Identification of 1-phenyl-4-cyano-5-aminopyrazoles as novel ecdysone receptor ligands by virtual screening, structural optimization, and biological evaluations

DR LI ZHANG (Orcid ID : 0000-0001-6319-1092)

Article type

: Research Article

Identification of 1-phenyl-4-cyano-5-aminopyrazoles as novel ecdysone agonists

by virtual screening, structural optimization, and biological evaluations

Xueping Hu^a, Xiaojuan Ma^a, Jialin Cui^a, Haishan Liu^a, Bin Zhu^b, Jin Xie^a, Pei Liang^b, Li Zhang^a*

^aDepartment of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China

^bDepartment of Entomology, China Agricultural University, Beijing 100193, China

Corresponding author:

Li Zhang,

E-mail: zhang_li@cau.edu.cn

Abstract

Ecdysteroids initiate the molting process in insects by binding to the ecdysone receptor (EcR), which is a promising

target for identifying insect growth regulators. This paper presents an in silico/in vitro screening procedure for

identifying new EcR ligands. The three-step virtual screening procedure uses a three-dimensional pharmacophore

model, docking and Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) rescoring routine. A novel

hit (VS14) with good binding activity against Plutella xylostella EcR was identified from a library of over 200 000

chemicals. Subsequently, the 1-phenyl-4-cyano-5-aminopyrazole scaffold and twelve ecdysone agonists were

synthesized. Their IC₅₀values against Plutella xylostella EcR ranged from 0.64 to 23.21 μM. Furthermore, a

preliminary analysis of the structure-activity relationship for novel scaffolds provided a basis for designing new

ligands with improved activity.

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/CBDD.13772

Keywords: ecdysone receptor; ecdysone agonists; virtual screening; fragment, structure−activity relationship

1. Introduction

Insect growth regulators (IGRs) are insecticides that mimic hormones and possess low mammalian toxicity,

selective inhibitory action and low insect resistance (Graf, 1993; Schneider et al., 2003). Ecdysone analogues belong

to the family of IGRs and bind to a heterodimeric protein complex formed by the ecdysone receptor (EcR) and the

ultraspiracle protein (USP), which results in premature molting, leading to death (Billas, 2009; Dhadialla et al., 1998;

Smagghe et al., 2012). Thus, EcR is considered a promising target for identifying IGRs for integrated pest

management (IPM).

Till date, five structurally diverse nonsteroidal ecdysone agonists (tebufenozide, methoxyfenozide,

chromafenozide, halofenozide, and fufenozide) have been successfully developed, which show potential for IPM

(Kramer et al., 2007; Le et al., 1996; Nauen et al., 2002; Retnakaran et al., 1995; Smagghe et al., 2012; Wing et al.,

1988; Yanagi et al., 2006; Yanagi et al., 2000; Zhang, 2005). These commercial agents share a prototypical

pharmacophore effective against Lepidopteran pests, which consists of a common dibenzoylhydrazine (DBH) core

structure and structurally diverse substituents on the phenyl rings. Other non-steroid/non-bisacylhydrazine

chemotypes, such as amidoketone (AMK) (Tice et al., 2003), oxadiazoline (OXA) (Hormann, 2004),

N-amino-imidazole (N-AIM) (Holmwood et al., 2009), tetrahydroquinoline (THQ) (Smith et al., 2003),

γ-methylene-γ-lactam (γ-ML) (Birru et al., 2010) and imidazothiadiazole (ITD) (Holmwood et al., 2009), were also

designed via scaffold hopping or high-throughput screening (Fig. 1(a)).

The crystal structures of the EcR/USP heterodimer, in complex with DBH from Heliothis virescens, revealed that

the ligand carbonyl groups and the unsubstituted amide nitrogen form hydrogen bonds with residues Thr343, Asn504,

and Tyr408 (Billas et al., 2003). The t-butyl and A-ring were located in a hydrophobic part of the cavity formed by

residues belonging to helixes H3, H5, H6, H11 and to the loops between helixes H6–H7 and H11–H12. It is

noteworthy that the hydrogen bond formed between the carbonyl group of the ligand and Asn504 was crucial not only

for stabilizing ligand binding, but also for preventing ligand escape (Hu, et al., 2018). To aid the development of lead

compounds, more X-ray crystallographic structures of the ligand binding domain of EcR/USP heterodimer

(EcR/USP-LBD), in complex with several potent ecdysone agonists, have been reported (Billas et al., 2003; Browning

et al., 2007; Carmichael et al., 2005; Iwema et al., 2007; Ren et al., 2014). The crystal structures showed that the side

chain of ponasterone A (PonA) shared the same space as the t-butyl and A-ring moieties of DBH (Fig. 1(b)). In

contrast, the steroid nucleus of PonA and the B-ring of DBH occupied the subpockets on both sides. Therefore, the

overlapping pocket can be considered a conserved region of the binding site. The compounds maintaining their

interaction with the overlapping pocket may also exhibit receptor binding activity.

In agrochemical discovery, virtual screening (VS) and structural optimization are powerful tools for identifying

novel scaffolds against different targets (Hao et al., 2016; Hao et al., 2012; Lin et al., 2019; Xiong et al., 2017; Xiong

et al., 2016; Zhao et al., 2010). To identify novel EcR ligands, Harada et al. applied shape-based virtual screening, and

revealed micromolar hits against two strains of insect cells (Harada et al., 2011). Recently, Horoiwa et al. performed

virtual screening based on the three-dimensional structure of the IMD-bound HvEcR using MM/PBSA, and five novel

EcR ligands (IC₅₀ranged from 9.1 to 100 μM) were successfully identified (Horoiwa et al., 2019). We also identified

a novel lead structure of an ecdysone analogue using virtual screening based on the binding mode of DBHs, and a

series of novel pyrazole amides were designed and synthesized (Deng et al., 2016; Deng et al., 2016; Hu et al., 2018).

However, the common characteristics of steroidal and non-steroidal ecdysone agonists have not been determined

using virtual screening. In this study, we have used the overlapping pocket as the pharmacophore element to design

novel lead compounds with promising activity. Using a combination of structure-based virtual screening, structural

synthesis, and biological evaluations, we obtained a fragment of 1-phenyl-4-cyano-5-aminopyrazole, which can be

used to develop potent ecdysone agonists. Our study provided valuable clues for the development of novel ecdysone

agonists to control lepidopteran pests.

2. Materials and methods

2.1 Database Preparation

The Specs database (http://www.specs.net) was selected for the screening purpose. First the database was washed

and the 3D structure of each compound was constructed using the MMFF94x force field using MOE (MOE2014).

Then, the low energy conformers for each compound were generated using Conformation Import methodology

implemented in MOE. Thus, the prepared database size was reduced to 202,919 unique compounds.

2.2 Pharmacophore Model Generation and Validation

X-ray crystal structures of the EcR-LBD complex with ecdysone agonists (PDB ID: 1R1K, 1R20, 1Z5X, 2NXX,

2R40, 3IXP) were used as input files for pharmacophore modeling using MOE. There are two types of ecdysone

agonists in the complexes (steroidal ecdysone agonist in 1R1K, 1Z5X, 2NXX and 2R40; nonsteroidal ecdysone

agonist in 1R20 and 3IXP). The pharmacophore features were created based on the pharmacophore query. The

important chemical features were identified based on the interaction of ecdysone agonists with active site residues.

Based on the comparison between pharmacophores generated from steroidal and nonsteroidal ecdysone agonists, two

common pharmacophore models were constructed. The generated pharmacophore models were validated using a test

database of 45 known ecdysone analogues (Minakuchi et al., 2003; Wheelock et al., 2006), which included 25

highly active DBHs, 14 DBHs with low activity, and 6 natural ecdysterones (Table S1).

2.3 Clustering and Drug-like Analysis

To further select a subset of lead-like compounds, compounds markedly outside the pesticide-likeness space were

removed by applying the pesticide-likeness criteria (Hao et al., 2011) as follows: molecular weight ≤ 435 Da, ClogP

≤ 6, number of H-bond acceptors (HBA) ≤ 6, number of H-bond donors (HBD) ≤ 2, number of rotatable bonds (ROB)

≤ 9, and number of aromatic bonds (ARB) ≤ 17. To ensure maximum chemical diversity, compounds were clustered

and one member per cluster kept. After clustering and drug-like analysis, 2,538 compounds were selected for further

evaluation using molecular docking.

2.4 Molecular Docking

A MOE docking program with default parameters was used to bind the selected ligands with receptor protein and

determine the correct conformation. The crystal structure of EcR-LBD complexed with BYI06830 (PDB code: 1R20)

was used for the docking studies.

2.5 Binding Free-energy Calculations

Each protein-ligand complex predicted using molecular docking was optimized and rescored using MM/PBSA

(Gohlke et al., 2003) (see details in Table S2 and Figure S1). Prior to the MM/PBSA calculation, the complex

structure from the docking study was further refined with the steepest descent algorithm beginning with 2,000 steps

and followed by the conjugated gradient algorithm for 3,000 steps using the AMBER12 package (Case, 2012). A 5 ps

production run in the NPT ensemble was performed for each system and the last frames were collected for the

following MM/PBSA rescoring.

2.6 Molecular Dynamic Simulation of VS14-bound EcR-LBD

The MD simulations were performed with AMBER 12. The VS14-bound EcR-LBD complex was parametrized

using AMBER03 and generalized AMBER force fields (GAFF) (Duan et al., 2003). The complex was solvated with

TIP3P water models (Jorgensen et al., 1983) in a 10 Å cubic box, and Na⁺ions were added to neutralize the net

charge of the system. The system was gradually minimized and then equilibrated for 140 ps. During the equilibration

phase, the NPT ensemble was used at 1 atmospheric pressure and 298 K. The cutoff for non-bonded interactions was

set to 10 Å. Finally, a production run was conducted for a total of 20 ns for the system using the PMEMD program

(Gotz et al., 2012; Salomon-Ferrer et al., 2013).

2.7 Chemicals and Instruments

Compounds (VS1-18) were purchased from commercial supplier (Specs database) and were used without further

purification. All laboratory reagents were acquired commercially and were of analytical grade. Melting points of the

compounds were determined using a X-4 binocular microscope (Fukai Instrument Co., Beijing, China) with

uncorrected values. The 1H nuclear magnetic resonance (NMR) and 13C NMR spectra of the purified target

compounds were determined at 25℃ using a Bruker AM-300 spectrometer at 300 MHz for 1H and 75 MHz for 13C.

CDCl3 or DMSO-d6 was used as the solvent and TMS as the internal standard. High resolution mass spectrometry

(HRMS) data were obtained on an FTICR-MS Varian 7.0T instrument.

General Procedure for the Synthesis of the core fragment H1. H1 was synthesized via a one-pot reaction

between phenylhydrazine and (ethoxymethylene)malonodinitrile in ethanol and the stirred mixture was refluxed for 12

h (Schenone et al., 2014). Then, the mixture was cooled at room temperature, and H1 was filtered under vacuum and

recrystallized from ethanol to obtain the pure products. Gray solid; yield 80.4%; m.p. 134.2137.3°C; 1H NMR (300

MHz, CDCl3) δ 7.64 (s, 1H), 7. 54–7.46 (m, 5H), 4.63 (s, 2H). 13C NMR (75 MHz, CDCl3) δ149.92, 141.31, 136.96,

129.98, 128.94, 124.26, 114.12, 75.98.

General Procedure for the Synthesis of the Target Compounds I1-12. According to the synthetic route of

Scheme 1, the chloride (1.1 equiv.) was added dropwise in the mixture of the H1 (1 equiv.) and Et₃N (1.2 equiv.) in

dichloromethane at 0℃. The reaction mixture was stirred overnight at room temperature. After that, the organic layer

was washed with 1 mol/L HCl solution, saturated sodium bicarbonate solution, and water, dried with anhydrous

sodium sulfate, filtered, and evaporated for purification. The target compounds were purified using column

chromatography on silica gel 60 (200-300 mesh, Merck).

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)acetamide (I1). White solid; yield 32.2%; m.p. 158.0160.0℃ (Lit m.p.:

155-156 °C); ¹H NMR (300 MHz, DMSO) δ10.53 (s, 1H), 8.26 (s, 1H), 7.65 – 7.33 (m, 5H), 2.02 (s, 3H). ¹³C NMR

(75 MHz, DMSO) δ 169.20, 142.46, 141.05, 137.46, 129.61, 128.99, 124.11, 112.88, 22.62. HRMS calcd for

C₁₂H₁₀N₄O (M+H)⁺: 227.0927, Found 227.0928.

1

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)isobutyramide (I2). White solid; yield 34.5%; m.p. 128.0130.0℃; H

NMR (300 MHz, DMSO) δ10.37 (s, 1H), 8.27 (s, 1H), 7.56 – 7.45 (m, 5H), 2.56 (dt, J = 13.7, 6.9 Hz, 1H), 1.02 (d, J

= 6.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO) δ 176.13, 142.35, 141.34, 137.41, 129.48, 129.03, 124.18, 112.75, 90.19,

34.14, 19.01. HRMS calcd for C₁₄H₁₄N₄O (M+H)⁺: 255.1240, Found 255.1237.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)cyclopropanecarboxamide (I3) ： Yellow solid ； Yield = 60.9%; m.p.

151.4~153.0℃ (Lit m.p.: 143-145 °C)；¹H NMR (300MHz，DMSO): δ10.77 (s, 1H), 8.28 (s, 1H), 7.58 (m, 2H),

7.54–7.47 (m, 3H), 1.85–1.72 (m, 1H), 0.89–0.81 (m, 2H), 0.80–0.72 (m, 2H); ¹³C NMR (75 MHz ， DMSO): δ

172.86, 142.39, 141.15, 137.51, 129.55, 128.99, 124.14, 112.86, 89.89, 13.75, 7.96；HRMS Calcd. for C₁₄H₁₂N₄O

[M+Na]⁺: 275.0903, Found: 275.0902.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)cyclopentanecarboxamide (I4) ： Yellow solid; Yield = 66.9%; m.p.

121.0~122.8℃；¹H NMR (300MHz，DMSO): δ 10.42 (s, 1H), 8.28 (s, 1H), 7.67 – 7.55 (m, 1H), 7.54 (s, 1H), 7.49

(m, 3H), 2.87 – 2.69 (m, 1H), 1.88 – 1.71 (m, 2H), 1.64 (m, 1H), 1.59 (s, 1H), 1.57 (s, 1H), 1.55 (s, 1H), 1.52 (m, 2H);

¹³C NMR (75 MHz，DMSO): δ 175.28, 142.34, 141.40, 137.44, 129.48, 129.01, 124.19, 112.82, 90.08, 44.17, 29.68,

25.57; HRMS Calcd. for C₁₆H₁₆N₄O [M+H]⁺：281.1397, Found: 281.1394.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)cyclohexanecarboxamide (I5) ： White solid; Yield = 68.6% ； m.p.

147.0~148.3℃；¹H NMR (300MHz, DMSO): δ 10.36 (s, 1H), 8.28 (s, 1H), 7.62–7.55 (m, 1H), 7.54 (s, 1H), 7.52 –

7.48 (m, 2H), 7.47 (d, J = 1.7 Hz, 1H), 2.33 (s, 1H), 1.71 (s, 4H), 1.60 (d, J = 9.6 Hz, 1H), 1.34 (d, J = 12.7 Hz, 1H),

1.27 (m, 2H), 1.17 (m, 2H); ¹³C NMR (75 MHz，DMSO): δ 175.06, 142.34, 141.38, 137.41, 129.48, 129.01, 124.16,

112.76, 90.14, 43.64, 28.76, 25.35, 25.00；HRMS Calcd. for C₁₇H₁₈N₄O [M+H]⁺:295.1553, Found: 295.1549.

1

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)benzamide (I6): Yellow solid; Yield = 75.1%; m.p. 181.5~183.1℃; H

NMR (300MHz, DMSO): δ 11.01 (s, 1H), 8.39 (s, 1H), 7.99–7.83 (m, 2H), 7.65 (m, 1H), 7.61–7.49 (m, 6H), 7.49–

7.41 (m, 1H); ¹³C NMR (75 MHz ， DMSO): δ 166.15, 142.57, 141.39, 137.59, 132.95, 132.18, 129.60, 129.02,

128.89, 128.00, 123.93, 112.82, 90.89；HRMS Calcd. for C₁₇H₁₂N₄O [M+H]⁺: 289.1084, Found: 289.1079.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-2-phenylacetamide (I7): White solid; Yield = 44.4%；m.p. 146.2~147.8℃

(Lit m.p.: 142-144 °C); ¹H NMR (300MHz，DMSO): δ 10.77 (s, 1H), 8.30 (s, 1H), 7.49 (m, 5H), 7.30 (t, J = 8.0 Hz,

3H), 7.23 (d, J = 6.9 Hz, 2H), 3.63 (s, 2H); ¹³C NMR (75 MHz，DMSO): δ 170.00, 142.47, 140.90, 137.34, 134.68,

129.47, 129.21, 128.91, 128.51, 126.93, 124.01, 112.81, 90.06, 42.02 ； HRMS Calcd. for C₁₈H₁₄N₄O [M+H]⁺:

303.1240, Found: 303.1236.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-3-phenylpropanamide (I8): White solid; Yield = 25.8% ； m.p.

141.9~143.4℃ (Lit m.p.: 128-130 °C); ¹H NMR (300MHz，DMSO): δ 10.57 (s, 1H), 8.27 (s, 1H), 7.48 (s, 3H), 7.41

(s, 2H), 7.25 (s, 2H), 7.20 (s, 3H), 2.85 (s, 2H), 2.56 (d, J = 42.2 Hz, 2H); ¹³C NMR (75 MHz，DMSO): δ 171.34,

142.50, 140.85, 140.71, 137.39, 129.57, 128.93, 128.49, 128.34, 126.21, 124.07, 112.84, 89.87, 36.76, 30.45；HRMS

Calcd. for C₁₉H₁₆N₄O [M+H]⁺: 317.1397, Found: 317.1393.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-4-phenylbutanamide (I9): White solid; Yield

= 24.3% ； m.p.

1

152.4~153.9℃; H NMR (300MHz，DMSO): δ 10.51 (s, 1H), 8.28 (s, 1H), 7.66–7.38 (m, 5H), 7.27 (t, J = 7.2 Hz,

2H), 7.16 (m, 3H), 2.57–2.47 (m, 2H), 2.30 (t, J = 7.0 Hz, 2H), 1.91–1.70 (m, 2H); ¹³C NMR (75 MHz，DMSO): δ

171.97, 142.41, 141.51, 141.21, 137.44, 129.55, 129.04, 128.44, 125.97, 124.27, 112.88, 90.00, 34.49, 34.28, 26.56；

HRMS Calcd. for C₂₀H₁₈N₄O [M+H]⁺: 331.1552,Found: 331.1551.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-2-cyclohexylacetamide (I10) ： White solid; Yield = 79.3%; m.p.

116.2~117.1℃；¹H NMR (300MHz，DMSO): δ 10.42 (s, 1H), 8.27 (s, 1H), 7.63–7.51 (m, 2H), 7.51–7.37 (m, 3H),

2.15 (d, J = 6.9 Hz, 2H), 1.56 (m, 6H), 1.25–0.99 (m, 3H), 0.86 (m, 2H); ¹³C NMR (75 MHz，DMSO): δ 171.42,

142.36, 141.27, 137.37, 129.48, 129.05, 124.42, 112.83, 90.13, 42.96, 34.75, 32.33, 25.82, 25.63；HRMS Calcd. for

C₁₈H₂₀N₄O [M+H]⁺: 309.1710, Found: 309.1705.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-2-cyclopentylacetamide (I11): White solid; Yield = 49.6%; m.p.

107.6~108.6℃；¹H NMR (300MHz，DMSO): δ 10.44 (s, 1H), 8.28 (s, 1H), 7.60 – 7.54 (m, 1H), 7.53 (s, 1H), 7.51

(m, 2H), 7.50–7.45 (m, 1H), 2.28 (d, J = 7.4 Hz, 2H), 1.66 (m, 2H), 1.61–1.36 (m, 5H), 1.08 (m, 2H); ¹³C NMR (75

MHz，DMSO): δ 171.80, 142.37, 141.25, 137.40, 129.49, 129.03, 124.31, 112.80, 90.14, 41.24, 36.56, 31.82, 24.54；

HRMS Calcd. for C₁₇H₁₈N₄O [M+H]⁺: 295.1553, Found: 295.1551.

N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)-3-cyclopentylpropanamide (I12): White solid; Yield = 60.1%; m.p.

92.0~93.7℃；1H NMR (300MHz，DMSO): δ 10.46 (s, 1H), 8.26 (s, 1H), 7.59–7.53 (m, 1H), 7.52 (s, 1H), 7.50 (m,

2H), 7.49–7.43 (m, 1H), 2.29 (m, 2H), 1.66 (s, 1H), 1.65–1.58 (m, 2H), 1.58–1.47 (m, 4H), 1.45 (m, 1H), 1.44–1.35

(m, 1H), 1.02 (m, 2H); 13C NMR (75 MHz，DMSO): δ 172.30, 142.37, 141.22, 137.44, 129.47, 129.01, 124.27,

112.81, 90.05, 38.97, 34.50, 32.03, 31.04, 24.78；HRMS Calcd. for C₁₈H₂₀N₄O [M+H]+: 309.1710, Found: 309.1709.

2.8 Ligand Binding Assay

The radioactive ligand binding assay was performed using the in vitro translated Plutella xylostella ecdysone

receptor (PxEcRB and PxUSP), according to previously reported methods (Graham et al., 2007; Minakuchi et al.,

2003; Tang et al., 2012). For the test compound, we first prepared 10,000 mg/L stock solution and then diluted it to

200 mg/L with buffer (2% DMSO). The [³H] PonA (95 ci/mmol, 1mci/ml, 1.05×10^-5mol/L, PerkinElmer Inc. Shelton

CA, USA) was diluted to 50 nM (0.5% ethanol). Briefly, 16 μL reaction mixture containing 3.2 μL test compound and

4.8 μL [³H] PonA was incubated for 90 min at 25°C. The reaction mixture was immediately filtered through a

nitrocellulose membrane (NC45, Merk Milipore) and was washed thrice with 3 mL ice-cold washing buffer. The

membrane was transferred into a glass vial containing 2 mL Aquasol-2 (PerkinElmer Inc.) and placed in an oscillator

for solvation. The radioactivity was then measured using a liquid scintillation counter (Hidex-300SL, 2 min/filter,

Hidex instrument, Finland). Three parallel experiments were performed simultaneously with different concentrations

of inhibitor to determine the IC₅₀values of the inhibitor compounds. IC₅₀value was obtained via curve ﬁtting using

the Prism program in the GraphPad software.

3. Results and Discussion

3.1 Pharmacophore Model Generation and Validation

A detailed structural analysis of steroidal ecdysone agonist binding sites and protein-ligand complex interactions

were investigated via the ligand interaction tool in MOE, which showed three regions: (1) binding region for steroidal

skeleton moiety containing a polar environment consisting of Glu309, Gln310, Thr343, Thr346, Arg383, Arg387,

Leu396, and Ala398; (2) a hydrophobic region of the alkyl chain group with Ile339, Met380, Met381, Met413,

Val416, Leu420, and Trp520 residues; (3) hydroxy oxygen of the alkyl chain group pointed towards Tyr408 and

Asn504 that formed hydrogen bonds with its side chain.

In the binding pocket of the nonsteroidal ecdysone agonists, three hydrophobic centers were derived from the

V-shaped hydrophobic pocket, and residues Ile339, Met380, Met381, Val384, Met413, Val416, Leu420, Leu500,

Met507, Leu511, and Trp520 contributed considerably to these hydrophobic interactions. The other important

pharmacophores were two hydrogen bond acceptor centers, which showed hydrogen bond interactions with Thr343

and Asn504, and one hydrogen bond donor center which showed hydrogen bond interactions with Tyr408.

Comparison of these two sets of pharmacophores revealed that two hydrophobic pharmacophores and three

pharmacophores that interacted with Thr343, Tyr408, and Asn504 were common to both steroidal and nonsteroidal

ecdysone agonist binding pockets. Compounds, the binding conformations of which in the proteins accommodated the

common pharmacophores, were expected to bind to both pockets simultaneously. Finally, two common

pharmacophore models were constructed: model_A, which is a structure-based pharmacophore, and model_B, which

is a ligand-based pharmacophore. In model_A (Fig. 2(a)), two of the hydrogen bond donor features, F1 and F3, were

located in the side chain of the key residues Thr343 and Asn504. One hydrogen bond acceptor, F2, was near the key

residue Tyr408. In addition, one hydrophobic feature was located in the t-butyl and alkyl chain overlap region. In

model_B (Fig. 2(b)), one hydrogen bond acceptor, F1, was located on the carbonyl and was complementary to

Thr343. Two hydrophobic features, F2 and F3, were located in the overlapping region to emphasize the hydrophobic

centers mentioned above. One hydrogen bond donor, F4, was located on the nitrogen atom, complementary to Tyr408.

Based on the results of previous studies, a test database of 45 known ecdysone analogues, containing 25 highly

active DBHs, 14 DBHs with low activity, and 6 natural ecdysterones, were used for the validation of the

pharmacophore models. The three-dimensional structures of the test database compound were modelled using MOE.

The test database was then screened against model_A and model_B, and the mapping modes of these compounds

were analyzed. All highly active compounds and natural ecdysterones were correctly mapped, whereas 50% of the

compounds with low activity could not be mapped using model_A. For model_B, 29 hits, including all the highly

active compounds, were retrieved (25 highly active compounds, 1 natural ecdysterone, and 3 compounds of poor

activity), whereas the remaining 22% of the hits corresponded to compounds of poor activity. Simultaneous use of

model_A and model_B for virtual screening may reduce the proportion of negative compounds and yield compounds,

the binding conformations of which in the binding site accommodated the common pharmacophores.

3.2 Compounds Obtained using Structure-based Virtual Screening

Virtual screening was performed in three steps: (i) pharmacophore screening, (ii) clustering and drug-like analysis,

and (iii) molecular docking and single-point MM/PBSA rescoring. Each member of the Specs database (202,919

compounds) was pharmacophore screened using model_A and model_B. In the initial step, 11,642 compounds were

selected for clustering and drug-like analysis. Our previous studies indicated that, in contrast to the MOE docking and

MM/GBSA methods, the MM/PBSA method was more suitable for predicting the binding free energy of both the

steroidal and nonsteroidal ecdysone agonists (Hu et al., 2017). Thus, 2,538 compounds were selected for further

evaluation using molecular docking and MM/PBSA rescoring. As shown in Table S2, the binding free energies

(ΔG_bind) calculated for all ligands binding to the EcR-LBD at less than -50 kcal/mol were obtained. The 18 selected

compounds (Fig. S2) were purchased from the commercial company for biological evaluation. The schematic of the

overall virtual screening procedure is shown in Fig. S1.

The binding activities of the 18 selected compounds from the virtual screening were measured using an

established ligand-binding assay (Tang et al., 2012). VS14 significantly inhibited the incorporation of [³H] PonA

(>80%) at their highest concentrations (40 mg/L) (Fig. 3(a)). VS14 were then evaluated to determine the IC₅₀, which

showed good binding activity, with an IC₅₀value of 10.21 μM (Fig. 3(b)).

Interestingly, the scaffolds of VS14 were different from the ecdysone analogues reported earlier. To obtain further

insights for structure optimization, the binding conformation of VS14 after 20 ns MD simulations with the ecdysone

receptor ligand binding domain was re-examined ((Fig. 1(b), Fig. S3). The nitrogen atom and carbonyl oxygen of

VS14 underwent hydrogen bonding to Asn504 and Tyr408 in a manner similar to those of DBHs at the same residues.

In addition to hydrogen bonding, VS14 also showed hydrophobic interactions with various residues in the ligand

binding site. The 1-phenylpyrazole was located in the overlapping hydrophobic pocket formed by Phe336, Ile339,

Met380, Met381, and Trp526. The piperazine ring showed hydrophobic interactions with Met413, Val416, Ile417,

Leu420, Leu500, and Gln503. Analysis of the predicted binding modes of VS14 revealed that

1-phenyl-4-cyano-5-aminopyrazole was the key fragment molecule for ligand binding. Therefore, a fragment of

1-phenyl-4-cyano-5-aminopyrazole, H1, was synthesized and its binding activity against PxEcR-USP was cautiously

assessed using a bioassay. As predicted, H1 showed binding activity against PxEcR-USP, although the activity was

relatively weak (IC₅₀= 252.3μΜ, Table 1). Nevertheless, the results verified that a new type of scaffold with binding

activity had been identified.

3.3 Core Fragment Optimization and Structure-activity Relationship Analysis

Synthetic structural optimization was performed to improve the potency of the core fragment H1. First, H1 was

docked into the binding domain of EcR using the DOCK module of MOE. Analysis of the electrostatic map around

the fragment molecule H1 revealed a negative region near Tyr408, thus extending a carbonyl group to form possible

hydrogen bond with Tyr408 on H1 (Fig. 4(a)). A hydrophobic cavity, which was formed by Val416, Ile417, Leu420,

Leu500, and Gln503, surrounded the carbonyl end (Fig. 4(b)). Therefore, an expansion was performed by substituting

various nonpolar groups with different hydrophobic effects at the carbonyl end to increase the binding activities

against PxEcR-USP. Finally, the 12 designed compounds were subjected to synthesis and bioassay.

The syntheses of 1-phenylpyrazole analogues I1-I12 were outlined in Fig. 5. Initially, H1 was obtained by reacting

phenylhydrazine with ethoxymethylenemalononitrile in refluxing ethanol for 12 h. Then, H1 was transformed into the

corresponding target compound via the amidation reaction, where the quality of the acid chloride was crucial. The

structures of I1-I12 were confirmed using HRMS, ¹H NMR and ¹³C NMR.

The binding activities of I2-I5, I7, I8, and I10-I12 were better than that of H1 (Fig. S4). The larger the hydrophobic

groups introduced by compounds I1-I5, the better the activity. Surprisingly, compound I6 showed extremely weak

binding activity (9.9%). However, addition of a methyl linker between the benzene ring and the carbonyl group did

increase the binding activity (I7, IC₅₀=23.21 μΜ, Table 1). However, the binding activities were reduced when the

methyl linker was replaced by ethyl (I8) or propyl (I9). Compared to that of I4, I11, and I12, the addition of a methyl

linker increased the binding activities. Finally, we obtained the best active compound I10 by adding a methyl linker

between cyclohexane and carbonyl (IC₅₀=0.64 μΜ).

To obtain insights into the binding activities of compounds I1-I12, their structure-activity relationships and

docking binding modes with EcR-LBD were analyzed carefully. Analysis of the predicted binding modes of the

synthesized compounds showed that the compounds exhibited similar binding modes with VS14 and H1. They all

formed hydrogen bonds with Tyr408 and Asn504, although the H-π interaction between synthesized compounds and

Met380 was increased significantly (Fig. 6). Comparison of compounds I2 and I5 revealed that the larger hydrophobic

group of I5 occupied more hydrophobic pockets, which may explain why the binding activity of I5 was better than

that of I2 (Fig. 6(a)). However, compared to that of I10, the cyclohexane of I5 did not insert properly in the

hydrophobic region composed of Ile417 and Leu420 (Fig. 6(b)), which decreased the affinity to EcR-LBD. Taken

together, our findings suggested that the active pocket of the EcR-LBD was large enough to easily accommodate

several varied chemical and big groups, which also provided a focus for designing agonists with improved potency in

the future.

3.4 In Vivo Insecticidal Activity

The insecticidal activities of all the synthesized compounds were evaluated against Plutella xylostella in vivo, and

the corresponding data are shown in Table 2. An initial screening at 400 mg/L with our synthesized 12 compounds

revealed 5 chemicals (I2, I4, I6, I7 and I12), which demonstrated significant larvicidal activities against Plutella

xylostella. The larvicidal activity was further evaluated by measuring the LC₅₀of these five chemicals. The larvicidal

activity of I12 (LC₅₀= 57.29mg/L) was slightly lower than that of tebufenozide (RH-5992, LC₅₀= 23.13mg/L).

Compound I7 with moderate binding activity (IC₅₀= 23.21 μΜ) demonstrated potential larvicidal activity (LC₅₀

=

102.7 mg/L). Although the compound I10 showed high binding affinities (IC₅₀= 0.64 μΜ) to the PxEcR-PxUSP

complexes, its larvicidal potency was weak. These results indicated that large hydrophobic groups can improve the

binding activity, but may reduce the penetrating ability of the compound, resulting in unsatisfactory insecticidal

activity. Given the observed binding activity and larvicidal activity, compound I7 is the most promising candidate for

further optimization. Probing the mechanism of action, as well as optimization of the potency, is currently underway

in our laboratory.

4. Conclusions

In the present study, we used a structure-based virtual screening approach to identify a novel and potent scaffold

against EcR. Furthermore, the chemicals applied in the synthetic structural optimization can be obtained easily. The

binding activities of four out of the twelve synthesized compounds (I5, I7, I10 and I11) against PxEcR-PxUSP were

determined to be relatively strong, and analysis of their structure−activity relationships provided insights regarding

their interactions with the active site, which may assist in improving the potency of the binding activity in future. This

study presents a novel class of ecdysone agonist 1-phenyl-4-cyano-5-aminopyrazole with insecticidal activities against

Plutella xylostella, which warrants further investigation. In addition, it offers an alternative approach for widespread

virtual screening for pesticide identification.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [grant numbers 21977114, 21907084].

Appendix A. Supplementary data

Supplementary data to this article can be found online.

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Table 1. Binding activity of VS14 and its analogues against PxEcR-PxUSP

N

R

N

H

N

Compounds

IC₅₀(μM)

R

N

O

VS14

I5

10.21±0.80

14.49±3.83

N

O

23.21±9.01

0.64±0.36

12.84±1.61

O

I7

I10

I11

H1

H

-

252.3±69.61

0.85±0.42

RH-5992

Table 2. Insecticidal activities (mortality) and LC₅₀values of 1-phenyl-4-cyano-5-aminopyrazoles against Plutella

xylostella

Compd.

Mortality (%) at different concentrations

(mg/L)

LC₅₀

LC₅₀95%

400

70

200

40

-

100

-

50

-

25

-

12.5

(mg/L)

VS14

-

I1

I2

60

-

100

20

85

-

50

-

30

-

87.41

40.89-186.8

I3

-

I4

100

60

90

-

54

-

19

-

10

-

67.19

40.64-111.1

I5

-

I6

100

95

90

84

-

57

45

-

20

15

-

83.88

53.21-132.2

92.19-114.5

I7

5

-

102.7

I8

60

-

I9

65

-

I10

I11

I12

RH-5992

60

-

50

-

100

92

100

60

95

40

87

17

55

-

57.29

23.13

32.42-101.2

13.52-39.59

24

(a)

OH

CN

HO

O

R

O

N

O

D

C

N

A

R¹

HO

H

O

N

OH

A

O

B

H

B

O

R⁴

R²

X

H

R³

O

AMK

γ-ML

PonA

DBH

F

X

S

HN

N

O

N

O

F

N

H

N

Cl

H

R

Cl

H

O

R¹

R²

O

N

O

ITD

N-AIM

THQ

OXA

Fig. 1. (a) Chemical structures of representative ecdysone receptor ligands. (b) Binding conformations of ponasterone

A (PonA, in black), dibenzoylhydrazine (DBH, in magenta), and VS14 (green) bound to the ecdysone receptor. The

EcR chains bound by PonA, DBH, and VS14 are represented by cyan, orange, and yellow colors, respectively.

Fig. 2. Space organization of the structure-based pharmacophore model (a) and ligand-based pharmacophore model

(b). The pharmacophore features are colored in cyan (hydrogen bond acceptor feature), magenta (hydrogen bond

donor feature), and green (hydrophobic groups). The steroidal (PonA and 20E, marked gray-green and yellow) and

nonsteroidal (BYI06830, marked brown) ecdysone agonists are mapped on the pharmacophore models.

(a)

150

100

50

0

VS1 VS2 VS3 VS4 VS5 VS6 VS7 VS8 VS9

VS10 VS11 VS12 VS13 VS14 VS15 VS16 VS17 VS18

Compound No.

RH-5992

(b)

160

140

120

100

80

VS14

IC₅₀=10.21 μM

R²=0.86

60

40

20

0

1

2

3

4

5

6

7

-20

Log concentration [nM]

Fig. 3. (a) Several virtual screening hits bound to the Plutella xylostella ecdysone receptor. Vertical axis indicates %

inhibition relative to PonA. (b) Binding activity of VS14 against in vitro-translated ecdysone receptor PxEcR-PxUSP

Fig. 4. Electrostatic map and fragment growth direction of fragment H1 with the EcR-LBD. The Hyd (hydrophobic)

regions are colored gray, negative regions (Acc) red, and positive regions (Don) blue.

N

O

NHNH₂

i

ii

R

N

+

N

H

O

NH₂

N

O

N

Cl

R

H1

I1-I12

I3 R=

I4 R=

I1 R=CH3

I6 R=

I2 R=

I7 R=

I5 R=

I8 R=

I9 R=

I12 R=

I10 R=

I11 R=

Fig. 5. Synthetic route for preparation of compounds I1- I12 and their precursor H1. Reagents and conditions: (i)

Ethanol, 12 h, reflux; (ii) CH₂Cl₂, Et₃N, room temperature, overnight.

Fig. 6. (a) Docking-predicted binding modes of compounds H1 (red), I2 (cyan), and I5 (yellow) in the EcR-LBD. (b)

Docking-predicted binding modes of compounds H1 (red), I5 (yellow), and I10 (green) in the EcR-LBD.

Article Doi

Identification of 1-phenyl-4-cyano-5-aminopyrazoles as novel ecdysone receptor ligands by virtual screening, structural optimization, and biological evaluations

DOI: 10.1111/cbdd.13772

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