Chinese Chemical Letters  2016, Vol.27 Issue (02): 251-255   PDF    
Target-based design, synthesis and biological activity of new pyrazole amide derivatives
Xi-Le Denga, Li Zhanga , Xue-Ping Hua, Bin Yina, Pei Liangb, Xin-Ling Yanga     
a Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China;
b Department of Entomology, College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China
Abstract: Based on the similarities in the conformation of VS008 (N-(4-methylphenyl)-3-(tert-butyl)-1-(phenylmethyl)-1H-pyrazole-5-carboxamide) and BYIO6830 (N'-(3,5-dimethylbenzoyl)-N'-tert-butyl-5-methyl-2,3-dihydro-1,4-benzodioxine-6-carbohydrazide) bound to the active site of the EcR subunit of the ecdysone receptor (EcR)-ultraspiracle protein (USP) heterodimeric receptor, a series of new pyrazole amide derivatives were designed and synthesized. Their structures were confirmed by IR, 1H NMR, 13C NMR and elemental analysis. Results from a preliminary bioassay revealed that two of the pyrazole derivatives exhibited promising insecticidal activity. Specifically, compounds 6e and 6i exhibited good activity against Helicoverpa armigera (cotton bollworm) at low concentration. Symptoms displayed by tebufenozide-treated H. armigera were identical with those displayed by its treated counterpart. 6i showed the same poisoning symptoms as those of tebufenozide. In addition, results from molecular docking result indicated that the binding modes of 6e and 6i at the active site of the EcR subunit of the heterodimeric receptor were similar to that of the bound tebufenozide.
Key words: Molting hormone     Pyrazole amide     Rational design     Bioactivity     Molecular docking    
1. Introduction

20-Hydroxyecdysone (20E),the molting hormone of several insects,initiates molting through the heterodimeric ecdysone receptor (EcR)-ultraspiracle protein (USP) receptor [1, 2]. Design of other ligands targeting the EcR-USP heterodimer remains a prominent approach toward the development of environmentally benign insect growth regulators [3]. For example,non-steroidal ecdysone agonists,dibenzoylhydrazine insecticides (DBHs),bind to the EcR subunit of the heterodimer and induce the insect molting. These agents,which bear the common dibenzoylhydrazine core structure,exhibit an excellent activity against lepidopteran pests but have no effect on mammals and environment [4, 5, 6, 7].

However,their relatively narrow spectrum of activity and emergence of DBH-resistant insects have prompted the efforts toward the design of compounds containing different structural motifs or identification of newer molecular targets. With the progress toward the structural resolution of target EcRs over last few decades,it is now possible to adopt a target structure-guided approach toward the design of new compounds with molting hormone activity. Such efforts have led to the identification of highly active and novel ecdysone agonists (I-IV,Fig. 1) in recent years [8, 9].

Download:
Fig. 1.Structures of new ecdysone agonists.

Previously,using a technique of virtual screen against the EcR-USP crystal structure,we obtained a compound library and identified ligands that interact with the EcR subunit of the heterodimer. Analysis of the binding results revealed that the identified pyrazole-based compound VS008 (N-(4-methylphenyl)- 3-(tert-butyl)-1-(phenylmethyl)-1H-pyrazole-5-carboxamide) has an active conformation that is similar to that of BYIO6830 (N0-(3,5- dimethylbenzoyl)-N0-tert-butyl-5-methyl-2,3-dihydro-1,4-benzodioxine- 6-carbohydrazide),a DBH analog (Fig. 2),at the target site of the EcR subunit [10]. Inspired by these observations,a series of pyrazole amides based on the lead compound,VS008,were designed and synthesized in this study. Subsequently,their insecticidal activity against a few lepidopterans was evaluated. Furthermore,interactions between these newly synthesized compounds and the target site on the EcR subunit were also investigated by molecular docking.

Download:
Fig. 2.The design strategy of target compound 6 based on the binding conformation of VS008 and BYIO6830 with EcR.
2. Experimental

Melting points of all compounds were determined on an X-5 binocular (Fukai Instrument Co.,Beijing,China),and were not corrected. 1H NMR and 13C NMR spectra were recorded on a Bruker AM-300 spectrometer with CDCl3 as the solvent and TMS as the internal standard. Chemical shifts were reported in δ (parts per million) values. IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometor (KBr presser method). Elemental Analysis was obtained with an ST-Carloerba. Co instrument. All the reagents were obtained commercially and used after further purification. VS008 was bought from J&K Scientific. Column chromatography purification was carried out using sillca gel.

2.1. General procedure for synthesis of compounds 6a-p

Pinacolone 1 (0.087 mol) and diethyl oxalate 2 (0.087 mol) were added dropwise to a solution of ethanol (= mL) containing thinly sliced sodium (0.047 mol) at 0 ℃. The mixture was stirred overnight at room temperature. Next morning,the mixture was acidified (pH 3.0,with 20% H2SO4) and filtered to remove the formed solid. The filtrate was extracted with dichloromethane,dried,and concentrated under vacuum to yield an orange red viscous liquid 3 (12.98 g; 74.6% y). A solution of the 1,3-diketone 3 (0.025 mol) in methanol (10 mL) was added dropwise to a cooled solution (0 ℃) of hydrazinobenzene (0.025 mol) in methanol (30 mL). The mixture was warmed to room temperature by stirring for an hour and then refluxed for 2 h. The resulting cooled mixture was concentrated under vacumm and the pyrazole ester 4 was obtained after purification by column chromatography (a 5% gradient of ethyl acetate in hexanes over a column of silica gel). To saponify the ester,a solution of 4 (0.007 mol) was combined with an aliquot of 6 mol/L NaOH(aq) (7 mL) and the mixture was stirred at 80 ℃. Ice water (50 mL) was added at the end of 2 h and the mixture was acidified (pH 1-2) with concentrated HCl. The formed solid was collected by filtration and the filter-cake was dried. The carboxylic acid was purified by recrystallization (methanol:water,1:1) to afford 5 (3.57 g; 83.2% yield).

The amide derivatives 6a-p were prepared through the acyl chlorides derived from 5. A solution of 5 (0.004 mol) in thionyl chloride (10 mL) was refluxed for 5 h [11] and then concentrated under vacuum. The formed crude acyl chloride was added dropwise to a cooled solution (0 ℃) of substituted aniline (0.004 mol) and TEA (0.008 mol) in dichloromethane (10 mL). The resulting mixture was stirred overnight at room temperature to produce the crude product,which was purified on a column of silica using a gradient of ethyl acetate in hexanes to afford the pure products 6a-p.

2.2. Insecticidal test of target compounds 6a-p

Their biological activities against Mythimna separata,Helicoverpa armigera and Pyrausta nubilalis were evaluated using the reference method [12, 13]. The poisoning symptoms of H. armigera treated with 6i were tested using the reference method [14].

2.3. Molecular docking

A Molecular Operating Environment (MOE) software [15] was used for the molecular docking. All synthesized compounds were built and optimized using theMMFF94 force field and charges. The low energy conformation of each compound was selected as the initial docking conformation. A crystal structure of ecdysone receptor (EcR) complexed with BYIO8346,which was obtained at 2.85Å ,was downloaded from the protein data bank (PDB ID: 3IXP). Waters and other solvent molecules (such as phosphatidylethanolamines) were removed and the modified structurewas protonated. The active site was defined by the residues with a radius of 6 Å around BYIO8346. The ligandmoleculeswere placed in the site with the Triangle Matchermethod and ranked with the London dG scoring function. 30 Docking poses per ligand molecules were retained and further refined by energy minimization in the pocket. Then theywere rescoredwith the GBVI/WSA dG scoring function,a force field-based scoring function,which was used to estimate the binding free energy of these ligands with EcR.

2.4. Molecular dynamics simulations

The molecular dynamics (MD) simulations were performed using the AMBER 12 program [16] by using the AMBER ff99SB force field. The protein was solvated using explicit TIP3P water models and the water molecules extended about 10Å from the protein atom in a cubic periodic box. Before the MD simulations,three energy minimization steps were applied to the system. First,the solute was kept fixed and waters and counterions were minimized. Second,the backbone atoms of the protein were fixed with the ligand,while side chains and other atoms were free to move. Finally,the entire system was fully minimized without any constraint. In each step,energy minimization was first performed using the steepest descent algorithm for 2000 steps and then the conjugated gradient algorithm for another 3000 steps.

The MD simulation was performed under periodic boundary conditions using the Sander module of the AMBER 12 program. First,the system was fixed to make the heating only for waters and counterions for 10 ps to ensure the solute was fully solvated; second,the whole system was gradually heated from 10 to 298 K by a weak-coupling method and equilibrated for 100 ps with the protein backbone fixed; last,the system was switched to a constant pressure equilibration for 20 ns. During the MD simulation,the particle mesh Ewald (pmE) algorithm was used to manage longrange electrostatic interactions with a cutoff distance of 10Å ,which was also used for the van der Waals (vdW) energy terms. All of the angles and bonds involving hydrogen atoms were constrained using the SHAKE algorithm. The binding free energy between ligands and receptor is computed as a sum of the gasphase molecular mechanics energies,and solvation energy,and conformational entropy based on the MM-PBSA method.

3. Results and discussion

The route adopted for the synthesis of the targeted compounds 6a-p is summarized in Scheme 1. Compound 3 is synthesized following a previously described protocol [11]. The pyrazole core of the key intermediate 4 is prepared by the cyclization of the 1,3- diketone moiety in 3 with phenylhydrazine. The saponification of 4,followed by the activation of the resulting carboxylic acid 5,and subsequent amidation using substituted anilines afford the targeted amides 6a-p in moderate yields.

Download:
Scheme 1.The synthetic route for obtaining the pyrazole-based analogs 6. Reagents and conditions: (a) sodium, ethanol, 0 8C, r.t. (overnight); (b) phenylhydrazine (1 equiv.), methanol, 0 8C (30 min), r.t. (1 h), reflux (2 h); (c) (i) NaOH, 80 8C (2 h), (ii) HCl; (d) (i) SOCl2, (ii) substituted aniline (1.0 equiv.), TEA (2.0 equiv.), DCM, 0 8C (10 min), r.t. (overnight).

The structures of the targeted compounds are confirmed through the analyses of their melting points,1H NMR spectra,13C NMR spectra,IR spectra,and elemental analysis. The 1H and 13C NMR spectra for compound 6e,6i are confirmed for instance. These data are included in the supporting information. The methyl protons of the t-butyl groups are observed at δ ~ 1.20 in the 1H NMR spectra of the compounds. Signals corresponding to the C-H and the N-H protons of the pyrazole ring in compounds 6a-p are observed at δ 6.85 and δ 8.66,respectively and signal for the protons on the benzene ring are observed at δ 6.63-8.96. In the IR spectra of compounds 6a-p,strong absorptions at 3200- 3400 cm-1,attributed to the presence of the secondary amide functionality,are observed. In addition,other strong absorptions bonds representing the carbonyl and unsaturated pyrazole groups are observed at ~1700 cm-1 and ~1500 cm-1,respectively. The elemental analyses of the compounds 6a-p are in good agreement with the theoretical data calculated based on the chemical formulae.

The insecticidal activity of these compounds against lepidopteran pests M. separata,H. armigera (cotton bollworm),and P. nubilalis are listed in Table 1. Compounds containing the t-butyl substituent at positions 2 (6g) and 4 (6i) relative to the aniline group exhibited superior activity. The increased activity observed for the 6g and 6i can be attributed to the hydrophobic effect of the t-butyl substituent. Other substituents at positions 2 and 4 are not as effective as t-butyl substitution. Amongst the analogs containing a substituent at position 3,the compound 6e,harboring a methoxy substituent,exhibits the highest observed activity. The factors contributing to the observed high activity can be the hydrophobic effect and the relatively small size of the methoxy substituent. It is likely that the bulky t-butyl substituent is not optimally accommodated at the active site of the EcR subunit. At 600 mg/mL,the insecticidal activity of compounds 6e and 6i is similar to that exhibited by the positive control tebufenozide. Even at lower concentrations,6e and 6i exhibit activity against H. armigera (see Table 2); this indicates that an electron-donating substituent is more desirable than an electron-withdrawing substituent to generate a compound with better insecticidal activity. It is worth mentioning here that the skeletal structure of this series of compounds (6a-p) is similar to that of the pesticide chlorantraniliprole,a compound that induces a convulsion-free gradual contraction,thickening,and shortening of the insect body by binding to the ryanodine receptor [17, 18]. In order to gain insights into the mode of action of the pyrazole based compounds,the effects of treating H. armigera with 6i are evaluated based on a method previously reported in literature [14]. Analysis of the results (Figs. S1 and S2 in Supporting information) reveals that symptoms,such as lipped head capsule and extrusion of the hindgut,exhibited by a cotton bollworm treated with 6i are similar to those exhibited by its tebufenozide-treated counterpart. These results indicate that it is more likely that,akin to tebufenozide,6i binds to the EcR subunit of the heterodimeric receptor [19] rather than the ryanodine receptor. The binding conformation of compounds 6e,6i,and tebufenozide are evaluated by docking these compounds into the active site pocket of the EcR subunit (Fig. 3a-c). Important amino acid residues forming hydrogen bonding interactions with tebufenozide are Asn504,Tyr408,and Thr343. The nitrogen atom of the pyrazole group for compound 6e establishes a hydrogen bond interaction with Tyr408 (Fig. 3b). For compound 6i,the amide group forming hydrogen bonding interactions with both Asn504 and Tyr408,while the nitrogen atom of the pyrazole group interacts with the Thr343 through a hydrogen bond (Fig. 3c). The docking results reveal that the binding conformations of 6e and 6i are similar to that of tebufenozide at the active site of the EcR subunit.

Table 1
Observed in vivo insecticidal activity (mortality) of pyrazole analogs (600μg/mL).

Table 2
Dosage-dependent in vivo insecticidal activity (mortality) of 6e, 6i, and tebufenozide against H. armigera.

Download:
Fig. 3.Hydrogen bonding interactions between the binding pocket in the target EcR subunit and the compounds (a) tebufenozide, (b) 6e, and (c) 6i. Carbons in 6e, 6i, and tebufenozide are represented by light blue spheres, oxygens as red spheres and nitrogens as dark blue spheres. H-bonds are indicated by blue dotted lines.

The calculated average free energies for the complexes of 6e and EcR,6i and EcR are -54.18 kJ/mol and -71.35 kJ/mol,respectively (Fig. S2). Under the same conditions,the calculated average free energy of binding of tebufenozide to the EcR subunit is determined to be -69.46 kJ/mol. These results show that the binding free energy values of compounds 6e and 6i results in the higher activity in the enzymatic assay. Further studies on the binding assay and structure-activity relationship are currently in progress.

4. Conclusion

In summary,based on the conformational similarities observed in the binding modes of VS008 and BYIO6830 at the active site of the EcR subunit of the heterodimeric receptor,a series of novel pyrazole amides were designed and synthesized. Bioassay-guided studies revealed that when compared to the activity displayed by the tebufenozide,two of the synthesized analogs,6e and 6i,displayed potent activity against lepidopteran pests M. separata,P. nubilalis and H. armigera even at low concentrations. Similarities in the observed symptoms when H. armigera was treated either with tebufenozide or the pyrazole analogs (6e or 6i) indicated that these compounds might have the same mode of action,i.e.,by binding to the EcR subunit of the ecdysone EcR-USP heterodimeric receptor. Furthermore,molecular docking and molecular dynamics studies revealed that the binding modes of 6e or 6i with the EcR subunit were similar to that of tebufenozide. These results gave useful guides to discovering new IGRs.

5. Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21272265) and the National High Technology Research and Development Program of China (No. 2011AA10A204).

References
[1] Y. Nakagawa, V.C. Henrich, Arthropod nuclear receptors and their role in molting, FEBS J. 276 (2009) 6128-6157.
[2] L.I. Gilbert, Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster, Mol. Cell. Endocrinol. 215 (2004) 1-10.
[3] R.J. Hill, I.M.L. Billas, F. Bonneton, L.D. Graham, M.C. Lawrence, Ecdysone receptors: from the Ashburner model to structural biology, Annu. Rev. Entomol. 58 (2013) 251-271.
[4] K.D. Wing, RH-5849, a nonsteroidal ecdysone agonist: effects on a drosophila cell line, Science 241 (1988) 467-469.
[5] T.S. Dhadialla, R.K. Jansson, Non-steroidal ecdysone agonists: new tools for IPM and insect resistance management, Pestic. Sci. 55 (1999) 357-359.
[6] G.R. Carlson, T.S. Dhadialla, R. Hunter, et al., The chemical and biological properties of methoxyfenozide, a new insecticidal ecdysteroid agonist, Pest Manag. Sci. 57 (2001) 115-119.
[7] Y. Sawada, T. Yanai, H. Nakagawa, et al., Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: part 2. Introduction of substituents on the benzene rings of the benzoheterocycle moiety, Pest Manag. Sci. 59 (2003) 36-48.
[8] G. Holmwood, M. Schindler, Protein structure based rational design of ecdysone agonists, Bioorg. Med. Chem. 17 (2009) 4064-4070.
[9] T. Harada, Y. Nakagawa, T. Ogura, et al., Virtual screening for ligands of the insect molting hormone receptor, J. Chem. Inf. Model. 51 (2011) 296-305.
[10] Y. Bin, Design, Synthesis and Bioactivity of Novel IGRs Lead Compound Based on EcR/USP Structure, (Master dissertation), China Agricultural University, Beijing, 2011.
[11] Y.F. Sun, H.L. Qiao, Y. Ling, et al., New analogues of (E)-β-farnesene with insecticidal activity and binding affinity to aphid odorant-binding proteins, J. Agric. Food Chem. 59 (2011) 2456-2461.
[12] W.L. Dong, J.Y. Xu, L.X. Xiong, X.H. Liu, Z.M. Li, Synthesis, structure and biological activities of some novel anthranilic acid esters containing N-pyridylpyrazole, Chin. J. Org. Chem. 27 (2009) 579-586.
[13] J.W. Zhang, Y.Q. Li, X.L. Yang, et al., Synthesis and bioactivities of nucleoside compounds containing substituted benzoyl carbamate thiourea, Chin. J. Org. Chem. 33 (2013) 305-311.
[14] X. Liu, L. Zhang, J.G. Tan, H.H. Xu, Design and synthesis of N-alkyl-N'-substituted 2,4-dioxo-3,4-dihydropyrimidin-1-diacylhydrazine derivatives as ecdysone receptor agonist, Bioorg. Med. Chem. 21 (2013) 4687-4697.
[15] Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2013.
[16] D.A. Case, T.A. Darden, T.E. Cheatham Ⅲ, et al., AMBER 12, University of California, San Francisco, 2012.
[17] M. Tohnishi, H. Nakao, T. Furuya, et al., Flubendiamide, a novel insecticide highly active against lepidopterous insect pests, J. Pestic. Sci. 30 (2005) 354-360.
[18] R. Nauen, Insecticide mode of action: return of the ryanodine receptor, Pest Manag. Sci. 62 (2006) 690-692.
[19] G. Smagghe, D. Degheele, Action of a novel nonsteroidal ecdysteroid mimic, tebufenozide (Rh-5992), on insects of different orders, Pestic. Sci. 42 (1994) 85-92.