New lead discovery of insect growth regulators based on the scaffold hopping strategy

Article abstract of DOI:10.1016/j.bmcl.2020.127500

Insect growth regulators (IGRs), which can interrupt or inhibit pest life cycles, are low-toxicity pesticides widely used in integrated pest management (IPM). Ecdysone analogues and chitinase inhibitors are familiar IGRs that have attracted considerable attention because of their unique modes of action and low toxicity to non-target organisms. To find new and highly effective candidate IGRs with novel mechanisms, D-08 (N-(4-(tert-butyl)phenyl)-2-phenyl-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) was chosen as a lead compound, and a series of novel heptacyclic pyrazolamide derivatives were designed and synthesized using the scaffold hopping strategy. The bioassay showed that III-27 (N-(2-methylphenethyl)-1-phenyl-1,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) had excellent activity against Plutella xylostella. Protein verification and molecular docking indicated that III-27 could act on both the ecdysone receptor (EcR) and Ostrinia furnacalis chitinase (Of ChtI) and is a promising new lead IGRs. The interaction mechanism of III-27 with EcR and Of ChtI was then studied by molecular docking. These results provide important guidance for the study of new dual-target IGRs.

Full text of DOI:10.1016/j.bmcl.2020.127500

Bioorganic&MedicinalChemistryLetters30(2020)127500
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New lead discovery of insect growth regulators based on the scaffold
hopping strategy
⁎
Biaobiao Jianga, Bingbo Guoa, Jialin Cuia, Yawen Donga, Li Cuib, Li Zhanga, , Qing Yangc,
⁎
Xinling Yanga,
a Innovation Center of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China
b Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
c State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Insect growth regulators (IGRs), which can interrupt or inhibit pest life cycles, are low-toxicity pesticides widely
used in integrated pest management (IPM). Ecdysone analogues and chitinase inhibitors are familiar IGRs that
have attracted considerable attention because of their unique modes of action and low toxicity to non-target
organisms. To find new and highly effective candidate IGRs with novel mechanisms, D-08 (N-(4-(tert-butyl)
phenyl)-2-phenyl-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) was chosen as a lead compound,
and a series of novel heptacyclic pyrazolamide derivatives were designed and synthesized using the scaffold
hopping strategy. The bioassay showed that III-27 (N-(2-methylphenethyl)-1-phenyl-1,4,5,6,7,8-hexahy-
drocyclohepta[c]pyrazole-5-carboxamide) had excellent activity against Plutella xylostella. Protein verification
and molecular docking indicated that III-27 could act on both the ecdysone receptor (EcR) and Ostrinia furnacalis
chitinase (Of ChtI) and is a promising new lead IGRs. The interaction mechanism of III-27 with EcR and Of ChtI
was then studied by molecular docking. These results provide important guidance for the study of new dual-
target IGRs.
Insect growth regulator
Scaffold hopping
Pyrazolamide
Ecdysone receptor
Chitinase
Molecular docking
Insect growth regulators (IGRs) can disrupt the normal development
of insects by mimicking juvenile hormone (JH) or molting hormone, or
interfering with chitin synthesis, and typically cause insects to fail to
develop into larval or pupal stages.1–3 The main advantages of IGRs
over conventional pesticides are their relative nontoxicity to mammals,
high selectivity and safety for non-target organisms. Owing to these
advantages, IGRs are regarded as ideal pesticides in the 21st century.
During the process of insect molting, the ecdysone receptor (EcR) and
chitinase can disturb the process of exfoliation, typically resulting in
larval or pupal mortality. Thus, ecdysone analogues and chitinase in-
hibitors are considered potential IGRs.4,5 However, the development of
IGRs has been limited due to the insufficient structural diversity of
ecdysone analogues and the absence of commercial chitinase in-
hibitors.6–8,9–13 Thus, using efficient strategies to design novel struc-
tures with higher IGRs activity is of great value to improve this lim-
itation.
developmental candidates is becoming an important tool in the search
for new agrochemicals.16
In our previous research, we found the compound D-08 (N-(4-(tert-
butyl)phenyl)-2-phenyl-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-
carboxamide) caused abnormal molting in Plutella xylostella and in-
duced typical symptoms of insect growth regulator action.17 The
bioactivity assay showed that D-08 had good insecticidal activity. To
discover a new effective IGRs agent, here, we chose D-08 as the lead
compound to design a series of novel heptacyclic pyrazolamide com-
pounds using the scaffold hopping strategy (Fig. 1). We first transferred
the carboxamide group from pyrazole position 5 to 3. A series of novel
title compounds were prepared by the acylation or condensation reac-
tion of pyrazole carboxylic acid 5 and the substituted amine (Scheme
1). The activities of all compounds against P. xylostella were assayed.
The potential mechanism of compound III-27 (N-(2-methylphenethyl)-
1-phenyl-1,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide)
was investigated by a molecular docking and a protein assay on the EcR
and chitinase (Of ChtI) proteins from P. xylostella and lepidopteran
pests (specifically, Ostrinia furnacalis), respectively. The results de-
monstrated that III-27 was inferred to bind strongly to the active
Scaffold-hopping, a medicinal chemistry method for molecular
backbone replacements, is an important and efficient drug-design
strategy to develop novel molecules with potent activity.14–16 Recently,
in crop protection research, scaffold hopping to identify promising
⁎ Corresponding authors.
E-mail addresses: zhang_li@cau.edu.cn (L. Zhang), yangxl@cau.edu.cn (X. Yang).
https://doi.org/10.1016/j.bmcl.2020.127500
Received 9 April 2020; Received in revised form 27 July 2020; Accepted 15 August 2020
Availableonline18August2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

B. Jiang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127500
is not as good as that of tebufenozide, which may prevent III-27 from
being totally absorbed by pests. However, this potential issue requires
further experimental data to be clarified. Further study for solving this
case, such as structure optimization and QSAR analysis, will be con-
ducted in our future work.
Additionally, our investigation of the growth and development of
treated P. xylostella indicated that the symptoms of developmental
malformation and stunted extrusion of the hindgut and cuticle fold
under III-27 treatment were similar to those induced by tebufenozide at
500 mg·L−1. However, the color of P. xylostella treated with III-27 was
different from the color of those treated with tebufenozide (Fig. 3),
which indicated that III-27 may differ in its mode of action from that of
tebufenozide.
Fig. 1. The design strategy of target compounds III.
pockets of EcR and Of ChtI. This present study indicated III-27 is a
potential dual-target IGR lead compound with a novel structure and
strong potential for further study and development.
We speculated other possible III-27 targets, including proteins re-
lated to molting, and conducted related target verification experiments.
The ecdysone receptor from P. xylostella and chitinase from a lepi-
dopteran pest (specifically, O. furnacalis) were chosen for conjecture
verification. The binding activities of III-27 were measured with a
competitive binding assay using [3H] PonA as the radioactive ligand.
The IC50 values of III-27, D-08 and tebufenozide toward EcR/USP were
The synthetic route of target compounds is shown in Scheme 1.
Compound 3 is synthesized following a previously described protocol.18
The pyrazole core of the key intermediate 4 is prepared by the cycli-
zation of the 1,3-diketone moiety in 3 with phenylhydrazine.19 The
pyrazole carboxylic acid 5 was obtained by the saponification reaction
of intermediate 4 as described in reference.18 Target compounds III-
01–III-25 were synthesized from compound 5 and a different sub-
stituted aniline in CH2Cl2 using Et3N as an alkali in acylation. Com-
pounds III-25–III-27 were prepared by condensation reaction between
intermediate 5 and the substituted benzylamine or phenethylamine in
CH2Cl2 at room temperature. The structures of all target compounds
were characterized by 1H NMR, 13C NMR, and HRMS, and physical data
are included in the Supplementary Material.
determined to be 4.31
0.66 mg·L−1 (Fig. 4a), 18.46
1.41 mg·L−1
and 0.19
0.2 mg·L−1 (Fig. S1). The results revealed that III-27 had a
better binding activity to EcR/USP than D-08, but lower than that of
tebufenozide. The inhibiting activity of III-27 to Of ChtI was measured
using a microplate reader, showing that III-27 had an excellent in-
hibition rate (0.29
0.01 mg·L−1) (Fig. 5a), which is higher than
those of D-08 (0.88
0.04 mg·L−1
)
and (GlcN)5
(13.42
0.39 mg·L−1) (Fig. S2). These results indicated that III-27
The in vivo bioactivity assay against P. xylostella, molecular docking,
ligand-binding assay on EcR/USP, and ligand-inhibiting assay on Of
ChtI are described in the Supplementary Material.
can act both on EcR/USP and Of ChtI.
Previous research revealed that the binding activity of di-
benzoylhydrazines (DBHs) was mainly dependent on the hydrophobic t-
butyl group interacting with the hydrophobic amino acid residues of
the ecdysone receptor.20–22 To clarify their binding modes, the com-
pounds D-08, III-27, and tebufenozide were selected to dock into the
binding pocket of EcR, respectively, revealing that the binding mode of
III-27 was similar to those of D-08 and tebufenozide in the active site of
EcR (Fig. 4b). The heptacyclic pyrazole ring of III-27 was located in the
hydrophobic region of the binding pocket, which occupied the same
position as the t-butyl substituent of tebufenozide (red circle), thus
providing a strong hydrophobic effect. The 2-methyl benzene ring of
III-27 (Fig. 4b) occupies a more hydrophobic position compared with
that of D-08. Moreover, the carbonyl oxygen atom of III-27 can form
key hydrogen bonds with Asn504 (Fig. 4b). Therefore, the binding ac-
tivity of III-27 was greater than that of D-08, but weaker than that of
tebufenozide (Fig. 4a and S1).
The crystal structure of compound III-19 (Fig. 2, CCDC 1990848)
was determined in a mixture of ethyl acetate and petroleum ether. X-
ray intensity data were recorded from a diffraction analysis (Rigaku
XtaLAB Pro; Rigaku Corp., Tokyo, Japan). The following features were
characterized for compound III-19: empirical formula, C22H23N3O;
formula weight, Mr = 345.43; crystal system, orthorhombic; space
group, P 21 21 21; hall group, P 2ac 2ab; cell parameters, a = 6.9716
(2) Å, b = 14.4363 (6) Å, c = 17.9794 (7) Å, α = 90°, β = 90°,
γ = 90°, V = 1809.52 (12) Å3, Z = 4, F(000) = 736.0,
Mμ = 0.079 mm−1; crystal structure modified consistency factor:
R = 0.0388 (3335), wR2 = 0.0928 (3896). The crystal structure data
are included in the Supporting Information (Tables S1 and S2).
The insecticidal activities of all synthesized compounds were pre-
liminarily evaluated against P. xylostella in vivo at 500 mg·L−1, and the
corresponding data are shown in Table 1. The commercial insecticide
tebufenozide was selected as a positive control. Three target com-
pounds (III-19, III-21, and III-27) exhibited higher insecticidal activ-
ities (> 90%) against P. xylostella than the lead D-08 (70%). In parti-
cular, at a dose of 200 mg·L−1, compound III-27 displayed 100%
insecticidal activity (Table 2). Therefore, III-27 was selected for further
testing at lower doses, and its LC50 value (64.13 mg·L−1) was com-
parable to that of the commercial insecticide tebufenozide
(LC50 = 33.83 mg·L−1) (Table 2 and Fig. S3). The analysis of pre-
liminary structure–activity relationships (SARs) indicated that when
R = 2-CH3, n = 0 and R = 2-CH3, n = 2, the change of the amide from
position 5 to position 3 improved insecticidal activity (III-19 > D-09,
and III-27 > D-11). However, when R = 4-t-Bu, n = 0 and R = 2-
CH3, n = 1, the activity was reduced slightly (III-25 < D-08, and III-
26 < D-10). Altogether, these data show types of substituents play
important roles in the activity of titled compounds. In particularly,
halogen groups had no positive effect on activity. However, the in vivo
biological activity of III-27 was also found to be lower than that of
tebufenozide, which may due to the difference in solubility between III
and 27 and tebufenozide. During the in vivo bioassay, we found that the
solubility of III-27 in an aqueous solution of 0.05% (w/v) Triton X-100
The compounds D-08, III-27, and (GlcN)5 were also docked into the
binding domain of Of ChtI to assess their binding modes. A typical
GH18 chitinase Of ChtI usually possesses a long, cleftlike catalytic do-
main with multiple binding subsites labelled +2, +1, −1, −2, −3,
−4, and −5, and catalysis always occurs between subsites +1 and
−1.23 Two hydrophobic cavities, S1 (formed by Tyr30, Phe61, Ala188,
Met215, Tyr217, and Trp372) and S2 (formed by Tyr149, Pro190,
Met215, and Tyr217), were extended near subsites −1 and +1. The
hydrophobicity of these two regions played a key role in this interac-
tion.19 The results of molecular docking revealed that the binding mode
of compound III-27 was similar to that of the substrate (GlcN)5, but
different from that of D-08 in the active site. We also found an overlap
in the hydrophobic regions S1 and S2 (Fig. 5b). The 2-methyl sub-
stituted benzene ring of III-27 was found locating at the mouth of the
S1 cavity, and the N-substituted benzene ring tended to extend toward
the S2 region (Fig. 5b). III-27 occupies more hydrophobic regions in S1
and S2 compared to D-08. This analysis also indicated that the hydro-
phobic regions of S1 and S2 were important for the binding affinity. The
carbonyl oxygen atom of the amide group of III-27 occupied subsite −1
and formed a hydrogen bond with Agr274; a pyrazole ring also existed
in subsites −1 and −2, formimg a π − π interaction with Trp107
2

B. Jiang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127500
Scheme 1. General synthetic procedure for target
compounds III-01–III-27. Reagents and conditions:
(a) EtOH, EtONa, 0℃ to room temperature (rt) (5 h);
(b) EtOH, ArNHNH2, rt (overnight); (c) (i) NaOH,
80℃ (2 h), (ii) HCl; (d) (i) SOCl2, (ii) substituted
aniline (1.2 equiv.), Et3N (2.0 equiv.), rt (over-
night); (e) (i) EDCI, HOBt, (ii) substituted benzyla-
mine and phenylethylamine (1.2 equiv.), rt (over-
night).
Table 2
Insecticidal activities of target compounds against P. xylostella at lower con-
centrations.
Compd Mortality(%)
LC50 (mg·L−1
)
200 mg·L−1 100 mg·L−1 50 mg·L−1 10 mg·L−1
III-19
III-21
III-27
62
31
11
62
/
/
47
/
/
100
42
13
64.13
(27.16–119.71)
D-11
TF#
57
19
92
/
/
100
61
35
33.83
(27.06–40.12)
#
(Tebufenozide).
Fig. 2. Crystal structure of compound III-19.
Table 1
Insecticidal activities of target compounds against Plutella xylostella at
500 mg·L−1
Compd
R
n
Mortality (%) Compd
R
n
Mortality (%)
500 mg·L−1
500 mg·L−1
III-01
III-02
III-03
III-04
III-05
III-06
III-07
III-08
III-09
III-10
III-11
III-12
III-13
III-14
III-15
III-16
III-17
2-F
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
22
19
37
33
22
26
30
33
33
19
19
22
26
14
14
22
11
III-18
III-19
III-20
III-21
III-22
III-23
III-24
III-25
III-26
III-27
D-08
D-09
D-10
D-11
TF#
4-OCH3
2-CH3
3-CH3
4-CH3
2-CH2CH3
3-CH2CH3
4-CH2CH3
4-t-Bu
2-CH3
2-CH3
4-t-Bu
2-CH3
2-CH3
2-CH3
/
0
0
0
0
0
0
0
0
1
2
0
0
1
2
22
100
41
92
30
33
37
30
33
100
70
67
60
100
100
0
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
Fig. 3. The abnormal molting and malformation of Plutella xylostella larvae
caused by (a) III-27, (b) tebufenozide, and (c) the control treatment at 96 h.
4-Br
2-CF3
3-CF3
4-CF3
2-OCF3
3-OCF3
4-OCF3
2-OCH3
3-OCH3
08, and it was also better than that of the control (GlcN)5 (Fig. 5a and
S2).
In summary, a series of novel heptacyclic pyrazolamide derivatives
were obtained based on lead compound D-08. The structures of the title
compounds were characterized by 1H NMR, 13C NMR, and HRMS.
Through the scaffold hopping strategy, we discovered a new type of
compound with an active skeleton. More importantly, compound III-27
showed potent insecticidal activity, good binding activity to EcR, and
excellent Of ChtI inhibition activity comparing with D-08. Therefore,
the scaffold-hopping drug-design strategy provided the promising po-
tential IGR lead compound III-27, which could be very valuable for the
CK
#
(Tebufenozide).
(Fig. 5b). The presence of these interactions stabilized the binding be-
tween Of ChtI and compound III-27. The results of inhibiting activities
showed that the activity of compound III-27 was higher than that of D-
3

B. Jiang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127500
Fig. 4. (a) IC50 values of the compound III-27
against PxEcR-PxUSP. (b) The predicted
binding modes of the compounds D-08
(yellow), III-27 (green), and tebufenozide
(blue) in the active site of ecydone receptor
(EcR). Hydrophobic regions are colored in
blue. (For interpretation of the references to
color in this figure legend, the reader is re-
ferred to the web version of this article.)
Fig. 5. (a) IC50 values of the compound
III-27 against Ostrinia furnacalis chitinase
(Of ChtI). (b) The predicted binding
modes of the compounds D-08 (yellow),
III-27 (green), and (GlcN)5 (brown) in
the active site of Of ChtI. Hydrophobic
regions are colored in blue. (For inter-
pretation of the references to color in this
figure legend, the reader is referred to
the web version of this article.)
further development of dual-target candidates. Molecular docking re-
sults showed hydrophobicity, hydrogen bonds, and an π − π interac-
tion play important roles in the interaction of III-27 with EcR and Of
ChtI, respectively.
6. Wing KD, Slawecki RA, Carlson GR. RH 5849, a Nonsteroidal ecdysone agonist: ef-
fects on larval lepidoptera. Science. 1988;241:470–472.
7. Dhadialla T, Retnakaran A, Smagghe G. Insect growth-and development-disrupting
insecticides. Compr. Mol. Insect Sci. 2005;6:55–115.
8. Takeo S, Hisamori D, Matsuda S, Vinetz J, Sattabongkot J, Tsuboi T. Enzymatic
characterization of the plasmodium vivax chitinase, a potential malaria transmission-
blocking target. Parasitol Int. 2009;58:243–248.
Declaration of Competing Interest
9. Dong Y, Jiang X, Liu T, et al. Structure-based virtual screening, compound synthesis,
and bioassay for the design of chitinase inhibitors. J Agric Food Chem.
2018;66:3351–3357.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
10. Dong Y, Hu S, Jiang X, et al. Pocket-based lead optimization strategy for the design
and synthesis of chitinase inhibitors. J Agric Food Chem. 2019;67:3575–3582.
11. Chen L, Zhou Y, Qu M, Zhao Y, Yang Q. Fully deacetylated chitooligosaccharides act
as efficient glycoside hydrolase family 18 chitinase inhibitors. J Biol Chem.
2014;289:17932–17940.
Acknowledgments
12. Jiang X, Kumar A, Liu T, Zhang KYJ, Yang Q. A novel scaffold for developing specific
or broad-spectrum chitinase inhibitors. J Chem Inf Model. 2016;56:2413–2420.
13. Chen L, Liu T, Duan Y, Lu X, Yang Q. Microbial secondary metabolite, phlegmacin B-
1, as a novel inhibitor of insect chitinolytic enzymes. J Agric Food Chem.
2017;65:3851–3857.
This work was supported by the National Natural Science
Foundation of China (No. 21877126 and No. 21977114). We appreciate
Miss Chen Ling’s contribution on English writing of this article.
14. Brown N. Bioisosteres and scaffold hopping in medicinal chemistry. Mol Inform.
2014;33:458–462.
15. Tung YS, Coumar M, Wu YS, et al. Scaffold-hopping strategy: synthesis and biological
evaluation of 5,6-fused bicyclic heteroaromatics to identify orally bioavailable an-
ticancer agents. J Med Chem. 2011;54:3076–3080.
Appendix A. Supplementary data
16. Lamberth C. Agrochemical lead optimization by scaffold hopping. Pest Manag Sci.
2017;74:282–292.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127500.
17. Jiang B, Jin X, Dong Y, et al. Design, synthesis, and biological activity of novel
heptacyclic pyrazolamide derivatives: a new candidate of dual-target insect growth
regulators. J Agric Food Chem. 2020;68:6347–6354.
References
18. Deng X, Zhang L, Hu X, Yin B, Liang P, Yang X. Target-based design, synthesis and
biological activity of new pyrazole amide derivatives. Chin Chem Lett.
2016;27:251–255.
1. Schneider MI, Smagghe G, Gobbi A, Vinuela E. Toxicity and pharmacokinetics of
insect growth regulators and other novel insecticides on pupae of hyposoter didy-
mator (hymenoptera: ichneumonidae), a parasitoid of early larval instars of lepi-
dopteran pests. J Econ Entomol. 2003;96:1054–1065.
19. Jiang J, Huang W, Zhai J, et al. ‘One-pot’ synthesis of 4-substituted 1,5-diaryl-1H-
pyrazole-3-carboxylates via lithium tert-butoxide-mediated sterically hindered
claisen condensation and knorr reaction. Tetrahedron. 2013;69:627–635.
20. Wheelock CE, Nakagawa Y, Harada T, et al. High-throughput screening of ecdysone
agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting
hormonal activity. Bioorgan. Med. Chem. 2006;14:1143–1159.
2. Schwartz L, Wolf D, Markus A, Wybraniec S, Wiesman Z. Controlled-release systems
for the insect growth regulator pyriproxyfen. J Agric Food Chem. 2003;51:5985–5989.
3. Sun R, Liu Y, Zhang Y, Xiong L, Wang Q. Design and synthesis of benzoylphenylureas
with fluorinated substituents on the aniline ring as insect growth regulators. J Agric
Food Chem. 2011;59:2471–2477.
21. Fujita T, Nakagawa Y. QSAR and mode of action studies of insecticidal ecdysone
agonists. SAR. QSAR. Environ. Res.. 2007;18:77–88.
4. Billas IML, Iwema T, Garnier JM, Mitschler A, Rochel N, Moras D. Structural
adaptability in the ligand-binding pocket of the ecdysone hormone receptor. Nature.
2003;426:91–96.
22. Hu X, Yin B, Cappelle K, et al. Identification of novel agonists and antagonists of the
ecdysone receptor by virtual screening. J Mol Graph Model. 2018;81:77–85.
23. Vaaje-Kolstad G, Horn S, Sorlie M, Eijsink V. The chitinolytic machinery of serratia
marcescens - a model system for enzymatic degradation of recalcitrant poly-
saccharides. FEBS J. 2013;280:3028–3049.
5. Pantoom S, Vetter IR, Prinz H, Suginta W. Potent family-18 chitinase inhibitors: x-ray
structures, affinities, and binding mechanisms. J Biol Chem. 2011;286:24312–24323.
4