Synthesis and Larvicidal Activity of Novel Thenoylhydrazide Derivatives

A pair of chemical isomeric structures of novel N-tert-butylphenyl thenoylhydrazide compounds I and II were designed and synthesized. Their structures were characterized by MS, IR, 1H NMR, elemental analysis and X-ray single crystal diffraction. The regioselectivity of the Meerwein arylation reaction and the electrophilic substitution reaction of N-tert-butyl hydrazine were studied by density functional theory (DFT) quantum chemical method. The larvicidal tests revealed that some compounds I had excellent larvicidal activity against Culex pipiens pallens. As the candidates of insect growth regulators (IGRs), the larval growth inhibition and regulation against Culex pipiens pallens were examined for some compounds, especially I1 and I7. Compounds I1 and I7 were further indicated as an ecdysteroid agonist by reporter gene assay on the Spodoptera frugiperda cell line (Sf9 cells). Finally, a molecular docking study of compound I7 was conducted, which was not only beneficial to understand the structure-activity relationship, but also useful for development of new IGRs for the control of mosquitos.

A solution of dilute aqueous sodium hydroxide (10%, 16 mmol) was added dropwise to the stirred solution of tert-butylhydrazine hydrochloride (16 mmol) in dichloromethane (10 mL) at 0 °C. The reaction mixture was stirred for 30 min, followed by adding dropwise the solution of 3-(substituted phenyl)-2-thenoyl chloride (4 mmol) in dichloromethane (10 mL) and dilute aqueous sodium hydroxide (10%, 4 mmol) at 0 °C simultaneously. Then the mixture was stirred at room temperature for 5 h and filtered to obtain a yellow solution. The organic filtrate was washed with water and dried with anhydrous magnesium sulphate overnight. After removing the solvent, the residues were purified by flash column chromatography (40 × 250 mm) on silica gel using a mixture of petroleum ether (60-90 °C) and ethyl acetate as the eluent (V petroleum ether /V ethyl acetate = 3/1) to obtain I and II.   58.34; H, 5.55; N, 9.07. Found: C, 58.65; H, 5.74; N, 9.09. White White                         Crystallography. Compound I7 was recrystallized from methanol to give colorless crystals suitable for X-ray single crystal diffraction. Cell constants at 294(2) K were determined by a least-square fit to the setting parameters of independent reflections measured on a Bruker SMART 51 1000 CCD area-detector diffractometer with a graphite-monochromated Mo Kα radiation (λ = 0.071073 nm) and operating in the phi and scan modes. The structure was solved by the direct method with SHELXS-97 52,53 and refined by the full-matrix least squares method on F2 data using SHELXL-97 53,54 . The empirical absorption corrections were applied to all intensity data. Computational Method. Structures of 2-furoic acid, 2-thenoic acid, and tert-butyl hydrazine were calculated with density functional theory (DFT) quantum chemical method by using Gaussian 09 program package 55 . Equilibrium geometries of all the three molecules were fully optimized at the B3LYP/6-311G (d, p) level of theory 56 . Vibrational frequencies, calculated at the same level, were used to determine the nature of the stationary points. The charge densities of all the atoms in the three molecules were acquired.

N-(tert-butyl)-3-(4-bromophenyl)thiophene-2-carbohydrazide
Bioassay. Larvicidal Activity. Assessments were made on a dead/alive basis. Evaluations are based on a percentage scale of 0-100, which 0 equals no activity and 100 equals total kill. The bioassay was repeated three times, and the result of bioactivity was the average of these replicates. The commercialized insecticide RH-5849 was tested as a control under the same condition. EXCEL2007 was applied to analyze bioassay data. The LC 50 values of some active title compounds were evaluated using logit analysis and the results were analyzed using the statistical data processing system (DPS, 10.15, Zhejiang, China).
Larvicidal Activity against Mosquito (Culex pipiens pallens). The larvicidal activity was evaluated at the preliminary test concentration of 10 μg mL −1 against the fourth-instar Culex pipiens pallens by the water immersion method 44 under conditions of (27 ± 2) °C, photoperiod of 10:14 (light:dark), and relative humidity 50-70%. All Scientific RepoRts | 6:22977 | DOI: 10.1038/srep22977 the test beakers containing twenty Culex pipiens pallens were evaluated 8 days after treatment. The results were recorded by average percentage mortality.
Larvicidal Activity against Oriental Armyworm (Mythimna separata). The larvicidal activity of the title compounds against oriental armyworm was evaluated by foliar application 19,21 . For the foliar armyworm tests, individual corn leaves were placed on moistened pieces of filter paper in Petri dishes. The leaves were then sprayed with the test solution at the preliminary test concentration of 200 μg mL −1 and allowed to dry. The dishes were infested with 10 fourth-instar oriental armyworm larvae. Percentage mortalities were evaluated 4 days after treatment.
Larvicidal Activity against Diamondback Moth (Plutella xylostella). The larvicidal activity of the title compounds against diamondback moth was tested by leaf-dip method 23,25 . Leaf disks (1.8 cm diameter) were cut from fresh cabbage leaves and then were dipped into the test solution at the preliminary test concentration of 200 μg mL −1 for 15 s. After air-drying, the treated leaf disks were placed in a Petri dish (9 cm diameter) lined with a piece of filter paper, and then 10 second-instar diamondback moth larvae were transferred to the Petri dish. Percentage mortalities were evaluated 6 days after treatment.
Larval growth inhibition and regulation against Culex pipiens pallens determined by water immersion method. The title compounds were dissolved in acetone and diluted to the required concentration of 10 μg mL −1 as test solutions. Every 20 second-instar Culex pipiens pallens larvae were weighted in 50 mL beaker with exact quantity of water and the tested compounds. The water immersion method was applied referring to the previous procedure 44 . The weight of the treated larvae was recorded after a 72 h treatment and the larvae were reared until pupation and adult emergence. The duration of pupal and larval stages, and adult eclosion rate were recorded.
Agonistic activity determined by reporter gene assay. Sf9 cells (derived from ovary of S. frugiperda and obtained from South China Agricultural University, China) were used for the reporter gene assay. Cells were cultured in HyClone SFX-Insect serum-free insect culture medium (Thermo Fisher Scientific, China) supplying with 5% fetal calf serum at 27.5 °C. A reporter plasmid pBmbA/hsp27/gfp, referred as ERE-b.act-GFP, was composed of seven copies of the ERE derived from D. melanogaster hsp27 promoter, and a promoter region of actin A3 gene of B. mori, which was followed by an EGFP coding gene. This plasmid was constructed for detecting ecdysone agonists dependent activation of transcription by Dr. Swevers et al. 57 .
The plasmid was purified by Omega Endo-free Plasmid Midi Kit (Omega Bio-Tek, Inc., China) after amplified in Escherichia coli according to the manufacturer. Then the plasmids were merged with MegaTran 1.0 (OriGene, Beijing, China) as complex and gently added into the cells (5 × 10 4 per well) in each well of 24-well plates. After incubation for 24 h, the test compounds (dissolved in DMSO) were diluted with culture medium and added into the cells for another 24 h incubation. The induced GFP fluorescence on the living cells was observed directly using Olympus CKX41 inverted microscope (Aizu, Japan). The micrographs were collected with ProgRes CF digital camera system (Jenoptik, Germany). Then, cells in each well were collected and were gently washed twice with 250 μL PBS and transferred to black 96-well plates at a density of 3 × 10 4 cells/mL. Fluorescence intensity of the cells was recorded by a 1420 Multilabel Counter Victor 3V (Perkinelmer, Massachusetts, America), and corrected by auto-fluorescence and background fluorescence in the absence of compounds 58 .
Molecular modeling and docking study. The ligand binding domain (LBD) of Culex pipiens EcR was constructed on the Swiss Model website (http://swissmodel.expasy.org/) [59][60][61] . The primary sequence was derived from the NCBI database (NCBI accession number: XP_001844581). The sequence of Culex pipiens EcR was aligned with HvEcR with an identity of 76.65% (shown in Supplementary data Fig. 1S). The chain D of the crystal structure of the ligand binding domain of HvEcR (PDB: 1R20) was selected as the template for homology modeling building 62,63 . The constructed modeling was evaluated by QMEAN, with the value of 0.63, as a reasonable modeling 64 . The downloaded structure was qualified and employed for the docking study. Molecular docking was performed using Surflex-Dock protocol in Sybyl 8.0 with the MMFF94 force field to evaluate the potential molecular binding mode between the synthesized compound and LBD of Culex pipiens EcR.
The three dimensional structure of EcR LBD was constructed and refined with MMFF94 by energy minimization and defined as a receptor. The active site was defined based on the ligand binding location of BYI06830 (the original ligand) with a radius of 10 Å. Once the compound was docked into the active site, a simulated annealing method was conducted following the default parameters and programs. Finally, 20 molecular docking poses were saved and ranked according to their dock score function. The pose with the lowest interaction energy was considered as the best one. The interaction energy of the docked LBD of EcR with compound was − 45.238 kcals mol −1 (pKd 6.54, crash − 0.32, and polar 2.29). To obtain more insights into the ligand-receptor interaction, the binding modes of compound I7 docked to the active site of EcR were exhibited and shown.

Results and Discussion
Chemistry. By the method of Meerwein arylation using copper(II)-catalyzed decomposition of diazonium salts, a series of 5-substituted phenyl-2-furoic acid were prepared in good yields in our previous work. The nucleophilic reaction with 2-furoic acid often exhibited a high regioselectivity at the 5-position of furan ring. When we change the reagent from 2-furoic acid to 2-thenoic acid, the nucleophilic reaction still kept a high regioselectivity, but the reactive position changed to the 3-position of thiophene ring (Fig. 2). It could be confirmed by the X-ray single crystal diffraction of compound 2 and all the coupling constants of H-4 with H-5 ( 3 J HH = 5.04 Hz) in the 1 H NMR data (see supplementary information for 1 H NMR spectra).
The DFT method was applied to calculate the optimized structures of 2-furoic and 2-thenoic acid. The charge density of all the atoms in both (Fig. 3) were obtained. The results showed that for 2-furoic acid, the charge density Scientific RepoRts | 6:22977 | DOI: 10.1038/srep22977 of C-5 (0.086) was positive, whereas that of C-3 (− 0.074) and C-4 (− 0.165) were negative, which meant that the nucleophilic reaction easily occurred at the C-5 position in 2-furoic acid. For 2-thenoic acid, the charge density of C-3 (0.011) was positive, while that of C-4 (− 0.076) and C-5 (− 0.279) were negative. It showed clearly that, compared to 2-furoic acid, the nucleophilic reaction selectively occurred at the C-3 position in 2-thenoic acid rather than C-5.
The same computational method was applied to calculate charge density of the two nitrogen atoms in the tert-butyl hydrazine (Fig. 4). The results showed that both of the nitrogen atoms were negative and the one connected with two hydrogen atoms had lower negative charge density (− 0.372) than that of the one connected with the tert-butyl group (− 0.320). Due to the bulky steric effect of the tert-butyl group and the low charge density, the electrophilic reaction was difficult to occur at the nitrogen atom connected to the tert-butyl group. That was the reason why all the title compounds I was the main product and had much better yield than compounds II.
The different effect factors, such as different substituted groups (Table S1), different bases (Table S2) and different molar ratio of reagents (Table S3), on the molar ratio of title compounds I and II were studied in this paper. It could be seen that the strong electron-withdrawing ability of the substituent led to the poor selectivity of the reaction. The sequence of the selectivity was 4-F < 4-NO 2 < 4-Cl < H < 4-CH 3 < 4-OCH 3 (Table S1). And the position of the substituted group also affected the reactive selectivity. The sequence was 4-Cl > 3-Cl > 2-Cl. The result of different bases on the selectivity showed that NaOH had the lowest effect on the molar ration of the title compounds (Table S2). The different molar ratio of reagents was also an important factor to the reactive selectivity. The excessive acyl chloride made the diacylhydrazide as the byproduct, and the optimized molar ratio of acyl chloride and hydrazine was 1:4 in this case (Table S3).
The crystal data are presented in Table S4 and Fig. 5, which give perspective view of compound I7. Some important bond lengths, angels, and torsion angles of compound I7 are given in Table S5. It could be seen from the X-ray single crystal analysis of I7 that the distance of C12-N2 (1.502(3) Å) and N1-N2 (1.426(3) Å) are equal   35 Å), and the single bonds C6-C7 and C10-C11 (1.487(4) and 1.499(3) Å) are shorter than the standard C-C single bond (1.54 Å), but longer than C-C double bond (1.34 Å). All of these clearly indicated that the p orbital of N1 atoms conjugated with the π molecular orbital and formed the delocalized π -bonds with the conjoint thiophene and benzene ring. But the p orbit of N1 seemed not to be conjugated with the π molecular orbital of the C11-O1 double bonds, which was explained by the bond length of C11-O1 (1.227(3) Å) that followed in the normal range of C-O double bond (1.19-1.23 Å).
In the crystal of the structure, C (1), C (2), C (3), C (4), C( 5) and C (6) formed a plane with the mean deviation of 0.0074 Å, defined as plane I; C (7), C (8), C (9), C( 10) and S (1) formed a plane with the mean deviation of 0.0035 Å, defined as plane II; O (1), C (11), N (1), and N (2) were nearly coplanar with the mean deviation of 0.0162 Å, defined as plane III; C (12), C (13), C (14) and C (15) (the tert-butyl group) were not coplanar, which were defined factitiously as plane IV with the mean deviation of 0.3825 Å (Fig. 5 and Table S6). Plane II, plane III and plane IV make a dihedral angle with plane I of 58.9°, 69.4° and 83.0°. Plane III and plane IV make a dihedral angle with plane II of 40.1° and 113.6°. And the dihedral angle between plane III and plane IV is 149.3°. The related data are summarized in Table S6.
Bioassay. Insecticidal Activity. The insecticidal results of the title compounds against Plutella xylostella, Mythimna separata, and Culex pipiens pallens were listed in Table 1. Mostly the insecticidal activity of compounds I was better than that of compounds II. The results indicate that the title compounds have significant promise for control of mosquitoes. For example, the LC 50 of compounds I1 and I7 against Culex pipiens pallens were 2.47 ± 0.14 μg mL −1 and 2.33 ± 0.12 μg mL −1 , which had the comparable activity with RH-5849 (2.52 ± 0.11 μg mL −1 ). The developmental effects on the mosquito larvae showed that the compounds can induce  the formation of nonviable larval-pupal intermediates (Fig. 6). Examination of the mosquito larvae treated by the title compounds revealed the presence of an ecdysial space between the epidermis and cuticle (Fig. 7). In some cases, a complete new cuticle appeared to be produced in response to treatment with the compounds, but the larvae failed to shed the head capsule and ecdyse and died trapped within the exuvium.
Some title compounds also exhibited significant insecticidal activity against Mythimna separate and Plutella xylostella. For example, the LC 50    Growth inhibition activities. Considering the regulation of ecdysteroid analogs on development, growth, and metamorphosis, the effects of the compounds I1 and I7 after a 72 h treatment on the weight gain of larvae and inhibitory rates (Table 2), the duration of the pupal and larval stages, and also the eclosion rate of the treated Culex pipiens pallens were evaluated at 10 μg mL −1 ( Table 3). The weight gain of larvae was inhibited by compounds I1 and I7, which gave inhibitory rates of 37.8 ± 2.9% and 39.1 ± 2.7%. The effects of compounds I1 and I7 on the duration of the pupal and larval stages were not obvious, but the rates of eclosion were only 58.2 ± 3.1% and 60.1 ± 2.8% after treatment by compounds I1 and I7. The results showed that compounds I1 and I7 possessed potent inhibitory activity on the growth and development of Culex pipiens pallens.
Reporter gene assay for the title compounds. To confirm the bioactivity of the title compounds as ecdysteroid agonists, reporter gene assay 57 was applied on compounds I1 and I7. The induced fluorescence intensity was evaluated (Fig. 8). The reporter plasmid had an ERE (ecdysone response element) derived from the Drosophila melanogaster hsp27 gene and a basal actin promoter derived from Bombyx mori, which was followed by a gfp gene and a termination signal. In this study, the plasmid was transfected into Sf9 cells in which endogenous EcR and USP were detected. This cell-based reporter gene system could evaluate whether or not the tested compounds act    Table 3. The Effect of I1 and I7 on the Growth and Development of Culex pipiens pallens a . # Data derived from the mean of three independent assays (means ± SE). The same letters in the same column indicate no significant difference of the means at p < 0.05 by Duncan's multiple-range test. † The concentration of I1 and I7 was 10 μg mL −1 . CK is the black control not containing the compounds.
on the receptor of EcR. After correction for auto-fluorescence, the fluorescence intensities of compounds I1 and I7 were higher than that of the negative control at 10 μg mL −1 , which was no significant difference between each other. Although their fluorescence intensities were lower than that of RH-5849. This result suggested that the title compounds induced transcription of the reporter gene and acted on EcR as agonists.
Molecular modeling and docking study. At least 4 residues in the LBD were involved in the interactions between the ligands and the receptor (Fig. 9). Compound I7 could form hydrogen bonds with Tyr323 and Asn419 in the LBD interacting with the imine and carbonyl groups of I7. The hydrophobic t-butyl and 3-phenyl-2-thiophene group of I7 were surrounded by the hydrophobic residues Tyr323 and Leu335, respectively. Meanwhile, the benzene ring of 3-phenyl-2-thiophene formed the π -π interaction with the residue Tyr323. The binding modes of I7 within the LBD provide detailed structural insights into the interaction between the title compounds and the receptor. The formation of hydrogen bonds in the ligand-receptor complex, the hydrophobic and π -π interaction between the compounds and the ecdysone receptor EcR, which played key roles in promoting the binding affinity of the compound to regulate and disrupt the growth or sterility of the insects. That could lead to and promote the death of the insects.

Conclusions
In summary, a pair of chemical isomeric structures of novel N-tert-butyl hydrazide containing 3-phenyl-2-thiophene moiety (I and II) were designed and synthesized. Their insecticidal tests indicated that most of the title compounds showed insecticidal activity against Plutella xylostella, Mythimna separata, and Culex pipiens pallens. Mostly the insecticidal activity of compounds I was better than that of compounds II. Some compounds I had excellent larvicidal activity against Culex pipiens. The title compounds functioned as ecdysteroid agonists, causing abnormal metamorphosis in insects and inducing transcription of the reporter gene. The observed biological effects on the mosquito larvae showed that the active compounds can induce the formation of nonviable larval-pupal intermediates. In some cases, mosquito larvae initiated molting and apolysis but failed to complete the molt and died trapped within the exuvium. The bioassay results showed that compounds I had great promise as a novel lead compound as insect growth regulators for further development.