Synthesis, Characterization, Biological Evaluation ...

Appl Biochem Biotechnol DOI 10.1007/s12010-015-1562-x

Synthesis, Characterization, Biological Evaluation and Docking Study of Heterocyclic-Based Synthetic Sulfonamides as Potential Pesticide Against G. mellonella Priyanka Sharma 1 & Sunil Thakur 2 & Pamita Awasthi 1

Received: 29 April 2014 / Accepted: 12 March 2015 # Springer Science+Business Media New York 2015

Abstract Juvenile hormone is an important hormone which controls the developmental process in the lepidopteran insects, hence, referred as insect growth regulator. Juvenile hormone binding proteins are the carrier of juvenile hormone from the site of secretion to the site of action and play vital role in juvenile hormone action. We have designed four different juvenile hormone analogs incorporating sulfonamide and heterocyclic moieties using computer-aided tools. All analogs (T3–T6) gave comparative energy profile in comparison to in use insect growth regulators like fenoxycarb (T2) and pyriproxyfen (T1). Further, theses analogs have been screened on biological model Galleria mellonella (wax moth) for their mortality rate. All analogs were evaluated using three different concentrations (1000, 1500, and 2000 ppm) and five different exposure periods (2, 4, 6, 8, and 10 h). In vivo study showed that analog N-(1-isopropyl-2-oxo-2-morpholino-ethyl) toluene sulfonamide (T6) and N-(1isopropyl-2-oxo-2-piperidino-ethyl) toluene sulfonamide (T4) exhibit the good larval mortality at lower concentration (1000 ppm) after 8 h exposure in comparison to pyriproxyfen (T1) and fenoxycarb (T2). The findings demonstrate the effectiveness and validity of the virtual screening approach (docking) and provide a starting point for the development of novel juvenile hormone analogs to counter G. mellonella. Keywords Juvenile hormone . Juvenile hormone analogs . Insect growth regulators . Juvenile hormone binding protein . G. mellonella . In silico screening . In vivo study

Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1562-x) contains supplementary material, which is available to authorized users.

* Pamita Awasthi [email protected] 1

Department of Chemistry, National Institute of Technology, Hamirpur 177005, India

2

Nematology, Institute of Environmental Science and Biotechnology, Hamirpur 177001, India

Appl Biochem Biotechnol

Introduction The most striking feature of the juvenile hormone of insect is the extraordinarily broad range of its developmental and physiological effects. Juvenile hormone (JH) plays an important role in the control of larval development and metamorphosis and various other aspects [1]. JH has been studied from several perspectives by researchers with interests in basic understanding as well as commercial applications. The agrochemical industry focused its attention on the chemical identity of naturally occurring hormones and their structureactivity relationships. This work carried out in direction of design of hormone mimic can be used as arthropod-specific pesticides. Juvenile hormone, different from animal hormones, with sesquiterpenoid skeleton consisting of three isoprene (5-C) units, a methyl ester group at C-1, and a 10, 11 epoxide at the other end (Fig. 1a). Detailed structure-activity relationships have been reported in literature on diverse insect species [2, 3]. Important features required for JH activity include the presence of both the methyl side branches and the terminal carbonyl group. Incorporation of aromatic rings at various locations within the carbon chain can further enhance its activity [4], and phenoxy derivatives of terpenoids are considered to belong to this category (Fig. 1b). It is well established that many potent mimic of JH, both natural and synthetic, have different chemical structures compared to the juvenile hormones of insect. Structure-activity relationships revealed that it is the polarity, stability, and chain lengths which contribute equally toward the activity of the analogs. Chemical synthesis has been the simplest and direct approach toward the development of new class of juvenile hormone analogs with juvenile hormone activity. Large number of juvenile hormone analogs (JHAs) have been synthesized in the literature and found to have potent JH activity against lepidopteran insects [5–12]. JH, hydrophobic hormone, is released from corpora allata and after passing through the plasma membrane binds to the receptor protein present in the cytoplasm or nucleus. The JH-receptor complex acts as a transcriptional regulator that either enhances or represses the expression of specific genes [13]. There are many “”JH-binding proteins (JHBPs) as well as degradative enzymes that bind JH with various affinities [1, 2]. In general, JH binding proteins are believed to have important roles in the distribution, protection, and delivery of the hormone to the target cells. Homology modeling revealed a strong sequence similarity in hemolymph JHBP of Lepidopteran insect species [14]. Lepidopteran low molecular weight JHBPs have been characterized biochemically. It has been found experimentally that low molecular weight JHBP are single polypeptide chain which can bind only to one JH molecule. Galleria mellonella act as severe honey comb pest and is the object of present study [15, 16]. Numerous experiments suggested that a conformational change occurs in JHBP of G. mellonella upon JH binding [17, 18]. The crystal structure of a JHBP molecule of wax moth, G. mellonella (Gm JHBP; PDB code: 2rck), has been solved showing two binding cavities: W cavity (diameter 6 to 10 Å) and E cavity (diameter 6 to 13 Å) [19]. As per photo affinity labeling experiments and crystallographic study, the JH binding site is located in the W cavity [20]. The calculated volumes of the W cavity is sufficient to accommodate JH homologs (JH I, JH II, and JH III) [19]. Number of analogs of JH is synthesized and studied extensively. Carbamide feature as well as aliphatic alkyl chain has been added to the parent skeleton. L-amino acid also has been added to the parent structure to enhance the interaction with the biological (target)


Fig. 1 a Natural juvenile hormones, b synthetic insect growth regulators (T1–T2), c structure of parent analog of designed series (T3–T6)

model [21, 22]. Heterocyclic rings as well as aromatic ring have been added at the terminal end of the analogs. All these moieties found to be potent for JH activity [11, 23–25]. To gain insight into the interactions between JH analogs and amino acid residues at the pocket site; various structural studies at the atomic level have been undertaken using computeraided tools [26–28]. Analogs have been designed considering the chemical nature of the pocket. Different functionalities have been added to the parent non-terpenoid skeleton. Sulfonamide functionality has been added to the parent skeleton because they are known to possess broad biological activities [29]. Different combinations of all these structural moieties in order to develop a potent analog of JH were a challenging task. Therefore, we propose to design JHAs having sulfonamide, aromatic, and heterocyclic features at the main chain (Fig. 1c). The work presented, here, is a part of pest management program carried out in our laboratory. In this paper, we presented the synthesis, characterization, biological evaluation, and docking study of different analogs of JHAs against JHBP of G. mellonella.


Materials and Methods Docking Study AutoDock 4.2 automated docking tool (The Scripps Research Institute La Jolla, CA 92037-1000, USA) is used for present study. The structures of analogs have been built using pymol software tool (www.pymol.com) and optimized using AMBER force field. Analog structures are saved as ligand.pdbqt format. The PDB file of the JHBP of G. mellonella (2RCK) has been downloaded from the protein data bank (www.rcsb.org) and saved in protein.pdbqt format. Grid box was generated around the binding cavity of the JHBP by selecting key amino acid residues THR 22, TYR 128 and 130 [19] [Supplementary Figure I]. The grids were chosen to be sufficiently large not to include the active site but also significant portion of the surrounding surface at receptor protein with grid points 69×67×69, along with grid spacing of 0.531 A0. Lamarckian genetic algorithm (LGA) protocol was applied using protein fixed: ligand flexible model [30]. Therefore, in totality, hundred search attempts were performed for each analog. All docking parameters were set as default. All the analogs were ranked according to their binding free energy (ΔGb in kcal/mol) and inhibition constant (Ki in μM) at 298.15 K. Overall free energy of binding (ΔGb) was composed of a sum of free energies measured for each analog pose and given as ΔGb ¼ intermolecular energy þ internal energy þ torsional energy − unbound system’s energy Ki ¼ exp ðΔG 1000Þ = ðR T Þ (where ΔGb is binding energy, R=1.98719 cal, and T=298.15 (temp in Kelvin). Combination of intermolecular + internal energy forms “dock energy”, while intermolecular + torsional energy forms “binding energy”. Same protocol was applied for all analogs. All simulations have been performed on Linux operating system with system properties (Intel(R) Pentium(R) D CPU 2.80 GHz, 4.0 GB of RAM).

General Methods and Material for Synthesis Solvents used were purchased from Sigma-Aldrich of 99 % purity grade. The progress of the reaction was monitored by thin layer chromatography (TLC). Melting points were determined on a hot-stage apparatus and are uncorrected. FT-IR spectra were recorded on Perkin Elmer 1600 spectrophotometer with the samples as compressed KBr pellets from 4000 to 400 cm−1. 1H and 13 C NMR spectra were recorded using a Brucker Avance 400 MHz spectrophotometer operating at room temperature in DMSO d6 as solvent. The electron spray ionization-mass spectroscopy analyses (ESI-MS) were carried out in positive ion modes using a Water Q of Micromass.

General Procedure for the Synthesis of Benzene Sulfonyl Valine Acid Chloride and p-Toluene Sulfonyl Valine Chloride A mixture of valine (11.7 g, 0.1 mol) and benzene sulfonyl chloride (17.6 g; 0.1 mol) and NaOH solution (1 N, 200 ml) was stirred together at 65–70 °C for 4 h [31, 32]. A clear solution


was obtained. The reaction mixture was cooled to 5 °C and treated with concentrated HCl to make it slightly acidic (pH 6.5). Benzene sulfonyl valine separated out as white crystals. It was recrystallized from hot water to give pure benzene sulfonyl valine (16.02 g; 69.95 %); m.p. 118–120°. Similarly, p-toluene sulfonyl valine was prepared (17.8; 73.25 % m.p. 128–138°. Further, benzene sulfonyl valine (1 g; 0.004 mol) was dissolved in dry benzene, and an excess of freshly distilled thionyl chloride (5.0 g) was slowly added to it and stirred at room temperature for 20 min. The reaction mixture was gently refluxed for 3 h. The solvent and excess of thionyl chloride were then distilled off under reduced pressure to give acid chloride of benzene sulfonyl valine. Similar procedure was followed for the preparation of p-toluene sulfonyl valine chloride [Supplementary Figure II]. These were used for next reaction without further purification. Synthesis of N-(1-Isopropyl-2-Oxo-2-Piperidino-Ethyl) Benzene Sulphonamide (T3) and Related Compounds (T3–T6) The synthesis of N-(1-isopropyl-2-oxo-3-aza-3-N-methyl-butanyl) benzene sulfonamide and related compounds (T3–T8) was accomplished along the following lines [Supplementary Figure II]. The acid chlorides of benzene and toluene sulfonyl valine prepared above were treated with piperidine at room temperature for 4 h to give desired products N-(1-isopropyl-2oxo-2-piperidino-ethyl) benzene sulphonamide (T3) and N-(1-isopropyl-2-oxo-2-piperidinoethyl) toluene sulphonamide (T4), respectively. Likewise, acid chlorides of benzene and toulene sulfonyl valine were treated with morpholine at room temperature for 4 h to give desired products N-(1-isopropyl-2-oxo-2-morpholino-ethyl) benzene sulphonamide (T5) and N-(1-isopropyl-2-oxo-2-morpholino-ethyl) p-toluene sulfonamide (T6), respectively. The structures of final products (T3–T6) were established by spectroscopic studies [Supplementary Table I].

Rearing of the Model Insect An artificial diet was developed during the year 2012–2013 for mass rearing of G. mellonella in our laboratory (temperature 27±1 °C, relative humidity 65±5 %, and 16:8 h scoto-photophase regime) and prepared by well-defined method with some modifications [33]. The diet consisted of the following ingredients on w/w basis: maize flour Zea mays—4 parts; wheat flour Triticum sp.—2 parts; wheat bran Triticum sp.—2 parts; dry yeast—1 part; milk powder—1 part; honey—4 parts, and glycerine—4 parts. In addition, antibiotic (Terramycin) and multivitamin (Supradin) each at 5 g per kg diet were also added to produce disease free and vigorous larvae. These ingredients were mixed thoroughly and stored in a tight container in refrigerator. This diet supported the growth and development of G. mellonella successfully for five generations with most suitable impact on its reproductive potential.

Formulations Tested Based upon literature review (different concentrations of pyriproxyfen like 100,000, 1000, and 3000 ppm against lepidopteran insects) [34–37]; different formulations of standards IGR pyriproxyfen (T1) and fenoxycarb (T2) have been prepared in acetone on w/v basis and designated as stock solution. Earlier, we prepared the concentrations—750, 500, and 250 ppm for all the analogs to check the effectiveness of analogs with respect to exposure period (in hours). None of the compound gave the good pest mortality at desired exposure


period (2–10 h). Three concentrations (2000, 1500, and 1000 ppm) were prepared from stock solutions of each formulation by adding distilled water and stored in cool dry conditions (0 °C) until their use. Similarly, the formulations of the four analogs have been prepared using the same stochiometry having concentrations 2000, 1500, and 1000 ppm. Two milliliter volume of each is used for the in vivo study. Larvae of forth instar of G. mellonella were collected and maintained for the treatment. Acetone solution (2 ml) containing the compound was poured on the filter paper in each petri dish and allowed to evaporate. Later, the counted number of larvae to be treated was transferred to the petri dishes and after applying the different concentrations of the analogs; mortality rate was noted for each of the replication set. Each treatment involved three replicates with each replicate containing ten insects. Same procedure is applied for all the analogs.

Statistical Analysis All data were grouped according to formulation (solution of analogs T1 to T6), concentration (C) and exposure time (I) and then subjected to analysis of variance. All data were statistically analyzed to calculate the critical difference (CD) at P≤0.05.

Results and Discussion Synthesis of N-(1-Isopropyl-2-Oxo-2-Piperidino-Ethyl) Benzene Sulfonamide and Related Compounds (T3–T6) Synthesis of N-(1-isopropyl-2-oxo-2-piperidino-ethyl) benzene sulfonamide and related compounds (T3–T6) has been achieved by the action of substituted amines with acid chlorides in dry benzene. N-(1-isopropyl-2-oxo-2-piperidino-ethyl) benzene sulfonamide and related compounds (T3–T6) have been found to be new in literature and fully identified on the basis of their spectral data (IR, 1H NMR, 13C NMR, and ESI-MS analysis). All the compounds have been obtained as pure solids [Supplementary Table II].

JHBP-JHAs Interactions Molecular docking has been employed based on the structure of JHBP which contributes to our understanding of the molecular mechanisms underlying selectivity of heterocyclic sulfonamides and suggested a possible path to design new IGRs. A docking screening model of JHAs with JHBP has been evaluated. The virtual screening hits were analyzed by considering their physical-chemical features, therefore, were focused to these compounds in the present article. Four synthetic analogs (T3–T6) and in use IGRs (T1 and T2) have been successfully docked into the hydrophobic cavity of JHBP of G. mellonella, and binding free energies of the complexes have been calculated using AutoDock 4.2 software [26–28, 38, 39]. The binding of JHAs with JHBP of G. mellonella and the best docking pose for each analog provided useful insights into the biological function of this protein. This knowledge is required for structural alteration of some of these ligands in order to modify the anti-JH properties. The energy profile was sequenced according to the binding energy profile (B.E. (ΔGb) Kcal/mol), docking energy (Kcal/mol), and inhibitory constant (Ki) (Table 1). All analogs exhibit low binding energy profile upon interaction with JHBP than fenoxycarb (T2) but higher than

Appl Biochem Biotechnol Table 1 Free energy of binding (ΔGb, Kcal/mol), docking energy (Kcal/mol), inhibitory constant (Ki in μM) and hydrogen bond interactions along with distances (A°) of IGRs (in use T1–T2 and synthesized T3–T6) with JHBP of G. mellonella Inhibition const

S. No. IDs

Binding energy

Docking energy

Torsional energy

(Kcal/mol)

(Kcal/mol)

(Kcal/mol)

−8.25

0.60

Binding interactions

Ligands Ki(µM)

Dista nces

Numb er in

(A)

cluster

2.781

99

1. T1

5.32 *

−7.18

O 2……. OH THR 22 O5……..O THR 22

2.

3.

4.

2.587

T2 136.43*

− 5.27

−0.69

1.19

C6=O ......HN LYS 218

1.779

42

7.74

−6.97

−8.94

1.49

C 5=O …..HN LYS 218

1.96a

39

12.33

−6.70

−9.64

1.49

C5=O…HN LYS 218

2.16

3

9.91

−6.83

−9.41

1.49

O……HN LYS 85

1.988

3

10.96

−6.77

−9.05

1.49

O……..HN LYS 218

2.073

23

T3

T4

5.

T5

6.

T6

pyriproxyfen (T1) (in use IGRs). Among all synthetic analogs (T3–T6), T3 and T5 gave the lowest binding energy profile (Fig. 2a). Total internal energy of all the analogs was compared. All the analogs (T1–T6) exhibit almost similar trend for internal energy profile. Uniform trend was not observed for the intermolecular energy profile for all the analogs (T1–T6). fenoxycarb (T2) exhibit the increase in intermolecular energy profile attributed for the rise in binding energy as well as docking energy (Fig. 2a, b). Torsional energy is associated with the degree of freedom of the analogs. Degree of freedom is directly related to the conformational change of the ligands inside the pocket. All the synthetic analogs (T3–T6) exhibit the increase in torsional energy profile (Fig. 2b). Inhibitory constant (Ki) vary in a similar manner as B.E. profile for all the synthetic analogs (T3–T6) and in use IGRs (T1–T2). Low is the value of inhibitory constant; more is the expected biological activity of the analogs. All the synthetic analogs bear comparable values of inhibitory constant. All the designed heterocyclic sulfonamide analogs (T3–T6) have common mode of interactions inside the binding pocket (Table 2). Analogs showed common bonding interaction with THR 22, LYS 218, and TYR 128,130 with in use IGRs (T1 and T2) [Supplementary Figure III (a & b)]. Comparing synthetic analogs (T3–T6), there occur the additional interactions with SER 129, HIS 207, and AGR 210, 214 inside the pocket (Table 2). Pyriproxyfen (T1) showed the leading behavior over all the analogs. It showed the unique binding interaction with PRO 146. This amino acid acts as strong codon having tendency to form six hydrogen bonds [40]. This type of interaction could be responsible for the leading nature of the analogs to be the best inhibitor. Further synthetic analogs (T3–T6) also exhibit the strong tendency to form six, five, and five hydrogen bonds with ARG, HIS, and SER amino


Fig. 2 a Binding energy profile of IGRs (T1–T6), b energy variations (intermolecular, internal, and torsional energy) of all the analogs with juvenile hormone binding protein of G. mellonella

acid, respectively, as they are being referred as mixed codons [40]. The formation of a ligandreceptor complex is controlled by the forces of molecular recognition. Any approach toward ligand design must be based on a firm understanding of these forces. These enthalpic intermolecular interactions can be divided into main groups: hydrogen bonding, electrostatic, steric, and hydrophobic interactions. The strength of the hydrophobic interactions of a protein is influenced by the shape of the pocket and the exposed surface area of the residues. Hydrogen bonding is a combination of electrostatic and steric effects. The role of H bonding seems to be more related to specificity, especially when the interaction is between charged groups [41].

Appl Biochem Biotechnol Table 2 Additional interactions (bold—common, normal—additional) of the IGRs (in use T1–T2 and synthesized T3–T6) with amino acid residues at the binding pocket of JHBP of G. mellonella S. No.

IDs

Additional interactions of the ligands with amino acid residues inside the binding pocket

1

T1

THR 22, LYS 85,218, PRO 146**, THR 148

2

T2

THR 22, TYR 130, LYS 218

3

T3

THR 22, SER* 129, HIS* 207, ARG** 210,214, LYS 218

4

T4

LYS 218, SER* 129, HIS* 207, ARG **210,214

5

T5

THR 22, LYS 39, 85,218, TYR 128, SER* 129, THR 148, HIS* 207, ARG**210,214

6

T6

LYS 39,218, TYR 128, 148, VAL149, SER* 129, HIS* 207, ARG **210,214

*Mixed codon **Strong codon

Hydrogen bond through entropy contributes more strongly to the free energy of binding. H bonding groups within a receptor site forms a unique 3D patterns. A ligand that can position complementary groups in the correct geometry will be able to bind to the receptor groups. Binding site will have a distinct electrostatic profile due to the differing electro negativities and bonding environments of the receptor atoms. Electrostatic interactions between a ligand and a receptor are localized and are responsible to a large extend for the enthalpy of binding. The affinity of the ligand will be enhanced if the pattern of the ligand partial charges can be made to complement that of the receptor. Complementary does not simply imply that positive charge on the ligand should be matched by the negative charge on the receptor. Complementary should be taken to imply a matching of the magnitudes of the charges. Electrostatic interactions play an important part in determining specificity. These interactions strongly contribute to the binding. Further, all the analogs (T3–T6) bear the comparable binding energy profile lying within the range of (ΔGb ≥2 Kcal/mol). Methylated analogs T4 and T6 bear the lower energy profile (Fig. 2a) as compared to non-methylated analogs T3 and T5. All the structural changes in conformation of synthesized analogs (T3–T6) are responsible for lower binding energy profile. Amongst all, analog T3 bears the lower energy profile having values −6.97 Kcal/mol.

In Vivo Analysis of Synthesized Analogs on G. mellonella Effect on Larval Mortality A significantly progressive increase in mortality was recorded with the increase in exposure period as well as concentration. The observations regarding effect of different concentrations of sulfonamide IGRs at different exposure periods on percent larval mortality of G. mellonella have been shown in Fig. 3 [Supplementary Table II]. None of the treatment was significantly effective at 1000 ppm concentration after exposure of 2 h with any larval mortality including commercial in use IGRs (T1 and T2). Although there was a progressive increase in mortality with increase in exposure period, overall rate of mortality remained low at this concentration. T1 shows maximum efficacy of 100.00 kills at 8 h exposure period followed by mortality of 100.00 % by T2 at same concentration and exposure. T6 and T4 with same concentration exhibit 100.00 and 66.67 % mortality at the exposure period of 8 h. Mortality did not exceed


Fig. 3 Effect of different concentrations (ppm) at different exposure period (in hours) of different JHAs (T1–T6) on larval mortality of G. mellonella

beyond 67.00 % in rest of the treatments for 8 hexposure period. Increasing the exposure periods up to 10 h, T4 attained the 90.00 % larval mortality followed by T5 and T3 at the same concentrations. Mortality did not exceed beyond 90.00 % in all other formulations. Fig. 4 a Physiological changes on G. mellonella larvae after applying 2000 ppm concentrations of N-(1- isopropyl-2-oxo-2-morpholino-ethyl) toluene sulfonamide (T6) at exposure period of 2 h in comparison to pyriproxyfen (T1) and fenoxycarb (T2), b physiological changes on G. mellonella larvae after applying 1500 ppm concentrations of N-(1-isopropyl-2-oxo-2-morpholino-ethyl) toluene sulfonamide (T6) at exposure period of 4 h in comparison to pyriproxyfen (T1) and fenoxycarb (T2), c physiological changes on G. mellonella larvae after applying 1000 ppm concentrations of N-(1-isopropyl-2-oxo-2-morpholino-ethyl) toluene sulfonamide (T6) at exposure period of 6 h in comparison to pyriproxyfen (T1) and fenoxycarb (T2)



Trends were similar for 1500 ppm concentration. T6 formulation was found to be least effective with 10.00 % kill at 2 h exposure time while T5 formulation at the same exposure period exhibited the 20.00 % mortality. While percent mortality in T5 enhanced from 20.00 %at 2 h exposure to 53.33 % at 6 h, T2 showed the enhancement from 53.33 to 100.00 % at 6 h followed by T1 from 46.67 to 100.00 % mortality at similar exposure periods. T3 also showed the increase from 16.67 to 76.67 % kill. Likewise, T4 exhibited the enhancement from 13.33 to 76.67 % kill at similar exposure period (6 h) at the same concentration. T5 and T6 were least effective registering 53.33 and 63.33 % mortality, and these values were insignificant to each other [Supplementary Table II]. Highest percent of larval mortality at 2000 ppm concentration up to 4 h exposure was shown by T1 (100.00 %), T2 (100.00 %) followed by T4 and T6 (100.00 %). Likewise, T3 and T5 also showed mortality 96.67 and 90.00 % at the same exposure period of 4 h at same concentrations. However, at 6 h exposure, all formulations showed the 100.00 % larval mortality. Results showed T6 to be the most effective sulfonamide formulation at all concentrations closely followed by T4 which proved to be more effective at the highest concentration of 2000 ppm at 4 h exposure period. T5 though less effective than T3 showed significantly improved efficiency against G. mellonella larvae as compared to standard T1 and T2. The mortality rate achieved by T3 and T5 at 2000 ppm concentration after 4 h exposure (96.67 and 90.00 %) was insignificant to each other [Fig. 4a–c].

Conclusions Thus, based on present study, N-(1-isopropyl-2-oxo-2-morpholino-ethyl) toluene sulfonamide (T6) and N-(1-isopropyl-2-oxo-2-piperidino-ethyl) toluene sulfonamide (T4) are suggested to be more effective IGRs in comparison to in use IGRs (T1 and T2). Therefore, it could be concluded that the aromatic substitution at one terminal and heterocyclic rings at B terminal with sulfonamide functionality initiates/stabilizes the process, but finally, its oxygen group at main chain and oxygen at p-position of nitrogen containing heterocyclic ring B are responsible for effective binding. Present study clearly indicates that these JH analogs (mimics of JH) could

Fig. 5 Comparison of in silico study and in vivo screening (2000 ppm at 4 h)


serve as the basis for future design to find new derivatives with multiple activities to counteract lepidopteran insects’ species. We are in progress on detailed investigation on lead compounds (T6, T4) and their environmental impact, which will be published in due course of time. Further, reasonably good correlation exists between in silico and in vivo activity for heterocyclic sulfonamide class of JHAs obtained from molecular docking study [Fig. 5]. These analogs made strong hydrogen bonding interaction with LYS 85 and LYS 218 amino acid residues of the JHBP of G. mellonella. Thus, docking analysis suggests that heterocyclic class of sulfonamide JHAs could act as potential IGRs. Further insights about the mode of interaction of the heterocyclic sulfonamide class of small JHAs with JHBP could provide guidance for the design of better analogs in the area of IGRs. Acknowledgments Research work reported in this manuscript is supported by the research grant number—SR/ FT/CS-078/2009 under SERC-DST (Fast Tract Project) Ministry of Science and Technology, Govt. of India. The authors would like to thank Director of National Institute of Technology, Hamirpur, India, for providing necessary laboratory facilities to carry out this work. We are also thankful to Director, Institute of Biotechnology and Environmental Science-Neri (Hamirpur) H.P for providing the help regarding biological evaluation of the analogs. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.

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