexene-H.Additional signals were found on the 13 C-NMR spectra of compound 8 at δ = 13.9 ppm due to carbon of methyl ester, 29.7 ppm due to cyclohexene-CH 2 , 39.6 ppm due to carbon of methylene ester, 169.9 ppm of cyclohexyl-CO, and 193.7 ppm of ester-CO.On the other hand, spectroscopic analyses supported the structure of 9, the IR spectra indicating a CN stretching peak at v = 2217 cm −1 .The 1 H-NMR spectra, which are in line with 13 C-NMR spectroscopy, revealed the signal (triplet) at δ = 1.51-1.54ppm due to -CH 2 CH 3 , and quartet signal at δ = 4.55-4.61ppm of -CH 2 CH 3 .In addition, 13 C-NMR (DEPT) distinguished the negative signal of methylene (CH 2 ) of 9 at δ = 34.2ppm and the others, CH 3 , CH or quaternary C have positive signals.

The molluscicidal activity of investigated compounds on Theba pisana applying the residual film assay
The residual film treatment was performed to evaluate the molluscicidal effects of pyridine derivatives on the terrestrial snail Theba pisana when compared to methomyl, the recommended compound (Tables 1 and  2).Results listed in Table 1 showed that the majority of the produced chemicals had remarkable effectiveness against T. pisana snails, with different levels of toxicity 7 days after treatment.The highest mortality rates for the tested snails were recorded by methomyl, followed by prop-2-en-1-one 2, and 1-thiocarbamoyl-2-pyrazoline 7 with values of (53.33, 73.33 and 86.67%), (46.67, 60.0 and 73.33%) and (40.0, 60.0 and 73.33%) at 50, 100 and 200 ppm concentrations, respectively.On the other hand, the synthesized compounds examined in this work did not cause death after the first day of treatment compared to methomyl, which showed a mortality rate of (6.67, 6.67, and 20.0%) at the same concentrations one-day post-treatment.After the third day, mortality percentages started to show up and progressively rose as treatment times and concentrations increased.The Median Lethal Concentration value (LC 50 in ppm) of each compound was estimated and is displayed in Table 2 In comparison to the methomyl reference compound, two of the tested compounds, 2 and 7, exhibited high molluscicidal efficacy towards T. pisana having LC 50 rates of 59.54 and 72.0 ppm, respectively, whereas that of methomyl was 44.14 ppm.Meanwhile, the least effective compounds, 2-pyrazoline 5, cyclohexanone 8, and 1-formyl-2-pyrazoline 3, had LC 50 values of (0.0, 0.0, and 0.0) and (212.56,226.50, and 431.65 ppm) after 3 and 7 days of treatment.

Table 1 Mortalities of Theba pisana adult snails exposed to different concentrations of certain compounds using residual film technique under laboratory conditions
The identical letters within the same column means that they are not significant LSD least significant difference (for tested compounds)  B. alexandrina and found that these compounds had promising molluscicidal activity.In addition, synthesized heterocyclic compounds have shown insecticidal activity [41][42][43].

Instrumentation and chemicals
The materials used in the current experiment were of analytical grade.The melting points that were not adjusted were estimated utilizing an APP Digital ST 15 melting point instrument.The Shimadzu-408 infrared spectrophotometer was applied to record the IR spectra that are presented in cm −1 .Using a Bruker AV-400 and JEOL ECA II 500 MHz spectrometer, the NMR spectra were acquired.Parts per million, or ppm, of chemical shifts, were determined with TMS as an internal reference.With System GmbH vario EL V2.3 1998 CHNS Mode, analysis of elements was carried out.

Collection of snails for testing
Adult white garden snails, Theba pisana, of approximately similar age and size, were obtained from infested citrus orchards in the Faisal region of the Suez Governorate, Egypt.The obtained samples were brought straight to the lab and placed in a glass terrarium containing moist clay soil and were covered using fabric netting.The snails were provided with fresh green lettuce (Lactuca sativa L.) leaves for about two weeks before treatment to allow for acclimatization until the commencement of the experiment.The rearing terrarium was cleaned regularly, and sick or dead snails were quickly removed.

Molluscicidal activity bioassays Residual film technique
Bioactivity experiments were carried out using the contact-based (residual film) technique described by Ascher and Mirian [44], to assess the obtained compounds toward the white garden land snail, T. pisana, in comparison with methomyl, the recommended pesticide, at different concentrations (50, 100, and 200 ppm) for each compound.Each compound was prepared as a stock solution in dimethyl sulfoxide (DMSO) before being diluted using distilled water to achieve the required concentrations.Two milliliters for each concentration of the compounds were added and then spread across the bottom of a petri dish, and then the dish was gently rotated in a circular motion.Within a few minutes, under room conditions, the water evaporated, leaving behind a thin layer of the compounds at the applied concentrations.Following that, five adult, healthy snails from the examined species were brought in and introduced to the specified concentration of the investigated materials.Each treatment, involving an untreated check, was carried out three times.The mortality percentages were computed and adjusted using the Abbott formula [45] after dead snail counts were conducted every day for seven days.

Data analysis
Mortality results from the molluscicidal test on T. pisana were analyzed using Costat software with one-way analysis of variance (ANOVA) and the least significant difference at 0.05 (LSD).Finney's probit analysis was utilized to determine the LC 50 and confidence intervals of 95% corresponding to the (upper and lower) confidence limits [46].

Conclusions
The novel chalcone 2-propen-1-one 2 was synthesized and reacted with different nucleophiles, binucleophiles, and active methylene reagents.The structures of the new heterocyclic compounds, 2-pyrazolines, and pyridine derivatives, were determined using elemental analysis with spectroscopic techniques including FT-IR and nuclear magnetic resonance spectroscopy.Our results indicate that the two compounds, prop-2-en-1-one 2, and 1-thiocarbamoyl pyrazoline derivative 7, exhibit a promising molluscicidal effect compared with the traditional chemical pesticide methomyl.Therefore, these compounds can be developed for use as molluscicides in control programs for these animals.

Fig. 1 Scheme 1
Fig. 1 Examples of reported and newly synthesized heterocycles

Table 2
Toxicity effect of certain compounds against Theba pisana snails using residual film technique under laboratory condition LC 50 50% lethal concentration, CL Confidence limits The mixture was cooled, put over crushed ice to be separated, and recrystallized from the ethanol.