Synthesis and insecticidal activity in vitro and vivo of novel benzenesulfonyl derivatives based on potent target subunit H of V-ATPase

Article abstract of DOI:10.1016/j.bmcl.2018.08.030

Two lead compounds with benzenesulfonamide were found through virtual screening based on the 3D structure of the subunit H of V-ATPase in previous study. 74 benzenesulfonyl derivatives were synthesized and their insecticidal activities were evaluated. The derivatives with propargyl substituents exhibit excellent insecticidal activities against Mythimna separata Walker. The LD₅₀ values of compounds A5.7 (28.0 μg·g^?1) and B5.7 (36.4 μg·g^?1) were significantly less than that of Celangulin V (344.0 μg·g^?1). Furthermore, Isothermal Titration Calorimetry (ITC) data indicate there is a strong binding affinity between A5.7 and V-ATPase Subunit H. These results demonstrate that it is a practical way to develop pesticides targeting at H subunit of V-ATPase.

Full text of DOI:10.1016/j.bmcl.2018.08.030

Accepted Manuscript
Synthesis and insecticidal activity in vitro and vivo of novel benzenesulfonyl
derivatives based on potent target subunit H of V-ATPase
Chaofu Yang, Xiaoting Li, Jielu Wei, Feng Zhu, Fangli Gang, Shaopeng Wei,
Yunlong Zhao, Jiwen Zhang, Wenjun Wu
PII:
S0960-894X(18)30706-6
https://doi.org/10.1016/j.bmcl.2018.08.030
BMCL 26010
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 July 2018
23 August 2018
25 August 2018
Please cite this article as: Yang, C., Li, X., Wei, J., Zhu, F., Gang, F., Wei, S., Zhao, Y., Zhang, J., Wu, W., Synthesis
and insecticidal activity in vitro and vivo of novel benzenesulfonyl derivatives based on potent target subunit H of
V-ATPase, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.08.030
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Graphical Abstract
Synthesis and insecticidal activity in vitro
and vivo of novel benzenesulfonyl derivatives
based on potent target subunit H of V-
ATPase
Leave this area blank for abstract info.
Chaofu Yang a, Xiaoting Li a, Jielu Wei a, Feng Zhu a,
Fangli Gang a, Shaopeng Wei b, Yunlong Zhao a,
Jiwen Zhang a,b*, Wenjun Wu b*
1 / 6

Bioorganic
journal homepage: www.elsevier.com
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Medicinal
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Letters
Synthesis and insecticidal activity in vitro and vivo of novel benzenesulfonyl
derivatives based on potent target subunit H of V-ATPase
Chaofu Yang a, #, Xiaoting Li a, #, Jielu Wei a, Feng Zhu a, Fangli Gang a, Shaopeng Wei b, Yunlong Zhao a,
Jiwen Zhang a, b*, Wenjun Wu b*
a College of Chemistry & Pharmacy, Shaanxi Key Laboratory of Natural Products & Chemical Biology, Northwest A&F University, Yangling 712100, Shaanxi,
People’s Republic of China
b Key Laboratory of Botanical Pesticide R&D in Shaanxi Province, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China
ARTICLE INFO
ABSTRACT
Article history:
Received
Two lead compounds with benzenesulfonamide were found through virtual screening based on
the 3D structure of the subunit H of V-ATPase in previous study. 74 benzenesulfonyl derivatives
were synthesized and their insecticidal activities were evaluated. The derivatives with propargyl
substituents exhibit excellent insecticidal activities against Mythimna separata Walker. The
LD50 values of compounds A5.7 (28.0μg.g-1) and B5.7 (36.4 μg.g-1) were significantly less than
that of Celangulin V (344.0 μg.g-1). Furthermore, Isothermal Titration Calorimetry (ITC) data
indicate there is a strong binding affinity between A5.7 and V-ATPase Subunit H. These results
demonstrate that it is a practical way to develop pesticides targeting at H subunit of V-ATPase.
Revised
Accepted
Available online
Keywords:
Subunit H of V-ATPase
Benzenesulfonyl derivatives
Insecticidal activities
Isothermal titration calorimetry
© 2018 Elsevier Ltd. All rights reserved.
Currently, 10-16% of global crop production is consumed by
pests, such as insects, fungi, bacteria.1 The more resistant the
pests are, the more urgent the need to find new pesticides.
Natural products are one of the most important sources to
generate pesticides with high-level insecticidal activity. They are
lead compounds and probes to find new targets. Based on the
target structures of lead compounds, many pesticides were
synthesized, including nicotine and scytalone dehydratase
inhibitors.2 Our laboratory has an ongoing research program
focused on Celastrus angulatus Maxim against insects. The β-
dihydroagarofuran derivatives with excellent insecticidal activity
have been isolated, determined, synthesized and biologically
evaluated.3-6 In particular, Celangulin V (1, Figure 1) is the
representative β-dihydroagarofurananalog and demonstrates
outstanding insecticidal activity. It is considered to act on the
vacuolar H+ -ATPase (V-ATPase) of the M. separata midgut
after oral administration.7 V-ATPase is a proton pump associated
with ATP transporters and provides energy for organelles and
membranes and present in almost all eukaryotic cells. 1 The V-
ATPase is made up of two complexes, the V0 and V1 complexe.8
The subunit H of V-ATPase is seated at V1 complex near the
interface between the V1 and V0 complex and has been proved to
silence ATP hydrolysis by free V1 through linking the stator and
rotary domains.9 In the previous work of our group, the subunit H
of V-ATPase of M. separate was purified and determined as
1807 bp full length which was similar to subunit H of yeast’s V-
ATPase.10
Applications of specific interactions between biological
macromolecules and their ligands are especially important in
drug discovery and development. Based on these interactions,
virtual screening techniques can facilitate the discovery of drug
candidates by discerning promising lead compounds and
reducing the number of experiments.11 Now application of
receptor-based rational drug design in agriculture has become an
important research field. 12-16 The subunit H model of V-ATPase
of M. separata (2, Figure 1) was set up based on known structure
of H subunit of yeast’s V-ATPase(1HO8).17With Diseovery
Studio software, active sites were defined and identified. Specs
data are virtually screened using homology model and molecular
docking of the subunit H of V-ATPase of M. separata.
Compounds 1 and 2 (3 and 4, Figure 1) with high scores
exhibited significant stomach poisoning activity against M.
separata with a series of symptoms, such as excitement,
convulsions and body fluids loss, which is similar to Celangulin
V (Detail in supplementary data). To investigate the effects of
substituents of benzene ring and amino groups of sulfonamide of
these two lead compounds on the insecticidal activity, we
synthesized 74 benzene sulfonamide derivatives, and
systematically evaluated their insecticidal activity against M.
separata.
———
* Corresponding author. fax: +86-029-87093987; e-mail:
nwzjw@nwsuaf.edu.cn; wuwenjun@nwsuaf.edu.cn
# These two authors contribute equally to this work.
2 / 6

Schemes 1 and 2 illustrate the synthesis of benzenesulfonyl
derivatives. Compound A1.1-5.9 were synthesized via a two-step
reaction using Sodium 4-hydroxy benzene sulfonate as raw
material. Compound B2.1-5.8 were synthesized through a two-
step reaction using Sodium 3-hydroxy benzene sulfonate as
starting materials. Key intermediates, compound A1-5 and B2-5,
were prepared by Williamson reaction in the presence of
anhydrous sodium carbonate in methanol. After the key
intermediates was refluxed in sulfoxide chloride with N, N-
dimethylformamide as catalyst, a series of benzenesulfonyl
derivatives (A1-5 and B2-5) were obtained by reaction of
intermediate chlorides with different anilines (R1-H, R4-H) in the
presence of 4-dimethylamino-pyridine (DMAP). Finally,
compounds A1.1-1.3 were treated with ammonia solution (25-
28%) or hydrazine hydrate at room temperature to obtain
derivatives A1.1.1-1.3.2 in moderate to good yields. All
1
synthesized products have been characterized by m. p., H-NMR
and 13C-NMR (see Supplementary data).
Figure 1. Structures of Celangulin V, subunit H, Compound 1 and compound 2
Scheme 1. Synthesis of benzenesulfonyl derivatives (A1.1.1-A1.3.2).
3 / 6

Scheme 2. Synthesis of benzenesulfonyl derivatives (A2.1-A5.9, B2.1-B5.8).
All the target compounds were screened for insecticidal
activity against 4th instar M. separata in vivo with the leaf dipping
method.3 Celangulin V and acetone were used as positive and
negative control, respectively. In the mortality bioassay, the
concentration of 10 mg·mL-1 was applied. The title compounds
with propargyl exhibited excellent insecticidal activity and toxic
symptoms like those caused by Celangulin V, which showed loss
of body fluid, soft and no response after they were taken orally
(Figure 2). It revealed that the target derivatives acted on the V-
ATPase subunit H in the midgut through a similar mechanism of
Celangulin V.
B5.7, B5.8 with 100% mortality. Next, A5.1-5.8 with propargyl
ether at 4 position bore slightly better potency than B5.1-5.8,
respectively. In order to investigate the structure-activity
relationships, A5.9 with sulfonate was synthesized by using A5.4
as template. The compound A5.9 showed no larvicidal activities
at 10 mg·mL-1. The only difference in the structures between
A5.9 and A5.4 was sulfanilamide and sulphonic acid ester, which
revealed that sulfonamide may be indispensable for insecticidal
activities of these compounds. These results indicated propargyl
ether and benzenesulfonamide were vital substituents for
pesticidal activity.
The bioassay results are summarized in Table 1. The removal
of the chlorine from the benzenesulfonyl fragment in A1.1.1-
1.3.1 resulted in a loss potency against M. Separata. Replacing
the aminocarbonylmethyl group with a hydrazylcarbonylmethyl
group (A1.1.2-1.3.2) or an alkyl group (B2.1-2.8) in the absence
of chlorine led to no potent activities. Meantime, the change of
ether position in A2.1-2.8 showed no potent activities. These
results indicated that the chlorinated aryl rings were very
important in vivo insecticidal activity. Interestingly, substituting
the alkyl group for a propargyl (B5.1-5.8) displayed much better
insecticidal activity than Celangulin V, especially B5.3, B5.4,
Subsequently, the median lethal dose (LD50) of active
compounds were evaluated.18 Most of the compounds with
propargyl exhibited extremely good insecticidal activities. The
LD50 of A5.7 and A5.8 within 24h were 28.0 and 36.4 μg·g-1
respectively, which were 10-fold potency improvement than that
of Celangulin V (344.0 μg·g-1). The LD50 of A5.3, A5.4, B5.3,
B5.4, B5.7 and B5.8 were also in the range of 43.6-51.3μg·g-1, 6-
8 folds more potent than Celangulin V, respectively. For
propargyl series A5.1-5.8 and B5.1-5.8, the insecticidal activity
of benzene sulfonamide derivatives with propargyl at position 4
of benzene ring was slightly stronger than at position 3. It could
4 / 6

be inferred the proper carbon quantity of their side chain at the
N-alkyl position was 3 and 4, and these compounds with
branched chain had better activities than those with linear chain.
ITC is the important technique that can resolve the enthalpic
19-21
and entropic constituents of binding affinity.
So ITC was
used for further quantitatively analyzing the interaction between
A5.7 and subunit H of V-ATPase at 25°C (Figure 3). It showed
that the ITC thermograms were characterized by negative peaks
in the plot of the corrected heat trace peaks versus time. This
indicated that the binding process is exothermic. As shown in
Figure 3, we can conclude that A5.7 binds to the subunit H of V-
ATPase based on the exothermic peaks. Moreover, along with
diminish of available binding subunit H, the magnitude of the
exothermic peaks continually decreased. A plateau in the
exothermic peaks occurred when the protein became saturated
with A5.7. One binding site model can well match the data in
Figure 3 B. The binding constant (Ka =5.11×106 M-1) suggested
that the binding affinity of A5.7 with V-ATPase subunit H was
Figure 2. The toxic symptoms of M. separata caused by benzenesulfonyl
derivatives
Note: (A) larvae before feeding;(B) convulsions of larvae;(C) body fluids loss
after 24h
stronger than JW-3 under the same conditions (the Celangulin V
derivative, Ka =2.97×105 M-1).
22
The magnitude of standard
molar enthalpy contribution was large enough to overcome this
unfavorable entropy term and make the overall reaction feasible
(ΔG = -38.29 kJ·mol-1, ΔH = -35.66 kJ·mol-1 and ΔS = 8.82
J·mol-1·K-1).
The bioassay results demonstrate that the variety of
substituted groups significantly affected the insecticidal activity
of the target compounds. Benzene sulfonamide derivatives with
propargyl possessed superior insecticidal activity against M.
separata to compounds with alkyl, aminocarbonylmethyl, or
hydrazyl- carbonylmethyl. It indicated that hydrophobic effects
that acted on the subunit H of V-ATPase, which agreed with the
result of ITC.
Table 1. Insecticidal activity of the target compounds against
the 4th instar larvaes of M. Separata
mortality SD (%)
Figure 3. ITC of A5.7 with the Subunit H of V-ATPase
Note: (A) Heat flows recorded upon injection of an A5.7 solution (0.2mM)
into a Subunit H of V-ATPase solution (3.5μM) at 25 °C. (B) Molar heat
values obtained through integration of the individual heat-flow signals as a
function of the mixing ratio concentration. The fitting parameters are
Ka=5.11×106 M-1, ΔG = -38.29 kJ·mol-1, ΔH = -35.66 kJ·mol-1 and ΔS =
8.823 J·mol-1·K-1 at 25 °C. The inset shows the raw data of the sequential
injections.
Compound
LD50(μg·g-1,24h)
漏%,10mg·mL-1,24h漐
acetone
0.0 0.0
0.0
344.0(326.7-361.4) a
Celangulin V
A1.1-1.3
A1.1.1-1.3.2
A2.1-2.8
A3.1-3.8
A4.1-4.8
A5.1
52.8 4.8
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
61.1 4.8
−
−
−
−
In conclusion, 74 novel benzenesulfonyl derivatives were
designed and synthesized in this study. The bioassay results
showed that benzenesulfonyl derivatives with propargyl are
promising insecticides with superior insecticidal activity than
Celangulin V. The toxic symptoms of M. separata and ITC data
indicated benzenesulfonyl derivatives with propargyl acted on
−
271.7(260.2-283.2)
A5.2
A5.3
44.4 4.8
100 0.0
100 0.0
69.4 4.8
33.3 8.3
100 0.0
100 0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
55.5 4.8
41.7 0.0
100 0.0
100 0.0
63.9 4.8
25.0 8.3
100 0.0
100 0.0
465.6(451.6-479.7)
45.8(44.1-47.5)
45.6(44.1-47.2)
216.5(206.7-226.4)
−
A5.4
A5.5
subunit H of V-ATPase. Remarkably, they were simply
synthesized and could be easily industrialized.
A5.6
A5.7
28.0(26.5-29.6)
43.6(41.0-46.2)
−
A5.8
Acknowledgments
A5.9
This work was financially supported by the National Key
B2.1-2.8
B3.1-3.8
B4.1-4.8
B5.1
−
Research
2017YFD0200506) and the National Natural Science Foundation
Development
Program
of
China
(No.
−
−
of China (21372185漓31371958).
313.1(298.2-328.0)
554.5(544.3-564.6)
51.3(49.0-53.6)
51.1(48.9-53.3)
227.9(214.2-241.6)
−
B5.2
A. Supplementary data
B5.3
B5.4
Supplementary data (experimental procedures and spectral
data) associated with this article can be found, in the online
version, at XXXXX.
B5.5
B5.6
B5.7
36.4(32.8-40.0)
50.9(48.8-52.9)
References and notes
B5.8
a The 95% confidence intervals (CI) are in parentheses.
“−” means that the LD50 was not measured.
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