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

Article abstract of DOI:10.1111/cbdd.13772

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 EcR ligands were synthesized. Their IC₅₀ values 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.

Full text of DOI:10.1111/cbdd.13772

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 Hua, Xiaojuan Maa, Jialin Cuia, Haishan Liua, Bin Zhub, Jin Xiea, Pei Liangb, Li Zhanga*
aDepartment of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
b Department 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 IC50 values 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.
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differences between this version and the Version of Record. Please cite this article as doi:
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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
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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 (IC50 ranged 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
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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
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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
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(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,
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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.
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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, 1.05×10-5 mol/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 IC50 values of the inhibitor compounds. IC50 value 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
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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
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(ΔGbind) 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 IC50 value 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.
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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 LC50 of these five chemicals. The larvicidal
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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
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N
R
N
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
O
O
I7
I10
I11
H1
H
-
252.3±69.61
0.85±0.42
RH-5992
Table 2. Insecticidal activities (mortality) and LC50 values of 1-phenyl-4-cyano-5-aminopyrazoles against Plutella
xylostella
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Compd.
Mortality (%) at different concentrations
(mg/L)
LC50
LC50 95%
400
70
200
40
-
100
-
50
-
25
-
12.5
(mg/L)
(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
100
92
100
60
95
40
87
17
55
-
57.29
23.13
32.42-101.2
13.52-39.59
24
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(a)
OH
CN
HO
O
R
O
O
N
O
D
C
N
A
R1
HO
HO
H
O
O
N
N
OH
A
O
B
H
B
O
R4
R2
X
H
R3
O
AMK
γ-ML
PonA
DBH
F
X
S
HN
N
N
O
N
O
O
F
N
N
N
N
N
H
N
N
N
Cl
H
R
Cl
H
O
R1
R2
O
N
O
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.
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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
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(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.
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N
N
N
O
NHNH2
i
ii
R
N
N
+
N
H
O
NH2
N
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.
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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.
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