Quantitative structure–activity relationship of substituted imidazothiadiazoles for their binding against the ecdysone receptor of Sf-9 cells

Article abstract of DOI:10.1016/j.bmcl.2017.10.013

Imidazothiadiazoles (ITDs) are a class of potent nonsteroidal ecdysone agonists with larvicidal activity. Previously, we performed the Hansch–Fujita type of quantitative structure–activity relationship (QSAR) analysis for ITD analogs (Yokoi et al., Pestic. Biochem. Physiol. 2015, 120, 40–50). The activity was reasonably explained by hydrophobicity and electronegativity of substituents on the imidazothiadiazole ring system. However, the limited data points (n = 8) hampered the examination of other physicochemical parameters. In the present study, we expanded the library of ITD congeners and evaluated their receptor-binding affinity using intact Sf-9 cells. The QSAR analysis for the expanded set revealed the significance of the third physicochemical parameter, the negative steric effect for long substituents. We also evaluated the larvicidal activity of the synthesized compounds against Spodoptera litura; however, it was not correlated to the binding affinity. The results obtained here suggests that the pharmacokinetic properties must be improved to enhance the larvicidal activity of ITDs.

Full text of DOI:10.1016/j.bmcl.2017.10.013

Accepted Manuscript
Quantitative structure–activity relationship of substituted imidazothiadiazoles
for their binding against the ecdysone receptor of Sf-9 cells
Taiyo Yokoi, Yoshiaki Nakagawa, Hisashi Miyagawa
PII:
S0960-894X(17)30988-5
https://doi.org/10.1016/j.bmcl.2017.10.013
BMCL 25342
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 August 2017
3 October 2017
7 October 2017
Please cite this article as: Yokoi, T., Nakagawa, Y., Miyagawa, H., Quantitative structure–activity relationship of
substituted imidazothiadiazoles for their binding against the ecdysone receptor of Sf-9 cells, Bioorganic & Medicinal
Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.013
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Quantitative structure–activity relationship of substituted imidazothiadiazoles for their
†
binding against the ecdysone receptor of Sf-9 cells
Taiyo Yokoi, Yoshiaki Nakagawa*, Hisashi Miyagawa
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
†
Dedicated to Professor Emeritus Toshio Fujita, who passed away on August 22, 2017.
*To whom correspondence should be addressed.
E-mail: naka@kais.kyoto-u.ac.jp
Keywords
Ecdysone, imidazothiadiazole, larvicide, Lepidoptera, QSAR
Abbreviations
2
1
0E,
20-hydroxyecdysone;
DAH,
diacylhydrazine;
DBU,
,8-diazobicyclo[5.4.0]undec-7-ene; DMF, N,N-dimethylformamide; EcR, ecdysone receptor;
ITD, imidazothiadiazole; PonA, ponasterone A; QSAR, quantitative structure–activity
relationship; USP, ultraspiracle
1

Abstract
Imidazothiadiazoles (ITDs) are a class of potent nonsteroidal ecdysone agonists with
larvicidal activity. Previously, we performed the Hansch–Fujita type of quantitative
structure–activity relationship (QSAR) analysis for ITD analogs (Yokoi et al., Pestic.
Biochem. Physiol. 2015, 120, 40–50). The activity was reasonably explained by
hydrophobicity and electronegativity of substituents on the imidazothiadiazole ring system.
However, the limited data points (n = 8) hampered the examination of other physicochemical
parameters. In the present study, we expanded the library of ITD congeners and evaluated
their receptor-binding affinity using intact Sf-9 cells. The QSAR analysis for the expanded set
revealed the significance of the third physicochemical parameter, the negative steric effect for
long substituents. We also evaluated the larvicidal activity of the synthesized compounds
against Spodoptera litura; however, it was not correlated to the binding affinity. The results
obtained here suggests that the pharmacokinetic properties must be improved to enhance the
larvicidal activity of ITDs.
2

Insects grow by shedding off their old exoskeleton and replacing it with a new one. This
process, known as molting or ecdysis, is regulated by 20-hydroxyecdysone (20E; Figure 1).
The molecular action of 20E is mediated by the heterodimeric complex of nuclear receptors:
ecdysone receptor (EcR) and ultraspiracle (USP). In the presence of 20E, the ternary complex
1
of 20E/EcR/USP activates the transcription of its target genes to trigger molting. Since the
2
0E-dependent molting is crucial to insects, disruptors of this process are good candidates for
insecticides that are harmless to vertebrates. However, 20E and the related steroids
collectively called ecdysteroids) share the highly hydroxylated, complex molecular
(
framework with multiple chiral centers, thereby hampering their practical use as insecticides.
OH
X
OH
S
N
O
N
Cl
R
N
N
O
N
Y
H
HO
HO
OH
X
HN
O
H
O
Diacylhydrazines (DAHs)
RH-5849: X = Y = H
R = OH: 20-hydroxyecdysone (20E)
R = H: Ponasterone A (PonA)
Imidazothiadiazoles (ITDs)
Tebufenozide: X = 3,5-(CH ) , Y = 4-Et
3
2
Figure 1. Chemical structures of ecdysone agonists.
In 1988, diacylhydrazines (DAHs; Figure 1) were serendipitously identified as
2
,3
nonsteroidal ecdysone agonists with larvicidal activity. This discovery made agricultural
chemists realize that nonsteroidal ecdysone agonists are druggable, and the subsequent lead
optimization efforts resulted in the development of 5 DAH congeners as agricultural
4
insecticides. Intriguingly, DAHs are highly toxic to Lepidoptera, but less toxic to Diptera and
3

5
Coleoptera. This specific toxicity results from the higher binding affinity of DAHs against
6
lepidopteran EcRs than those from other insect orders. X-ray crystal structures of EcR/USP
complexes imply that the structural plasticity of lepidopteran EcRs could be essential for the
7
perception of DAHs with high affinity.
The discovery of DAHs also stimulated the exploration of completely different
chemical class of ecdysone agonists. Among various nonsteroidal chemotypes screened to
5
date, imidazothiadiazoles (ITDs; Figure 1) are remarkable in that they achieved nanomolar
8
potency in an ecdysone-inducible gene expression system. These authors also provided the
possible binding mode of ITDs against EcR, which is somewhat different from that of DAHs.
In their report, however, any experimental procedures and target insect species were not
described. The synthesized compounds were not chemically characterized, either.
Previously, we synthesized ITD congeners to confirm their potency as ecdysone
9
agonists. As is the case with DAHs, ITDs are highly specific to lepidopteran EcRs, and the
binding affinity of ITDs with fluoroalkyl group as substituent X reached nanomolar level in
Sf-9 cell. The Hansch–Fujita type of quantitative structure–activity relationship (QSAR)
1
0
analysis disclosed that hydrophobicity and electronegativity of substituent X are important
to exhibit high binding affinity. However, the number of the compounds used for the analysis
was limited (n = 8), and was insufficient for revealing the involvement of other
physicochemical properties. Herein, we report the updated results of our QSAR study for
ITDs.
4

Scheme 1. Synthetic route to compound 9. Reagents and conditions: (a) Br
RT, 18 h, 87%; (b) 2-chlorophenacyl bromide, EtOH, reflux, overnight, 73%; (c) Me
MeOH/H O, microwave, 100°C, 10 min, 87%; (d) POCl , DMF, 70°C, 1 h, 97%; (e)
EtO) P(O)CH CO Et, LiCl, DBU, MeCN, RT, 2 h, 85%; (f) isopropylamine, AlMe
CH Cl /hexane, 35°C, 1 day, 81%.
2
, NaOAc, AcOH,
2
NH,
2
3
(
2
2
2
3
,
2
2
To expand the compound set for QSAR analyses, we newly prepared a congeneric
series of ITDs with various substituents on the imidazothiadiazole ring (3–9). Of these,
9
compounds 3–8 were synthesized according to the conventional method. Compound 9 was
synthesized following the procedure illustrated in Scheme 1. Treatment of
2
2
-amino-1,3,4-thiadiazole with bromine gave compound 20, which was condensed with
-chlorophenacyl bromide to afford compound 21. This was subjected to a nucleophilic
aromatic substitution reaction with dimethylamine under microwave heating to give
compound 22. Vilsmeier–Haack formylation of 22 cleanly furnished aldehyde 23, which was
transformed to α,β-unsaturated ester 24 via Horner–Wadsworth–Emmons reaction. Finally,
5

11
aminolysis of ester 24 with the aid of trimethylaluminum gave N-isopropylamide 9. The
authenticity of the synthesized compounds was confirmed by spectroscopic analyses (see
Supplementary Materials).
The synthesized compounds were then subjected to a competitive binding assay in
lepidopteran Sf-9 cells, wherein binding affinity of each compound was evaluated as the 50%
3
inhibition concentration [IC50 (M)] for binding of the reference ligand, [ H]ponasterone A.
12,13
The results are summarized in terms of the reciprocal logarithmic values (pIC50; Table 1). All
compounds synthesized in the present study (3–9) exhibited higher binding affinity than the
unsubstituted one (1), and some compounds (4, 7, 8) were more potent than the natural insect
molting hormone, 20E (18). Across compounds with linear alkyl substituents, stepwise
3
enhancement in the activity was observed for CH , Et, and n-Pr groups (2–4), whereas n-Bu
group (6) decreased the activity. Among compounds with branched alkyl chains, hydrophobic,
bulky i-Bu (7) and t-Bu (8) groups are more favorable than i-Pr (5) group. Compound with
3 2
N(CH ) group (9) are about 5 times less potent than that with i-Pr group (5) despite the
similar shapes between these two substituents. Compounds with fluoroalkyl groups (13–15)
were tens to hundreds of times more potent than those with the corresponding non-fluorinated
alkyl groups (2–4), demonstrating the importance of electronegative fluorine atoms.
6

Table 1. Biological activity and physicochemical parameters of synthesized ITDs.
X
S
N
Cl
N
N
HN
O
Compounds
Substituent (X)
H
Physicochemical parameters
Binding activity [pIC50 (M)]
Larvicidal activity
[pLD50 (mmol/larva)]
< 4.00 (0%)
a
b
I
c
d
e
d,f
No.
1
Clog P
3.74
4.24
4.77
5.30
5.17
5.83
5.70
5.57
σ
∆L
Obsd.
Calcd.
5.15
5.49
6.12
6.69
6.89
7.04
7.25
7.14
g
0.00
0.00
0.88
2.07
3.00
2.02
4.17
2.98
2.03
4.79
g
g
2
CH
3
−0.04
−0.01
−0.01
0.01
5.44
< 4.30 (0%)
3
Et
6.23 ± 0.12 (2)
7.15 ± 0.01 (2)
6.78 ± 0.20 (2)
6.37 ± 0.08 (2)
7.35 ± 0.13 (2)
7.07 ± 0.24 (2)
< 4.00 (0%)
< 4.00 (0%)
< 4.48 (0%)
< 4.00 (0%)
< 4.00 (0%)
< 6.00 (0%)
4
n-Pr
i-Pr
n-Bu
i-Bu
t-Bu
5
6
−0.04
−0.03
−0.07
7
8
7

9
1
N(CH
3
)
2
4.28
4.44
2.44
2.26
4.63
5.00
5.23
2.48
4.51
−1.21
1.00
0.06
0.25
0.49
0.59
0.40
0.41
0.39
-
2.05
2.17
2.03
2.26
1.24
2.67
3.48
-
6.10 ± 0.01 (2)
5.68
6.83
4.81
4.93
8.17
8.37
8.39
-
< 4.30 (0%)
< 4.30 (0%)
< 4.00 (0%)
< 4.30 (0%)
g
g
g
g
0
SCH
3
7.39
g
11
S(O)CH
3
4.79
g
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
SO
2
CH
3
4.76
g
h
h
CF
CF
CF
3
2
2
8.03
5.15 ± 0.01 (2)
4.94 ± 0.04 (2)
4.45 ± 0.01 (2)
g
CF
3
2
8.35
h
CF
CF
3
8.36 ± 0.13 (3)
i
g
RH-5849
6.44
4.41
i
g
Tebufenozide
20E
-
-
8.81
-
6.47
i
-
-
6.78
-
inactive
inactive
i
1
PonA
-
-
8.05
-
a
b
Calculated by CLOGP program (BioByte Corp., Claremont, CA, USA). Taken from ref.
14 c
Calculated for the energy-minimized
d
structures (see Supplementary Materials). Mean ± standard deviation. Values in parentheses are the number of replications.
e
f
Calculated by Eq. 3. Percentages in parentheses are the proportion killed at the corresponding dose. Taken from ref. Reevaluated in
g
9 h
i
this study. Taken from ref.
12
8

We also evaluated larvicidal activity [pLD50 (mmol/larva)] of the synthesized
compounds against the Oriental leafworm, Spodoptera litura (Table 1). Unfortunately, none
of the newly synthesized compounds (3–9) gave pLD50 values, although the highest dose of
compound 8 was only 1/100 those of other compounds due to its limited solubility in
dimethyl sulfoxide. The larvicidal activity of compounds with fluorinated alkyl groups
9
13–15), which were evaluated in our earlier publication, were measured again to confirm the
(
reproducibility. They showed the moderate larvicidal activity with small standard deviation.
Compound 13, which showed the highest larvicidal activity among the ITD congeners, is
approximately 20 times less potent than the commercialized ecdysone agonist, tebufenozide
(
17).
To identify the physicochemical factors governing the ligand–receptor interaction,
we performed the Hansch–Fujita QSAR analysis. We previously obtained Eq. 1 for the
9
binding affinity of a small set of compounds (1, 2, 10–15) :
pIC50 = 1.519 (± 0.315) ∆Clog P + 3.923 (± 1.643) F + 4.872 (± 0.361)
n = 8, s = 0.349, r = 0.985, F2,5 = 81.754
(1)
where ∆Clog P is the hydrophobic parameter which represents the Clog P value of a
compound relative to that of the unsubstituted compound [∆Clog P = Clog P (X) − Clog P
1
H)], and F is the electronic parameter defined by Swain and Lupton. In this and the
5
(
following equations, values in parentheses are the 95% confidence intervals of the regression
coefficients, n is the number of compounds, s is the standard deviation, and r is the correlation
9

coefficient. Fm,n−m−1 is the value of ratio between regression and residual variances, where m
is the number of independent variables. Equation 1 gave the satisfactory correlation; however,
the number of parameters used to derive the equation was two (∆Clog P and F), which is not
statistically favored. In general, at least 5 compounds are required per parameter.
We reanalyzed the binding affinity of the expanded set of compounds (1–15) to
obtain Eq. 2:
pIC50 = 1.386 (± 0.296) Clog P + 4.157 (± 1.365) σ
I
– 0.405 (± 1.515)
(2)
n = 15, s = 0.431, r = 0.947, F2,12 = 52.280
where σ
I
is the inductive component of Hammett substituent constant σ, which by definition is
with F also yielded the similar
equivalent to Swain–Lupton F. Indeed, the replacement of σ
I
correlation (s = 0.447, r = 0.943). The intercept was much smaller than that in Eq. 1, because
here we utilized intact Clog P instead of ∆Clog P. The significance of Eq. 2 was justified
above 99.9% by F-test; however, the standard deviation was still large compared to the
maximal standard deviation in the data set (0.24). In our previous QSAR study, the binding
affinity of DAHs with various para-substituents Y (Figure 1; X = 2-Cl) was reasonably
1
explained by hydrophobic, electronic, and steric parameters. This experience prompted us to
6
add a steric parameter, formulating Eq. 3:
pIC50 = 1.626 (± 0.316) Clog P + 4.914 (± 1.292) σ – 0.315 (± 0.263) ∆L – 0.930 (± 1.326)
I
(
3)
1
0

n = 15, s = 0.352, r = 0.968, F3,11 = 54.497
17
where ∆L represents the STERIMOL length parameter for a substituent relative to hydrogen.
Equation 3 indicates that hydrophobic and inductively electron-withdrawing substituents
enhance the binding affinity, while long substituents not. The physicochemical parameters and
the calculated pIC50 values by Eq. 3 are listed in Table 1. Below we discuss the implications
of the above QSAR model in detail.
The large, positive coefficient of the Clog P term in Eq. 3 indicates that the
hydrophobic substituents drastically enhance the binding affinity. It is generally accepted that
the slope of log P term implies the location of the ligand binding site, since log P corresponds
to the desolvation free energy of a compound upon its partitioning from the aqueous phase
1
8
into the 1-octanol phase. The slope of 1.0 means the complete desolvation of water
molecules from the ligand, suggesting the ligand is completely buried in the receptor, while
the slope of 0.5 means the half-desolvation, suggesting that the ligand binds to the surface of
the receptor. In Eq. 3, the coefficient (1.626) of the Clog P term is much greater than 1.0. This
might suggest that the binding cavity of ITDs is located inside the receptor and composed of
hydrophobic amino acid side chains, wherein hydrophobic interaction is predominant.
The positive coefficient of the σ
electron-withdrawing substituents are favorable for the ligand–receptor interaction. Two
assumptions are possible for this positive contribution of the σ term: (1) the
electron-withdrawing effect toward the imidazothiadiazole ring system enhances the binding,
2) the electronegative substituent participates in the interaction with electropositive residues
I
term in Eq. 3 means that inductively
I
(
11

of the EcR. If the assumption (1) is true, the resonance electronic effect should be significant;
however, addition of the resonance component, σ , did not improved the correlation. We
R
therefore concluded that the assumption (2) is reasonable, which means that the
electronegative atoms (e.g. fluorine) participate in the electrostatic interaction with the
receptor.
The negative coefficient of the ∆L term in Eq. 3 indicates that long substituents are
sterically unfavorable for the binding. Replacement of the ∆L term with other steric
parameters, such as substituent width (∆B
5
w
) and volume (∆V ), did not improve the
correlation. This negative steric effect is prominent in the case of compounds with linear alkyl
chains. With elongation of the carbon chain, the binding affinity showed a gradual increase
for 1–4, but suddenly dropped for 6. These results suggest the presence of the receptor wall
that limits the binding of long substituents.
Equation 3 rationally explains the change in binding affinity of ITDs. A notable
example is the activity difference between compounds 5 and 9: compound 5 with i-Pr group is
5
times more active than compound 9 with N(CH
3 2
) group. As shown in Table 1, these two
substituents are almost equivalent in terms of σ and ∆L. By contrast, the Clog P value of
I
compound 5 is about 8 times higher than that of compound 9. This difference in
hydrophobicity leads to the higher binding affinity of compound 5 than that of compound 9.
Figure 2 shows the possible binding model of ITDs based on the above QSAR
9
model and our previous study. Holmwood and Schindler reported the possible binding mode
8
of ITDs, although they did not provide any experimental data supporting it. According to
their structures, ITDs and DAHs roughly share the same binding position, and substituent X
1
2

of ITDs and tert-butyl group of DAHs are closely located. In the crystal structure of Heliothis
virescens EcR/USP complex bound to a DAH analog (PDB: 1R20), the tert-butyl group is
7
surrounded by hydrophobic amino acid residues, such as F336, M413, L511, and L518. In
particular, F336 seems to function as a cap that separates the ligand binding cavity from the
bulk water, limiting the size of the ligand molecule. Furthermore, located at the edge of the
binding pocket is T340 that is able to form hydrogen bond with electronegative atoms like
fluorine. These observations are in good accordance with the present QSAR results.
Figure 2. The possible binding model of imidazothiadiazoles against the receptor.
As shown in Table 1, compounds with fluoroalkyl groups (13–15) were
moderately toxic to S. litura, while the other compounds (1–12) showed no substantial
1
3

activity. This is probably due to the metabolic detoxification in insect body after application.
Compounds with alkyl and sulfide moieties (2–8, 10) can undergo the oxidative metabolism
to lower the insecticidal activity. It is likely that the presence of fluorine atom is effective not
only for improving the intrinsic activity but also for suppressing the metabolic detoxification.
Meanwhile, there is no clear correlation between the larvicidal and binding activity among
compounds with fluoroalkyl groups (13–15). Instead, the larvicidal activity is negatively
correlated to the molecular hydrophobicity. Increased hydrophobicity seems to act as a
limiting factor of their concentration at the target site. Further synthetic efforts, especially to
improve their pharmacokinetic properties, are required to bring their larvicidal potency into
the practical level.
In summary, the present QSAR study identified the key physicochemical factors
of ITDs that are important for the ligand–receptor interaction: molecular hydrophobicity,
inductive electron-withdrawing effects, and substituent length. Comparison of the binding and
larvicidal activities demonstrated the importance of the fluoroalkyl substituent on the
imidazothiadiazole ring, in terms of improving their intrinsic activity and metabolic stability.
These findings provide a valuable information for the rational design of novel ecdysone
agonists.
Acknowledgements
We are grateful to Ishihara Sangyo Kaisha, Ltd. for providing eggs of Spodoptera litura. We
also thank Ms. Karin Nishimura (Graduate School of Engineering, Kyoto University) for
conducting mass spectrometric analyses. This work was supported in part by JSPS
1
4

KAKENHI Grant Numbers 16K07625 (Y.N.) and 17J01486 (T.Y.). T.Y. is a recipient of JSPS
Research Fellowship for Young Scientists.
Supplementary Materials
Detailed experimental procedures and chemical characterization of new compounds are
provided in the Supplementary Materials.
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