Tyrosinase enzyme mediated: Synthesis and larvicidal activity of 1,5-diphenyl pent-4-en-1-one derivatives against Culex quinquefasciatus and investigation of Ichthyotoxicity against O. mossambicus

Larvicidal activity of 1,5-diphenylpent-4-en-1-one derivatives were synthesized via grindstone method, tyrosinase enzyme used as a catalyst of this process. This method offers high yields with mild reaction conditions. Synthesized compounds were conformed from FTIR, 1 H NMR, 13 C NMR, mass spectral and elemental analysis. In this study, a total of 17 compounds (1a–1q) were synthesized, and their larvicidal and antifeedant activities were evaluated. Compound 1i (1-(5-oxo-1,5-diphenylpent-1-en-3-yl)-3-(3-phenylallylidene)thiourea) was extremely active (LD 50 : 12.09 µg/mL) against Culex quinquefasciatus compared with temephos and permethrin, whereas compounds 1i at 100 µg/mL generated 0% mortality within 24h against Oreochromis mossambicus in an antifeedant screening, and Ichthyotoxicity was determined as the death ratio (%) at 24 h. The compounds 1a, 1e, 1f, 1j, and 1k were found to be highly toxic whereas the 1i was not toxic in antifeedant screening. Therefore, 1i was found to have a high larvicidal activity against C. quinquefasciatus, and was non-toxic to non-target aquatic species. Molecular docking studies also supported the nding that 1i is a potent larvicide with more binding energy than the control (-10.0 vs. -7.6 Kcal/mol) in the 3OGN protein. The lead molecule is very important to larvicidal properties and insecticides. N, 10.52%; found: C, 76.68; H, 6.80; N, 10.51%. Ar ring), 135.2, 134.4, 116.1, 20.6 (6C, Ph ring), 134.6, 128.1, 55.6, 50.1, 14.4; EIMS(m/z): 399.15(M + ,27%); Anal. Calcd. for C 25 H 22 N 2 OS: C, 75.35; H, 5.56; N, 7.03 %; found: C, 75.30; H, 5.60; N, 7.04 %.


Introduction
In the broadest sense, human beings are part of nature; however, our activity is often understood and interpreted as a separate and unique category from the rest of the natural phenomena. It is both the legal and moral obligation of every human to protect planet earth by undertaking activities that would prevent contaminating our planet and thereby protect it for future generations. For instance, as a chemist in chemical industries or academia, one could focus on protecting nature by employing green chemistry so as to produce various chemical and pharmaceutical active ingredients. Out of several green chemistry methodologies, grindstone chemistry technique is one of the simple methodology consist of preparing chemical compounds. Toda et al., was developed the chemical reactions carried out by simply grinding or triturating the solids together 1 . We will now turn our focus towards Mannich reactions, which are a muchstudied type of reaction in the organic and medicinal chemistry domains 2 .
Mannich type reactions face signi cant challenges at present, major challenges such as reaction time, reaction conditions, toxicity, catalyst requirements and separating the purify of nal product(s). Other challenges include synthetic methodologies such as ultrasound or microwave irradiation, the usage of Lewis acids or bases, or the usage of solubilizing agents or surfactant-type catalysts 3 . In addition, some of the known green trends in Mannich reactions consist of ball-milling without solvents 4 , using ionic liquid mediums 5 , using ionic liquids reinforced with nanoparticles 6 , or the application of enzymes under bio-catalytic conditions 7,8 . However, present study focus grindstone green chemistry method was overcome the above challenges for preparation of Mannich base derivatives.
Mosquitoes are a very important transmission vector for several diseases, particularly malaria 9,10 . These types of diseases have an economic and social impact all over the word. Among mosquito species, Culex quinquefasciatus is particularly associated with various disease vectors in several regions. Control of mosquitos presents a substantial challenge, and currently mosquito inhibitors such as Permethrin 11 , organophosphates 12 , fenthion 13,14 , chlorpyrifos 15-17 , temephos 18,19 , di ubenzuron 20 and methoprene 21 are used; Fig. 1 details the compositions of commercial insecticides. However, the usage of chemical insecticides causes bigger challenges and various potential environmental problems, such as widespread development of resistance and disrupted natural biological control systems 22,23 . These problems require overcoming new mosquito larvae inhibitors, and give rise to a strong need to improve green methodologies so as to address these challenges; this need can be met through Mannich base condensation reactions. Mannich base synthesis is one of the best tools for the preparation of green synthesis. For this reason, we selected 1,5-diphenylpent-4-en-1-one derivatives, which are environmentally safe and match well with 1,3-diaryl-2-propen-1-ones (chalcones, Fig. 1) and with mosquito larvicidal properties 24 . In this study, we sought to develop a simple and e cient grindstone chemistry methodology that can overcome the above identi ed challenges and limitations so as to obtain novel water-soluble and nontoxic Mannich base derivatives.

Results And Discussion
Chemistry A one-pot multicomponent of title compounds was achieved using grindstone green chemistry method. The synthetic route outline was represented in Scheme 1. The proposed mechanism for the formation of Mannich base derivative was displayed in Scheme 2. Copper containing materials like coppertri ate 25 , copperacetate 26 , copperbromide 27 and copper nanoparticles 28 plays a vital role in Mannich base reaction. One-pot multicomponent Mannich reaction was synthesized via various enzymes catalyzed such as, trypsin 29 , lipase 30,31 and protease 32 . Present study copper containing tyrosinase enzyme was used as a catalyst for the synthesis of N-Mannich base (1a-1q) derivatives. The title compounds were synthesized using the catalysts trypsin, lipase, protease, CuCl 2 .2H 2 O, and tyrosinase enzyme with yields of 64%, 72%, 68%, 84%, and 92%, respectively. The use of the Tyrosinase enzyme green catalyst, instead of CuCl 2 .2H 2 O, increased the yield of the Mannich derivatives to 92% and reduced the reaction time. The optimization of reaction conditions and catalysts were presented in Tables 1 and 2. The obtained compounds were analyzed via FT-IR, 1 H, and 13 C NMR spectra. The key assignments of the compounds showed signi cant bands at 3170. 23-3176.54, 2595.45-2599.98 and 1710.68-1716.70 cm -1 in the IR spectrum, conforming towards the -NH, -C = N and -C = O groups, respectively. The ¹H NMR showed signals at δ 8.03-9.70, 3.82-4.81 and 2.40-2.98 ppm, indicating -NH, 4-CH, and -CH 2 protons, respectively. The  ppm, which conforms to -C = O, -CH, and -CH 2 atoms, respectively. The mass spectra and elemental analysis were used to satisfy with the conformation of all compounds. Biological activity A total of 17 compounds (1a-1q) were tested against second instar C. quinquefasciatus larvae and the toxicity of the title compounds was evaluated in the marine sh Oreochromis mossambicus. Toxicity was de ned as ratios of deaths (%) at 24h. Structure activity relationships showed that the nal compounds contain 1,5-diphenylpent-4-en-1-one with different types of amines, thus producing larvicidal activity and toxicity based on the formation of chemical compositions. Compound 1i showed more larvicidal activity relative to other compounds, with an LD 50 of 12.09 ± 0.23 µg/mL, which was better than that of the controls temephos (LD 50 of 17.74 ± 0.01µg/mL) 33 and permethrin (LD 50 of 21.40 ± 0.02 µg/mL). The compound 1a induced 80% mortality at 100 µg/mL and its LD 50 value was 59.45 ± 0.02 µg/mL, whereas the antifeedant induced 100% mortality at 100 µg/mL and had a LD 50 value of 13.23 µg/mL; this is due to the compound presence of the hydrazine group, which showed full toxicity against O. mossambicus ngerlings within 15 min of screening.
Compounds 1b, 1g, and 1p induced 40% mortality at 100 µg/mL, whereas the antifeedant induced 20% mortality at 100 µg/mL due to the compound presence of the benzylidenehydrazine group, indicating that they are less toxic than compound 1a. Moderate activity was observed from compound 1c, which reached 40% mortality at 100 µg/mL, whereas the antifeedant reached 0% mortality at 100 µg/mL due to the presence of the (3-phenylallylidene) hydrazine group; therefore it is less toxic than compounds 1b, 1g, 1p, and 1c. Compound 1d and 1o induced 0% mortality at 100 µg/mL in both the larvicidal and antifeedant screening due to the presence of the 5-hydrazonopentanal and 1-benzylideneurea groups; thus they exhibited no active and no toxic behavior.
Compound 1k also induced 0% mortality at 100 µg/mL in larvicidal screening, and was highly toxic in antifeedant screening, inducing 100% mortality with a LD 50 value of 13.78 µg/mL due to the presence of the p-toluidine group. Compounds 1h and 1e induced 60% mortality at 100 µg/mL in larvicidal screening, and it induced LD 50 values of 65.85 and 66.25 µg/mL due to the presence of the 1-benzylidenethiourea and phenylhydrazine groups. Compounds 1f and 1j induced a mortality rate of 80% with an LD 50 values of 58.10 and 58.49 µg/mL in larvicidal screening, whereas they induced 100% mortality in antifeedant screening due to the presence of aniline and naphthalen-2-amine, respectively. Compounds 1m and 1n induced a mortality rate of 80% with an LD 50 values of 56.77 and 56.16 µg/mL in larvicidal screening, whereas they induced 0% mortality in antifeedant screening due to the presence of benzamide and urea, respectively. Compounds 1q and 1l induced a mortality rate of 20% in larvicidal screening, whereas they induced 20% mortality in antifeedant screening due to the presence of methylamine and acetamide, respectively. Therefore, the above analysis indicates that the compound li was signi cantly active in larvicidal and displayed less toxicity in antifeedant screening. The percentage of mortality and LD 50 values are presented in Table 3 and Table 4.

Culex quinquefasciatus larval growth regulation
To explore the impact of 1,5-diphenylpent-4-en-1-one formulations on C. quinquefasciatus larvae growth, metamorphosis, and production, we exposed the larvae to compound 1i for 72 hours. Table 5 summarizes the effects of compound 1i impact on larval weight and growth inhibition. When subjected to 10 µg/mL of compound 1i, the eclosion rate and time of the pupal and adult periods of administered C. quinquefasciatus is calculated, and the ndings are seen in Table 6. Compound 1i had a growthinhibition score of 41.36 % and suppressed larval weight development. Furthermore, compound 1i had little effect on the duration of the adult and pupal periods, but it did result in a 55 percent eclosion rate.
Compound 1i hindered the production and growth of C. quinquefasciatus larvae, according to these ndings.  The Autodock Vina program was used to assess the docking behavior between compounds 1i, permethrin and temephos with the 3OGN protein. Compound 1i displayed more binding a nity (-10.0 kcal/mol) than other compounds and permethrin (-9.7kcal/mol) and temephos (-7.6 kcal/mol) with the 3OGN protein.

Antifeedant Activity
The antifeedant activity was screened via 10, 25, 50 and 100 µg/mL concentrations of the tested samples and evaluated for marine ngerlings (O. mossambicus). Mortality caused by the compounds was evaluated as ratios (%) of the numbers of dead vs. live ngerlings. Table 2 summarizes the results.
The method followed was described previously 8 .

Larval growth inhibition and regulation
The regulation and inhibition of larval growth in C. quinquefasciatus by compound 1i (10 µg/mL) were analysed via the water-immersion method 34 .

Preparation of ligands
The ligand molecules (1a-1q) were drawn via Chemdraw 12.0 and energy was minimized by using the MM2 force eld in Chem3Dpro software. The ligand molecules were then saved in Protein Data Bank (PDB) format and further used for molecular docking studies.

Preparation Of Receptor
The 3D crystal structure of mosquito odorant binding protein (PDB ID: 3OGN) was downloaded from Protein Data Bank. The water molecules and inbound co-crystallized ligands were removed from the receptor using the Discovery Studio 2019 program. The receptor was energy minimized via the SWISS PDB Viewer program. The receptor was then used for molecular docking evaluation.

Identi cation Of Binding Pocket
The binding pocket of the target protein was recognized by using inbound co-crystallized ligands via the Discovery Studio 2019 Program. Residues of the amino acids Tyr10, Leu15, Leu19, Leu73, Leu80, Met84, Ile87, Ala88, Met91, His111, Trp114, His121, and Phe123 were situated in the binding pocket.

Docking
The interaction of binding modes between compounds 1a-1q, permethrin, temephos and the mosquito odorant binding protein was assessed using molecular docking studies via Autodock vina 1.1.2.
software 35 . The selection of docking grid box was based on the active amino acid residues situated on the binding pocket. The search grid of the 3OGN protein was stable with the dimensions sizes x: 22, y: 20, and z: 22 with center_x: 18.681, y: 49.66, and z: 11.409, with a spacing of 1.0 Å 36 . The value of exhaustiveness was set to 8 and the interactions were visually examined using the Pymol and Discovery studio 2019 programs.

Statistical analysis
The mean of the results (LD 50 values) was calculated based on at least three independent evaluations and the standard deviations (SD) were calculated using Microsoft Excel.

Conclusions
In this study, we identi ed the most effective and easily prepared larvicidal active Mannich base derivatives synthesis by way of the grindstone method by using Tyrosinase enzyme as a catalyst; this method is economical and produces good coating and high yield. These compounds were investigated using larvicides against Culex quinquefasciatus and toxicity screening against non-target aquatic species of ichthyotoxicity activity. A total of 17 compounds were screened, and compound 1i was found to be the most active among them (LD 50 = 12.09 µg/mL) against Culex quinquefasciatus compared with permethrin, and also induced 0% mortality within 24 h against Oreochromis mossambicus in an antifeedant screening. Molecular docking was carried out with all compounds 1a-1q and the controls temephos and permethrin against the 3OGN protein, and the docking score was the best for compound li. Therefore, our results indicate that compound li was the best insecticide, and these compounds may serve as a prospective foundation for emerging ecologically important bioactive compounds, as well as eco-friendly pesticides and biopharmaceuticals.

Con icts of Interest
The authors declare no con ict of interest.

Author Contributions
C.S synthesis of compounds and docking result analysis; D.A and S.A methodology of biological activity analysis; preparation; R.S chemical data analysis, A.I investigation total work chemistry and Biology. All authors were contributions preparation of through writing-original draft. Figure 1 Target molecules and commercial insecticide.   Recyclability of Tyrosinase enzyme catalyst

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