Design, synthesis and biological activity of novel substituted pyrazole amide derivatives targeting EcR/USP receptor

Article abstract of DOI:10.1016/j.cclet.2016.02.009

In order to discover highly active ecdysone analogs, a series of new substituted pyrazole amide derivatives were obtained using structure-guided optimization method and further screened for their insecticidal activities, in the basis of the core structures of the two active compounds N-(3-methoxyphenyl)-3-(tert-butyl)-1-phenyl-1H-pyrazole-5-carboxamide (6e) and N-(4-(tert-butyl)phenyl)-3-(tert-butyl)-1-phenyl-1H-pyrazole-5-carboxamide (6i), previously presented by us. The chemical structures of the title compounds were identified by spectral analyses. The preliminary bioassay results indicated that one among the synthesized pyrazole derivatives, compound 34, endowed with good activity against Mythimna Separata at 10 mg/L, which was equal to that displayed by the positive control tebufenozide. In addition, examples of molecular docking and molecular dynamics studies demonstrated that 34 may be the potential inhibitor to EcR and its docking conformation was similar to that of tebufenozide. In addition, increasing the hydrophobic effect and considering the suitable bulk effect on pyrazole ring are beneficial to the inhibiting activity to EcR and activity in vivo.

Full text of DOI:10.1016/j.cclet.2016.02.009

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CCLET-3596; No. of Pages 5
Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Design, synthesis and biological activity of novel substituted pyrazole
amide derivatives targeting EcR/USP receptor
Xi-Le Deng a, Jin Xie a, Yong-Qiang Li b, De-Kai Yuan a, Xue-Ping Hu a, Li Zhang a,
*
,
Qing-Min Wang b, Ming Chi a, Xin-Ling Yang a,
*
a Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
b State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China
A R T I C L E I N F O
A B S T R A C T
Article history:
In order to discover highly active ecdysone analogs, a series of new substituted pyrazole amide
derivatives were obtained using structure-guided optimization method and further screened for their
insecticidal activities, in the basis of the core structures of the two active compounds N-(3-
methoxyphenyl)-3-(tert-butyl)-1-phenyl-1H-pyrazole-5-carboxamide (6e) and N-(4-(tert-butyl)phe-
nyl)-3-(tert-butyl)-1-phenyl-1H-pyrazole-5-carboxamide (6i), previously presented by us. The chemical
structures of the title compounds were identified by spectral analyses. The preliminary bioassay results
indicated that one among the synthesized pyrazole derivatives, compound 34, endowed with good
activity against Mythimna Separata at 10 mg/L, which was equal to that displayed by the positive control
tebufenozide. In addition, examples of molecular docking and molecular dynamics studies
demonstrated that 34 may be the potential inhibitor to EcR and its docking conformation was similar
to that of tebufenozide. In addition, increasing the hydrophobic effect and considering the suitable bulk
effect on pyrazole ring are beneficial to the inhibiting activity to EcR and activity in vivo.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 25 December 2015
Received in revised form 23 January 2016
Accepted 2 February 2016
Available online xxx
Keywords:
Moulting hormone
Pyrazole amide
Bioactivity
Molecular docking
Molecular dynamics simulation, Insect
growth regulators
1. Introduction
agonists have been rationally designed using target structure-
guided approaches. Additional efforts that include structural
Molting hormones, such as 20-hydroxyecdysone (20E), are
compounds that regulate the molting of insects [1,2]. Molting or
ecdysis is a critical aspect of insect growth. The binding of 20E to
the ecdysone (EcR)–ultraspiracle protein (USP) heterodimer
during insect development results in ecdysis. One prominent
approach toward the development of environmentally benign
insect growth regulators has been to design ligands that target the
EcR–USP heterodimer. For example, the non-steroidal ecdysone
agonists, dibenzoylhydrazines (DBHs), bind to the EcR subunit of
the heterodimer and induce insect moulting. These agents, which
bear the common dibenzoylhydrazine core structure, display
outstanding activity against lepidopteran pests while having no
negative effects on mammals and the environment [3–6].
modifications of previously identified agonists may led to the
identification of other highly active and novel ecdysone agonists in
recent years [9] (I, Fig. 1). Hence, that modification of the structure
designed by target ecdysone receptor is a success way to design
new ecdysone agonists for pest controls.
In the course of our research efforts, we had previously identified
two compounds, 6e and 6i (II, Fig. 1), which displayed high ecdysone
agonistic activities, and an active conformer of these compounds
was similar to that of tebufenozide [10]. Attribute to the
hydrophobic effect of the t-butyl and methoxy substituent in
phenyl ring and their excellent activity of 6e and 6i, so we retained
themethoxyandt-butylsubstituentsonaromaticamideatpositions
3 and 4, and then modified the substituent groups in the pyrazole
ring. Inspired by these facts, we focused on designing and
synthesizing novel substituted pyrazole amide derivatives based
on the lead compounds 6e and 6i in this study to discover highly
active ecdysone analogs (Fig. 2) and a range of lepidopteran
biological activities was ascribe to this series. Additionally, selected
examples of molecular docking and molecular dynamics studies
were evaluated to study the mechanisms of these derivatives with
active site of the EcR subunit of the heterodimeric receptor.
Recently, a series of novel ecdysone agonists with non-steroidal
or non-dibenzoylhydrazine structures and displaying highly
insecticidal activity have been identified [7,8]. These ecdysone
*
Corresponding authors.
E-mail addresses: zhang_li@cau.edu.cn (L. Zhang),
yangxl@cau.edu.cn (X.-L. Yang).
http://dx.doi.org/10.1016/j.cclet.2016.02.009
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: X.-L. Deng, et al., Design, synthesis and biological activity of novel substituted pyrazole amide
derivatives targeting EcR/USP receptor, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.009

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2.2. Molecular docking study
Compounds 6e, 6i, 34 and tebufenozide were built and
optimized using the MMFF94 force field and charges in Molecular
Operating Environment (MOE) software [13]. The first low energy
conformation of every compound was selected to dock into the
active pocket using the Induced Fit protocol. A crystal structure of
ecdysone receptor (EcR) complexed with BYIO8346 (PDB ID: 1R20)
was modified and protonated. Waters and phosphatidylethanola-
mines were deleted. The active site was set by the residues with a
˚
radius of 6 A around BYIO8346. The docked small molecules were
Fig. 1. Chemical structures of known potential ecdysone agonists.
placed in the site with the Triangle Matcher method and ranked
with the London dG scoring function. Thirty conformations with
the docking poses were output and further rescored with the GBVI/
WSA dG scoring function. The docking complex with the rational
binding mode and higher scoring would be studied further for
molecular dynamics simulation.
2.3. Molecular dynamics simulation
The molecular dynamics (MD) simulation were finished using
the AMBER 12 software [14]. EcR receptor was solvated using
explicit TIP3P water models and the water molecules were filled in
˚
the range of 10 A bound to the protein atom in a cubic periodic box.
AMBER ff99SB force field was used for EcR and gaff force field was
assigned to the small molecules. Energy minimization was first
performed using the steepest descent algorithm for two thousand
steps and then the conjugated gradient algorithm for another three
thousand steps before the MD simulation. Then three dynamics
simulation steps were applied to the whole system. First, only
waters and counterions were heated for 10 ps to ensure the solute
was entirely solvated; second, the system was slowly heated from
10 to 298 K by a weak-coupling method and equilibrated for 100 ps
except the protein backbone; finally, the full system was executed
to a constant pressure equilibration for 20 ns. The binding free
energies between ligands and EcR were computed, as a sum of the
gas-phase molecular mechanics energies, and solvation energies,
and conformational entropy, based on the MM-PBSA method.
Fig. 2. Design strategy of target compounds 7–8, 13–14 and 21–38 through
modifying the substituent on pyrazole of lead 6e and 6i.
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 spectra and 13C NMR were recorded on a Bruker
AM-300 (300 MHz) spectrometer with CDCl3 or DMSO-d6 as the
solvent and TMS as the internal standard. Chemical shifts were
reported in d (parts permillion) values. IR spectra were recorded on
a Perkin Elmer Spectrum 100 FT-IR spectrometor (KBr presser
method). High resolution mass spectrometry (HRMS) data were
obtained on an FTICR-MS Varian 7.0 T FTICR-MS instrument. All the
reagents were obtained commercially and used after further
purification.
3. Results and discussion
The synthetic procedures for substituted pyrazole amide
derivatives are depicted in Schemes 1–3. Condensation of the
phenylhydrazine with malonaldehydic acid 1 yields the compound
2. Bromination of 2 with NBS gives the compound 3, which
undergoes cyclization with a methyl acrylate to afford the bromo-
pyrazoline 4 [15]. The oxidation of 4 affords the pyrazole ester 5
[16], which is subjected to saponification and amidation to afford
the target amides 7 and 8 in moderate overall yields (Scheme 1). A
different method is followed to install a trifluoro methyl group on
2.1. General procedures for synthesis and Insecticidal test of
compounds 7–8, 13–14 and 21–38
The general procedures for synthesis of compounds 7–8, 13–14
and 21–38 were listed in the Supporting information. Their
biological activities against Mythimna separate, Helicoverpa armi-
gera and Pyrausta nubilalis were evaluated using the reference
methods [11,12].
Scheme 1. Synthetic procedure for title compouds 7–8.
Please cite this article in press as: X.-L. Deng, et al., Design, synthesis and biological activity of novel substituted pyrazole amide
derivatives targeting EcR/USP receptor, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.009

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3
Scheme 2. Synthetic procedure for title compounds 13–14.
the pyrazole ring (Scheme 2). The 1,3-diketone compound 10 is
synthesized from 9 following a previously described protocol
[17]. The trifluromethyl-substituted pyrazole core of the key
intermediate 11 is prepared by the cyclization of the 1,3-diketone
moiety in 10 with phenylhydrazine [17]. Subsequently, oxidation
of 11 and the amidations of the resulting carboxylic acid 12 yield
the desired trifluromethyl-substituted pyrazole amides 13 and 14
in moderate yields [18]. The synthesis of the alkyl-substituted
pyrazoles 21–38 is initiated with the compounds 15 following a
previously described protocol [19] (Scheme 3). The pyrazole core of
the key intermediate 18a–e is prepared by the cyclization of the
1,3-diketone moiety in 17a–e with phenylhydrazine (Scheme 3).
The saponification of the ester 18a–e affords the carboxylic acid
19a–e, and aromatic halogenation of 19a–e gives the chlorine
substituted compound 20a–e [20]. Amidations of the carboxylic
acids (containing pyrazole and chloro-pyrazole motifs) using
appropriately substituted anilines obtain the target compounds
21–38 in moderate yields.
HRMS data of the target compounds are in good agreement with
the theoretical data calculated based on the chemical formulae.
The insecticidal activities of all title compounds were evaluated
against lepidopteran pests (M. separata, H. armigera, and P. nubilalis)
invivoandthecorrespondingdataareshowninTable1. Inthisstudy,
we focused on the relative small sizes and suitable hydrophobic
effects of the substituents at the modification positions on the
pyrazole ring. Therefore, a series of alkyl substitutes, trifluoro
methyl group, and bromine atom at position 3 were taken into
consideration, as well as hydrogen atom and chlorine atom at
position 4. While keeping the methoxy on phenyl ring at positions 3,
compounds
7 and 29, exhibited superior activity against
M. separate. The compounds 26, 27 harboring ethyl or n-propyl
substituents at position 3 and chlorine atom at position 4 on the
pyrazole ring displayed promising activity against H. armigera, and
P. nubilalis. Respectively, retaining the substituents as t-butyl at
position 4 of the phenyl substituent, higher activity was observed
with compound 14, 34, 36, and 38 against M. separate. Moreover,
compound34evenexhibited excellentactivitiesagainst H. armigera,
and P. nubilalis, containing 3-methyl and 4-chloro substituent on
pyrazole ring. In particular, the superior activity of compound 34
against M. separata at lower doses was comparable to that displayed
by the positive control tebufenozide (see Table 2). This indicates that
different sizes of substituents on the pyrazole ring contribute
significantly to the insecticidal activities in vivo.
The structures of the targeted compounds are confirmed
through the analyses of their melting points, 1H NMR spectra,
13C NMR spectra, IR spectra, and HRMS. These data can be found in
the Supporting information. Signals corresponding to the C–H and
the N–H protons of the pyrazole ring in target compounds are
observed at about
spectra of the compounds, respectively signals as the C–H protons
d
ꢀ 6.84–6.68 and
d
ꢀ 7.97–8.71 in the 1H NMR
of t-butyl substituent and methoxy substituent in phenyl ring are
Molecular docking is an effective method used in structure-
guided design, due to its ability to elucidate the possible binding
conformations for ligands to bind into EcR/USP receptor. Moreover,
the characterization of binding process, such as hydrogen bonding
interactions and hydrophobic effect, plays a critical role in rational
design of ecdysone analogs [7,8]. In order to gain insights into the
mode of action of the substituted pyrazole ring, molecular docking
observed at about
on the benzene ring are observed at
d
ꢀ 1.33 and
d
ꢀ 3.79 and signal for the protons
d
6.63–7.73. In the IR spectra of
target compounds, strong absorptions at 3200–3400 cmꢁ1, func-
tionality, are observed. In addition, other strong absorptions bonds
representing the carbonyl and unsaturated pyrazole groups are
observed at 1600–1700 cmꢁ1 and 1530 cmꢁ1, respectively. The
Scheme 3. Synthetic procedure for title compouds 21–38.
Please cite this article in press as: X.-L. Deng, et al., Design, synthesis and biological activity of novel substituted pyrazole amide
derivatives targeting EcR/USP receptor, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.009

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Table 1
as that of tebufenozide. To analyze the binding affinity of small
molecules to EcR, molecular dynamics simulations were per-
formed for EcR complexed with compound 34 and tebufenozide,
respectively. The calculated binding free energy is ꢁ64.8 kJ/mol for
the complex of compound 34 and EcR, which is nearly close to that
(ꢁ69.66 kJ/mol) for the complex of tebufenozide and EcR (Fig. S1 in
Supporting information). These data further showed compound 34
might have binding affinity with EcR. We will carry out the
[3]PonA-labeled binding experiments as enzymatic assay testing
EC50 on EcR/USP to confirm the above supposition in future work.
In addition, our previous study showed that the binding modes
of 6e or 6i with the EcR subunit were similar to that of
tebufenozide [10]. Here, we aligned and compared the equilibrat-
ing conformations for 6e, 6i and tebufenozide in the pocket of EcR
(Fig. 3c). The pyrazole rings of 6e, 6i located in the hydrophobic
region in the binding pocket, which had the similar position as the
t-butyl substituent of tebufenozide at the binding pocket (red
cycle). Moreover, the hydrophobic t-butyl group interacting with
the hydrophobic residues of EcR is a key factor affecting the
hydrophobic parameters of the ligands, which is also important to
the binding affinity and activity in vivo of DBHs [21,22]. The better
in vivo activity observed for the 6e and 6i may also be attributed to
the hydrophobic effect of the substituted pyrazole. Then, we
analyzed the superimposed conformations between tebufenozide
and 34. Fig. 3d showed that the pyrazole ring of 34 almost fully
occupied in the position where the hydrophobic t-butyl group of
tebufenozide exited (red cycle). So increasing the hydrophobic
effect and considering the suitable bulk effect on pyrazole ring,
such as 3-methyl and 4-chloro substituents on pyrazole ring, will
be beneficial to the inhibiting activity to EcR.
Observed in vivo insecticidal activity (mortality) of pyrazole analogs.
Compounds. R1
R2
X
Mortality (%) (Conc. 600 mg/L)
Mythimna Helico
Pyrausta
nubilalis
Separata
verpa
armigera
7
3-OCH3
–Br
–H 100
50
35
60
45
13
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
–CF3
–H
–H
–H
50
15
45
70
50
21
–CH3
–CH2CH3
65
70
22
65
70
23
–CH2CH2CH3 –H
40
45
24
–CH(CH3)2
–CH3
–H
35
40
25
–Cl 30
–Cl 50
45
65
26
–CH2CH3
100
100
25
100
100
35
27
–CH2CH2CH3 –Cl 30
28
–CH(CH3)2
–C(CH3)3
–Cl 70
–Cl 100
29
15
20
8
4-C(CH3)3 –Br
–H
20
40
55
14
4-C(CH3)3 –CF3
4-C(CH3)3 –CH3
4-C(CH3)3 –CH2CH3
–H 100
25
35
30
–H
–H
30
20
15
50
60
70
31
45
55
32
4-C(CH3)3 –CH2CH2CH3 –H
50
65
33
4-C(CH3)3 –CH(CH3)2
4-C(CH3)3 –CH3
–H
30
45
34
–Cl 100
–Cl 35
100
30
100
45
35
4-C(CH3)3 –CH2CH3
36
4-C(CH3)3 –CH2CH2CH3 –Cl 100
10
20
37
4-C(CH3)3 –CH(CH3)2
4-C(CH3)3 –C(CH3)3
–Cl 60
–Cl 100
100
30
40
38
75
80
Tebufenozide
100
100
Table 2
Dosage-dependent in vivo insecticidal activity (mortality) of 7, 14, 29, 34, 36, 38 and
tebufenozide against Mythimna Separata.
Compd.
Mortality (%) at different concentrations (mg/L)
600
200
100
50
10
4. Conclusion
7
100
100
100
100
100
100
20
20
nta
nt
nt
nt
nt
nt
60
nt
nt
60
14
nt
Through this study, a series of novel substituted pyrazole
amides derivatives were obtained by structure-guided optimiza-
tion method in basis of the core structures of the two active
compounds N-(3-methoxy phenyl)-3-(tert-butyl)-1-phenyl-1H-
pyrazole-5-carboxamide (6e) and N-(4-(tert-butyl)phenyl)-3-
(tert-butyl)-1-phenyl-1H-pyrazole-5-carboxamide (6i). In partic-
ular, the preliminary assays for insecticidal activity indicated that
one of the compounds of the series, 34, showed excellent activities
against lepidopteran pests, such as M. separate, that was
particularly comparable to that displayed by tebufenozide.
Selected examples from molecule docking and molecular dynam-
ics studies demonstrated that compound 34 could be a potential
inhibitor against EcR/USP receptor which binding conformation
was similar to that of tebufenozide. Particularly, the comparison of
the binding conformations indicated the increasing of hydrophobic
effect and the suitable substituent effects of pyrazole ring were
essential for the inhibiting activity to EcR and activity in vivo. These
29
20
nt
nt
34
100
40
100
nt
100
nt
36
38
60
nt
nt
Tebufenozide
100
100
100
a
nt = not tested.
was considered to study the interactions of these compounds with
EcR. The binding conformation of compound 34 and tebufenozide
with the important residues of EcR were shown in Fig. 3a and b. The
carbonyl oxygen of the amide group for compound 34 established a
hydrogen bonding interaction with Tyr408 (Fig. 3a). The residues
Asn504, and Thr343 were formed in the hydrogen bonding
interactions with tebufenozide (Fig. 3b). Tyr408 is the important
residue to ensure small molecules to keep the inhibiting activity to
EcR. The docking results showed that compound 34 may be the
potential inhibitor to EcR and its docking conformation was similar
Fig. 3. Hydrogen bonding interactions between the residues at the binding pocket of the EcR subunit and compounds (a) 34 and (b) tebufenozide. Carbons in tebufenozide and
34 are represented by blue spheres. Oxygens and nitrogen atoms in both the structures are displayed as red and dark blue solid spheres, respectively. H-bonds are indicated by
green dotted lines. (c) The comparison of the binding conformation between tebufenozide (in brown), 6e (in pink) and 6i (in blue). (d) The comparison of the binding
conformation between tebufenozide (in brown) and 34 (in red).
Please cite this article in press as: X.-L. Deng, et al., Design, synthesis and biological activity of novel substituted pyrazole amide
derivatives targeting EcR/USP receptor, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.009

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Please cite this article in press as: X.-L. Deng, et al., Design, synthesis and biological activity of novel substituted pyrazole amide
derivatives targeting EcR/USP receptor, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.009