Azobenzene-benzoylphenylureas as photoswitchable chitin synthesis inhibitors

Article abstract of DOI:10.1039/c6ob02813f

Benzoylphenylureas (BPUs) are used as synthetic insect growth regulators for inhibiting chitin synthesis. Merging insecticidal BPUs with photoswitchable azobenzene generated photoresponsive chitin synthesis inhibitors. A prepared azobenzene-benzoylphenylurea can be activated upon irradiation with UV light, and shows 6-fold and 2-fold activity difference to armyworm (Mythimna separata) and German cockroach (Blattella germanica) sulfonylurea receptors, respectively. This is the first example of a photoswitchable BPU insecticide. The generation of such a photoresponsive BPU insecticide allows for modulation of the insecticidal activity by light, and may facilitate the spatiotemporal control over the sulfonylurea receptor and the mechanistic study of this kind of insecticide.

Full text of DOI:10.1039/c6ob02813f

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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ARTICLE
Azobenzene-Benzoylphenylureas as Photoswitchable Chitin
Synthesis Inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
Xue Tian, Chao Zhang, Qi Xu, Zhong Li and Xusheng Shao*
Abstract: Benzoylphenylureas (BPUs) are used as synthetic insect growth regulators for inhibiting the chitin synthesis.
Merging insecticidal BPUs with photoswitchable azobenzene generated the photoresponsive chitin synthesis inhibitors.
One prepared azobenzene-benzoylphenylurea can be activated upon irradiation of UV light, which shows 6-fold and 2-fold
activity difference to armyworm (Mythimna separate) and German cockroach (Blattlla germanica) sulfonylurea receptor,
respectively. This is the first example of photoswitchable BPU insecticide. The generation of such photoresponsive BPU
insecticide allows for modulating the insecticidal activity by light and may facilitate the spatiotemporal control over the
sulfonylurea receptor and the mechanism study of this kind of insecticide.
DOI: 10.1039/x0xx00000x
www.rsc.org/
their superiorities, BPUs have detrimental effects to beneficial
species when dispersed administrated, such as bees, aquatic
organisms and natural predators, which led to the ban of
Introduction
Light has been subtly used to control the chemical or biological
events owing to the fast development of photoresponsive small or
macro- molecules.1-4 To achieve such optical modulation, much
efforts are recently focused on the development of small
photoresponsive molecules by hybrid of a photoresponsive moiety,
such as a photoswitch5-7 or a photoprotecting group,8-11 with a bio-
functional molecule. These photosensitive entities can undergo
activity change (activated or inactivated) with the aid of light, which
flufenoxuron in the European Union in 2011.18
Most recently, we reported a first example of a photoswitchable
neonicotinoid insecticide by incorporating of imidacloprid with
azobenzene, which enable the reversible manipulation of an
insecticide performance.24,25 As continued interests in this area,
herein, we reported the photoswitchable benzoylphenylurea-
azobenzene analogues and their photochemical and photophysical
properties and bioactivities. The principle behind these
provide
mechanism of toxicology and pharmacology.12,13 It facilitates to
understand when and where (spatiotemporal resolution)
a new technology for investigating the underlying
photoswitchable pesticides involves the coupling of
a
photoswitchable azobenzene with benzoylphenylurea molecules.
The trans-cis isomerization occurred at the presence of light
accompanied with the change of the activity. This photo-regulated
process is reversible with ‘switch-on’ of the activity.
a
bioactive molecule takes action. When used in drugs, it had the
advantages such as avoidance of side effects, antiresistance and
high specificity.14-16 Therefore, the generation of photoresponsive
bioactive molecules offers substantial advantages for studying and
manipulating the chemical or biological process of interest. Here,
we chemically modified insecticidal benzoylphenylureas with
photoswitchable group so as to make their actions dependent on
light.
Results and discussion
Benzoylphenylurea (BPUs) insecticides draw intensive attractions
about 40 years due to their excellent activities, low toxicity to
nontarget organisms, low frequency of resistance development and
interesting mode of action.17,18 They are insect growth regulators
(IGRs) and are extremely potent to leaf-eating insects and their
larvaes. BPUs block insect’s ability to turn into an adult through
inhibiting chitin synthesis and hindering the normal molting of
insects.19-21 Recent research revealed that BPUs bind with
sulfonylurea receptor (SUR),20,22,23 a group of ABC-transporters that
correlate with the disruption of the cuticle chitin synthesis. Despite
Scheme 1. Molecular Design of photoswitchable benzoylphenylureas chitin
synthesis inhibitors.
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isomer ratios and half-life time of thermal relaxation of cis-isomer
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DOI: 10.1039/C6OB02813F
were collected in Table 1. All the compounds showed typical
absorption spectrum of azobenzene. As an example, the UV-Vis
spectra changes of B3 upon irradiation were shown in (Figure 1,
left). The maximum absorption wavelength of π-π* transition (λtrans-
of trans configuration was 347 nm and the maximum
max π-π*)
absorption wavelength of n-π* transition (λcis-max
of cis
n-π*)
configuration was 437 nm. Upon irradiation at 430 nm, the reversal
spectra changes were detected and this process can be switched
forth and back for many times without any significant photo
bleaching (Figure 3, right). Substituent positions do not have any
significant influence on the maximum absorbance wavelength. The
meta-compounds (A2 and B2) have shorter maximum absorbance
wavelength than the corresponding ortho- and para- ones (A1, A3，
B1 and B3).
1.5
0
s
10
20
30
40
50
s
s
s
s
s
0.10
0.05
1.0
0.5
0.0
1.2
0.8
0.4
0.0
1
min
1.5 min
min
3
400
500
300
400
500
0
1
2
3
4
5
6
7
8
Cycle Number (n>6)
Wavelength(nm)
Scheme 2. Synthetic procedure for photoswitchable azobenzene-
benzoylphenylureas derivatives.
Figure 1. UV-Vis spectra of B3 before to after irradiation (left) and cycle
performance of compound B3 (right).
Molecular design. In order to obtain a photoresponsive ligand, the
Table1. Maximum absorbance wavelength (λmax, nm), ratio of trans and cis
isomers and the rate of thermal relaxation of azobenzene-benzoylphenylureas
derivatives
most commonly-used strategy is to decorate a bioactive precursor
by
a
photosensitive moiety.13 The biggest challenge is the
maintenance of precursor’s activity when introducing large
photoresponsive structural components. Therefore, proper
selection of photoswitchable units, anchor points and attachment
of spacer are needed to take into careful consideration based on
deep analysis of structure activity relationships.13 The basic skeleton
of BPU insecticides contains three primary fragments: the middle
urea moiety, the benzoyl and phenyl units at both ends (Figure
1).18,26 We used azobenzene here as the photoswitchable elements
due to its widest application, easy chemical modification and good
photochemical properties.27 Azobenzene was appended at both
ends of BPUs, respectively, as a replacement of benzoyl or phenyl
parts generating two series of photoswitchable benzoylphenylurea-
azobenzene analogues (Scheme 1).
λ
π-π*
Nonirradiated
λ
n-π*
irradiated
Compd.
t1/2 (h)
max
max
(trans, nm)
(cis, nm)
trans:cis
89:11
trans:cis
51: 49
A1
A2
A3
B1
B2
B3
B4
B5
B6
B7
332
430
31
345
87
315
330
328
315
347
357
321
370
311
87:13
89:11
90:10
89:11
86:14
82:18
87:13
99:1
435
436
444
430
437
440
449
455
429
68:32
76:24
16:84
14:86
7:93
59
292
128
88
17:83
44:56
---
29
0.3
251
Synthesis. Two synthetic methodologies were adopted here
according to structural features of the target compounds.
Isocyanate 2 and acyl isocyanate 7 were prepared from aniline 1
and 2,6-difluorobenzamide 6 through reacting with triphosgene,
98:2
41:59
Taking wavelength at isosbestic point as detection wavelength of
HPLC, the percentage contents of cis and trans configurations were
determined. The trans isomer was dominant for all the compounds
before irradiation with percentage ranging from 82% to 99% (Table
1). When subjected to light irradiation at 365 nm, different
isomerization efficiency was observed for different compounds,
which correlated closely with substituent patterns and positions.
Compounds A1-A3 showed low conversion rates, indicating that
replacement of phenyl at this position do not favor the
transformation from trans to cis. Irradiation of B1-B4 resulted in the
production of more than 80% cis-isomers. Half-life time of thermal
respectively. Oxidation of aniline
1
by oxone provided
nitrosobenzene compounds 3, which were then reacted with
phenylenediamine 8 or aminobenzamide 4 through Mills reaction
generating amide- or amine- substituted azobenzene analogues 5
or 9, respectively. Finally, condensation of azobenzene 5 with 2, or
9 with 7 furnished target compounds A1-A3 and B1-B7, respectively
(Scheme 2).
Optical Properties. The photoisomerization properties of
synthesized compounds were investigated by UV-Vis and HPLC
analysis. The maximum absorbance wavelength (λmax), trans and cis-
2 | J. Name., 2012, 00, 1-3
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relaxation of cis-isomers was calculated according to absorbance control over insecticidal activity. The activity of B3 was about 5-fold
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DOI: 10.1039/C6OB02813F
changes over time. The thermal stability of cis-isomers varied a lot lower than that of teflubenzuron.
with half-life times ranging from 0.3 h to 345 h. Testing trans:cis Sulfonylurea Receptor Affinity. BPUs inhibit the chitin synthesis. It
ratios of B6 using presented method failed due to its fast cis-to- is now generally accepted that the effects of BPUs are dependent
trans isomerization (t1/2 = 0.3 h) caused by the strong electron- on their interactions with sulfonylurea receptors and therefore
withdrawing effects of nitro substituent. Considering isomerization inhibit the chitin synthesis.20 To investigate activity difference in the
efficiency and thermal stability, compounds B2-B4 were most target level, the activity difference of B3 before and after irradiation
suitable for further activity evaluation.
to cockroach SUR was evaluated according to previously reported
procedures.22,23,28 Glibenclamide was used as comparison as it is a
Table 2. Insecticidal activity against armyworm (Mythimna separate Walker) of potent ligand of SUR. The IC50 of trans-B3 is about 13.01 μM, which
target compounds.
is a bit lower than that of Glibenclamide (IC50 = 24.94 μM) (Figure 2,
right). After irradiation, about two-fold activity enhancement (IC50
24.45 μM) was observed, which is similar with the activation trend
obtained by in vitro assay against armyworm (Mythimna separate).
Mortality (500 mg L-1, %)
Nonirradiated Irradiated
=
Compd.
A1
0
0
Table 3. Insecticidal activity evaluation of B3 against armyworm (Mythimna
separate Walker)
A2
0
0
0
0
Mortality (%) or LC50 (mg L-1)
B3
A3
mg L-1
Nonirradiated
100
Irradiated
100
B1
0
0
100
B2
75
100
0
59
100
0
50
100
86
100
100
96
B3
10
B4
3
56
B5
0
0
2
32
87
B6
B7
0
---
0
1
0.5
9
73
0
2
55
teflubenzuron
100
LC50
2.766
0.08
0.422
---
teflubenzuron
Insecticidal Activity. BPUs are extremely active to the larvae but
less toxic to the adult by inhibiting chitin synthesis. The efficacy
difference of synthesized photoswitchable BPUs was evaluated
against armyworm (Mythimna separate) larvae (Table 2).
Replacement of benzoyl end with azobenzene led to complete loss
of the activity irrespective of irradiation or not. This observation
was in accordance with previous SAR studies in which the optimal
substituents on benzoyl end are two ortho-fluorine or one ortho-
chlorine.18 Any other modifications would have detrimental effects
on the activity. However, the phenyl end was much tolerant to
decorations and even permitted large substituents such as diphenyl
ether unit. The requisite for retaining high activity upon chemical
modifications at phenyl part is the maintenance of high
hydrophobicity and electron-withdrawing ability. Therefore,
appending of azobenzene at phenyl position generate the ortho-,
meta- and para-substituted BPU-azobenzene derivatives B1, B2 and
B3, respectively. Installation position at phenyl part has significant
influence on the activity. Ortho-compound B1 was inactive and
meta-one B2 had moderate activity. Para-substituted compound B3
showed best activity with 100% mortality at 500 mg/L for both
photo isomers. Further activity screening proved a 6-fold activity
increase upon irradiation of UV light (365 nm) (Table 3). This
indicated that B3 can be activated by light which enables the optical
40
20
Glibenclamide
B3 (dark)
B3 (light)
40
20
0
0
2
4
6
0
5
10
15
1-NPN (μM)
Ligand (μM)
Figure 2. Binding of 1-NPN ligand to SUR (left) and inhibition of 1-NPN to SUR
binding of glibenclamide and B3 before and after irradiation (right).
Conclusions
In conclusion, we have developed photoswitchable BPU
insecticides via coupling of azobenzene with core BPU
structure. Replacement of phenyl part of BPU with para-
substituted azobenzene maintained the high activity of BPU
and conferred photoswitchable properties. This is the first
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photoresponsive BPU insecticide described to data. Bioassays
towards armyworm and SUR revealed that the designed
photoswitchable BUP had good activity in the dark, which can
be converted into the more active form upon exposure to UV
light. As small photoresponsive molecules have potentials for
spatiotemporal activity modulation, we anticipate that such
photoswitchable insecticide will find applications in optical
control over sulfonylurea receptor activity or insect behaviors
with high specificity in the near future, which is now in
progress in our lab.
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Acknowledgements
This work was financial supported by National Natural Science
Foundation of China (21372079, 21472046), Shanghai Pujiang
Program (14PJD012) and Science and Technology Commission
of Shanghai Municipality (16391902300).
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One prepared azobenzene-benzoylphenylurea can be activated upon irradiation of UV light, which
shows 6-fold and 2-fold activity difference to armyworm (Mythimna separate).