Inhibition of chitin synthesis by 5-benzoylamino-3-phenylisoxazoles with various substituents at two benzene rings and their larvicidal activity

Abstract

N-(3-Phenylisoxazol-5-yl)benzamides (5-benzoylamino-3-phenylisoxazoles: IOXs) with various substituents at two benzene rings were synthesized, and the chitin synthesis inhibition was measured in the cultured integumentary system of Chilo suppressalis. Larvicidal effects against C. suppressalis and Spodoptera litura were also examined, and the larvicidal activity in terms of the 50% lethal dose (LD₅₀) was determined for some compounds. Among IOXs with various substituents at the benzoyl moiety, 2,6-difluoro-substituted (2,6-F₂) benzoyl analogs showed the highest chitin synthesis activity. The larvicidal activities against C. suppressalis and S. litura were 1/138 and 1/35 that of diflubenzuron, a representative benzoylphenylurea-type insecticide, respectively. In a further study, 2,6-F₂ benzoyl analogs with various substituents at the phenyl moiety, such as Br, CF₃, CN, OEt, Ph, and alkyls (CH₃, Et, i-Pr, n-Bu, and t-Bu), were synthesized, and their chitin synthesis inhibition in the Chilo integument and their larvicidal activity against S. litura were quantitatively measured. The introduction of bulky CF₃ and t-Bu at the phenyl moiety of 2,6-F₂ benzoyl analog favorably enhanced the larvicidal activity against S. litura.

Introduction

Since insects grow by repeated molting, molting disruptors can be insecticides. This type of insecticide is categorized as an insect growth regulator (IGRs),¹⁾ in which ecdysone agonists, juvenile hormone agonists, and chitin synthesis inhibitors (CSI) are included. About half a century ago, benzoylphenylurea (BPU)-type compounds (Fig. 1) were found to inhibit chitin synthesis in insects.^2–5) The first BPU compound published was Du19111 (Fig. 1)²⁾; the structure–activity relationship was then intensively studied to develop diflubenzuron, an agricultural insecticide (Fig. 1).⁶⁾

Fig. 1. General and representative structures of BPUs.

After the discovery of diflubenzuron, various BPUs were developed as insecticides/acaricides, most of which have a 2,6-F₂ benzoyl (A-ring) moiety.^5,6) One BPU with a 2,6-F₂ benzoyl moiety, chlorfluazuron, was discovered by ISHIHARA SANGYO KAISHA, LTD. in Japan (Fig. 1).⁷⁾ The substitution at the phenyl (B-ring) moiety of chlorfluazuron is not only at the para- but also the meta-position. Lufenuron and teflubenzuron, with multiply substituted phenyl moieties, are also registered as insecticides/acaricides (Fig. 1).^5,8)

After the development of various BPUs, structurally different CSIs, such as LY131215,⁹⁾ buprofezin¹⁰⁾ and etoxazole,¹¹⁾ (Fig. 2) were reported. The benzoyl moiety of LY131215 is substituted with 2,6-(OCH₃)₂, and the benzene ring of buprofezin is unsubstituted. With respect to etoxazole, the 2-phenyl ring has 2,6-difluoro, and the 4-phenyl ring has 2-ethoxy-4-tert-butyl substituents.

Fig. 2. Structures of various CSIs.

Previously we synthesized various thiadiazole (TD)-type compounds (Fig. 3) and quantitatively analyzed the substituent effect at both the benzoyl (X₁, X₂) and phenyl moieties.¹²⁾ As a result, 2,6-F₂ substitution at the benzoyl moiety was detrimental to the activity, but 2,6-(OCH₃)₂ and 2-OCH₃ substitutions were the best benzoyl substitutions in TDs, which is different from that of BPUs. Etoxazole analogs with various substituents at both benzene rings were synthesized and the substituent effects were quantitatively analyzed, resulting in the conclusion that molecular hydrophobicity is important for the acaricidal activity.¹³⁾

Fig. 3. Structures of thiadiazole (TD), isoxaben, and isoxazole (IOX).

We recently reported that compounds with an isoxazole (IOX) ring,¹⁴⁾ which were designed from the structure of cellulose synthesis inhibitor isoxaben¹⁵⁾ and TD-type chitin synthesis inhibitors,^9,12) inhibit chitin synthesis (Fig. 3). With further study, the substituent effect at the phenyl moiety (B-ring) of IOX with 2,6-dimethoxybenzoyl moiety on the chitin synthesis inhibition was quantitatively analyzed using the classical quantitative structure–activity relationship (QSAR).¹⁶⁾ QSAR results indicated that the less-bulky substituent at the B-ring is favorable to the inhibition of chitin synthesis. With respect to the hydrophobicity, there is an optimum value. This QSAR result is like that for the phenyl ring (B-ring) of BPUs.

2,6-F₂ substitution is favored as the A-ring substitution for BPUs, and most of the BPUs marketed in the agricultural field carry a 2,6-F₂-substituted benzoyl (A-ring) moiety as shown in Fig. 1. On the other hand, it is unknown whether the substituent effect at the A-ring moiety of IOXs are same as that of BPUs. The aim of this study is to examine the substituent effects at the A-ring moiety of IOXs by measuring their chitin-synthesis inhibition in the cultured integument.¹⁷⁾ With further study, 2,6-F₂ benzoyl IOX analogs with various substituents at B-ring were synthesized to measure the activity. The larvicidal effect of synthesized IOXs was also examined against Spodoptera litura and Chilo suppressalis.

Materials and methods

1. Chemicals

Compounds 1–3, 5–16 were newly synthesized in this study according to the previously reported method (Supplementary materials).^14,16) Structures were identified by ¹H-NMR (AVANCE-400, BRUKER; Supplementary materials), elemental analysis, and HRMS (Orbitrap Exploris 240, Thermo Fisher Scitentific; Supplementary materials). Elemental analysis was performed at the Center for Organic Elemental Analysis of Graduate School of Pharmaceutical Sciences, Kyoto University. Melting points were measured by Yanaco melting point apparatus (Yanaco, Kyoto, Japan). Synthetic procedures are shown in the Supplementary files. IOXs were dissolved in DMSO to perform the bioassay. Piperonyl butoxide (PB) was purchased and dissolved in EtOH to prepare 100 mM stock solution.

2. Chitin synthesis inhibition

Chitin synthesis inhibition was quantitatively measured using the Chilo cultured integument system reported previously.^14,16,17)

3. Larvicidal activity test

Eggs of C. suppressalis were kindly given by Sumitomo Chemical. An artificial diet¹⁸⁾ containing PB (100 µM) for C. suppressalis was prepared (ϕ7 cm Petri dish). The 3rd instar larvae were put on the PB-containing diet 1 hr before the application of compounds. Compounds were topically applied to the dorsal part of larvae as a DMSO solution. After rearing for 5 days, the dead larvae were counted to determine the mortality. The dose–response relationship was drawn for compound 5 as shown in Supplementary Fig. S1, and the 50% lethal dose (LD₅₀ [mmol/insect]) was determined (Table 1). Since the procedure for the larvicidal test against C. suppressalis is cumbersome, the larvicidal test against C. suppressalis was not continued.

Table 1. Inhibition of chitin synthesis and larvicidal effect by IOXs with various substituents at the benzoyl (A-ring) moiety.

No.	X1	X2	Inhibition of chitin synthesisa) [pIC50 (M)]	Larvicidal activity (with PB)b) [pLD50 (mmol/insect)]
				C. suppressalis	S. litura
1	H	H	<4.00 (38%)	<4.00 (0%)	<3.70 (0%)
2	F	H	5.40	<4.00 (29%)	3.88
3	OMe	H	<4.00 (39%)	<4.00 (34%)	<3.70 (0%)
4	OMe	OMe	5.83	<4.00 (25%)	<3.70 (0%)
5	F	F	7.04	4.50	4.99±0.15
	Diflubenzuron		7.72c)	6.64d)	6.53e)

^a) Values in parentheses mean the inhibition % for the chitin synthesis at the corresponding maximum concentration.

^b) Values in parentheses mean the mortality at the corresponding maximum dose.

^c) From Ref. 20.

^d) From Ref. 21.

^e) From Ref. 22.

Eggs or larvae of S. litura were purchased from Sumika Technoservice (https://www.sc-sts.co.jp/). An aliquot (1 µL) of PB (100 mM EtOH solution) was applied to the dorsal part of 3rd instar larvae of S. litura before the application of test compounds. After keeping larvae for 1 hr, DMSO solutions (1–2 µL) of test compounds was topically applied on the dorsal part of larvae. Ten larvae were used for each dose. A 50% lethal dose (LD₅₀) was determined from each dose–response relationship, and the reciprocal log value of LD₅₀, pLD₅₀, was used as the index of larvicidal activity; these values are listed in Table 1 and 2. The results of larvicidal test and the dose–response relationship are shown in Supplementary Fig. S2.

Results

Previously, we examined the substituent effect at the phenyl moiety (B-ring) on the inhibition of chitin synthesis for the 2,6-(OMe)₂-substituted compounds.¹⁶⁾ In this study, the substituent effect at A-ring was examined. As shown in Table 1, 2,6-F₂-substituted compound (5) was 18 times more potent than the corresponding 2,6-(OMe)₂-substituted compound (4), but it is 1/5 that of the corresponding BPU, diflubenzuron. The pIC₅₀ value was determined for 2-F analog (2), but unsubstituted (1) and 2-OMe benzoyl (3) analogs were very weak as shown in Table 1. The substituent effect at the A-ring moiety of IOXs was close to that of BPUs.¹⁹⁾

The most potent compound 5 in the chitin synthesis inhibition showed larvicidal activity against C. suppressalis and S. litura, and then the LD₅₀ values of compound 5 were determined from each dose–response curve (Supplementary Figs. S1 and S2). The larvicidal activity of compound 5 was 1/138 and 1/35 that of the representative BPU, diflubenzuron, against C. suppressalis and S. litura, respectively. Since S. litura is more sensitive than C. suppressalis in the larvicidal test, S. litura was used in the following larvicidal test. The LD₅₀ value of 2-F analog (2) against S. litura was determined, but unsubstituted (1), 2-OMe (3)- and 2,6-(OMe)₂ (4)-substituted benzoyl analogs were inactive as shown in Table 1.

Since 2,6-F₂ benzoyl analog (5) is more effective (over 10 times) than the corresponding 2,6-(OMe)₂ benzoyl-type compound (4) in chitin-synthesis inhibition, the substituent effects at A- and B-rings were thought to be close to those of BPUs. Thus, 2,6-F₂ benzoyl analogs with various B-ring moieties were synthesized and chitin-synthesis inhibition was quantitatively measured as shown in Table 2. Substituents such as CH₃ (9) and Et (10) were as effective as Cl (4). Although CF₃ (8) was unfavorable as a B-ring substituent in 2,6-(OMe)₂ benzoyl series, it was favorable in 2,6-F₂ benzoyl series.

Table 2. Inhibition of chitin synthesis and the larvicidal activity of 2,6-F₂ benzoyl-type compounds with various substituents at the B-ring^a).

Compounds		Inhibition of chitin synthesis pIC50 (M)	Larvicidal activity [pLD50 (mmol/insect)]b)
No.	Y		C. suppressalis	S. litura
6	H	5.90	n.d.	3.59
5	Cl	7.04	4.50	4.99 ±0.15
7	Br	6.76	n.d.	5.25±0.37
8	CF3	6.97	< 5.00 (18%)	5.44±0.24
9	CH3	6.93	n.d.	<4.70 (20%)
10	Et	6.88	n.d.	5.23±0.32
11	i-Pr	n.d.	n.d.	5.55±0.19
12	n-Bu	6.44	n.d.	< 4.70 (10%)
13	t-Bu	n.d.	n.d.	5.32±1.11
14	OEt	n.d.	< 5.00 (0%)	5.41±0.18
15	CN	6.28	n.d.	<5.70 (10%)
16	Ph	n.d.	n.d.	5.25±0.03

^a) n.d. not determined.

^b) Values in parentheses are mortality at the corresponding doses.

Since 2,6-F₂ benzoyl analogs with various B-ring moieties showed high inhibition activity in chitin synthesis, their larvicidal test was performed against S. litura (Table 2). The insecticidal activity in terms of pLD₅₀ was determined for compounds with Br (7), CF₃ (8), Et (10), i-Pr (11), t-Bu (13), OEt (14), and Ph (16); however, the enhancement of the larvicidal activity relative to Cl (5: pLD₅₀=4.99) was only 2–3 times (pLD₅₀=5.23–5.55). Interestingly, the introduction of CF₃ (8) was detrimental for the inhibition of chitin synthesis in the 2,6-(OMe)₂ benzoyl series,¹⁶⁾ but it was effective for the larvicidal activity in the 2,6-F₂ benzoyl series. Compound 8 (CF₃) showed slightly higher larvicidal activity than compound 5 (Cl). The introduction of another bulky substituent t-Bu (13) that is unfavorable in the 2,6-(OMe)₂ benzoyl series¹⁶⁾ was also effective in the 2,6-F₂ benzoyl series. Compound 12 (n-Bu) and 15 (CN), which had lower chitin synthesis–inhibition activity, did not show larvicidal activity against S. litura. On the other hand, compound 9 (CH₃), which had strong chitin synthesis-inhibitory activity, did not show larvicidal activity against S. litura. The activity of unsubstituted compound 6 was 1/20 that of compound 5 in chitin synthesis inhibition, and 1/30 that of compound 5 in larvicidal activity against S. litura. None of the 2,6-(OMe)₂ benzoyl-type compounds with various substituents at the 3-phenyl moiety (B-ring)¹⁶⁾ showed larvicidal activity against S. litura. Mortality against S. litura is 0% even at the highest dosage (dose=0.0002 mmol/insect: log 1/dose=3.7).

Discussion

As shown in Table 1, 2,6-F₂ substitution at the A-ring of IOXs is better than 2,6-(OMe)₂ and 2-OMe substitutions, which is close to the structure–activity relationship for BPUs.¹⁹⁾ Even though IOXs were designed from isoxaben (Fig. 3) and LY131215⁹⁾ (Fig. 2), both of which have 2,6-(OMe)₂ substitutions at the benzoyl (A-ring) moiety, 2,6-(OMe)₂ substitution was not the best for IOXs. Unsubstituted (H: A-ring) compounds were inactive in all BPUs, TDs, and IOXs, but the substituent effects of 2-OMe and 2,6-F₂ were different among them. 2-OMe (3) substitution was the most effective in TDs, but it is detrimental in IOXs¹⁶⁾ and BPU.¹⁹⁾ The structure–activity relationships for the benzoyl moiety (A-ring) are summarized in Table 3. Electron-withdrawing groups seem to be better in BPUs and IOXs, and electron-donating groups seem better in TDs. Since the substituents at the benzoyl moiety (A-ring) of IOXs are limited, it is difficult to discuss the difference in substituent effects among three series (BPU, TD, and IOX).

Table 3. Comparison of substituent effects on the in vitro activity among three different chemistries, BPUs, TDs and IOXs.

X1	X2	Inhibition of chitin synthesis [pIC50 (M)]a)
		BPU	TD	IOX
H	H	<6.00 (0%) b)	<5.30 (10%)	<4.00 (38%)
F	H	6.82	6.14	5.40
OMe	H	6.07, <6.00 (26%)b)	7.27	<4.00 (39%)
OMe	OMe	7.22	7.23	5.83
F	F	7.69	<5.30 (42%)	7.04

^a) Values in parentheses are inhibition % at the corresponding concentrations.

^b) Inhibition of cuticle formation. The thickness of new cuticle was measured under microscope.

Previously, we demonstrated that the intramolecular hydrogen bond can be made between OMe and NH at the benzoyl (A-ring) moiety in BPU, which is detrimental for the inhibition of cuticle formation.^19,23) Thus, we proposed that the coplanarity between benzene ring and amide bond is not favored for the activity. On the other hand, the introduction of substituents at both ortho-positions of the benzoyl ring (A-ring) makes the perpendicular conformation between benzene ring and amide bond as shown in Fig. 4, which is favored for the activity. As shown in Fig. 4, the intramolecular hydrogen-bond formation between OMe oxygen and amide NH is thought to be possible also in IOX, suggesting that compound 2 (2-OMe) is inactive. On the other hand, the 2-OMe analog of TD is very potent, which is different from that of BPU and IOX. The formation of an intramolecular hydrogen bond may not be possible for TD analogs from the present structure–activity relationship study. In the case of TD analogs, the amide bond –CONH– possibly tautomerizes to –C(=N)OH and formulate the hydrogen-bond to S, which may be difficult in BPU and IOX analogs as shown in Fig. 5.

Fig. 4. Intramolecular hydrogen bond formation for 2-OMe analogs of BPU (A) and IOX (B). The di-ortho substituted benzene ring is perpendicular to the amide structure.

Fig. 5. Tautomerization of TD compounds and possible intrarmolecular hydrogen bond formation.

In our previous study, the substituent effect at the phenyl (B-ring) moiety of 2,6-(OMe)₂-type IOXs was quantitatively analyzed as shown in Eq. 1.¹⁶⁾

$$\begin{array}{l}{\text{pIC}}_{50}=1.612E_{\text{s}}+2.728\pi -0.800{\pi }^2+5.949 \hfill \\ n=15,s=0.429,r=0.909,{\text{F}}_{3,11}=17.488,{\pi }_{\text{opt}}=1.7\hfill \end{array}$$ (1)

Equation 1 means that the bulky substituents at the para-position in terms of E_s²⁴⁾ were unfavorable in the 2,6-(OMe)₂ benzoyl-type IOX series of compounds. With respect to hydrophobicity (π), there is an optimum value (π_opt=1.7). Therefore, the binding pocket surrounding the substituent of the phenyl moiety is hydrophobic and small. We constructed the three-dimensional structure of insect CHS1 using AlphaFold2 and performed docking simulations with IOXs as ligands against this structure (Supplementary material Figs. S3–S6). As a result, IOXs were estimated to bind to a large cavity that serves as the pathway for chitin polymers. In the estimated binding poses, the para-substituents of the B-ring were found to be in contact with a narrow space composed of several hydrophobic residues, which was consistent with the results of Eq. 1. However, the substituent effect at the phenyl moiety (B-ring) of 2,6-F₂ benzoyl-type IOXs (Table 2) is very different from that of 2,6-(OMe)₂-type compounds. The introduction of bulky substituents such as CF₃ and t-Bu at the B-ring of IOXs was detrimental in 2,6-(OMe)₂ benzoyl-type IOXs,¹⁶⁾ as seen from Eq. 1. However, the high activity of 2,6-F₂ benzoyl analogs with compounds with bulky substituents such as CF₃ (8) and t-Bu (13) cannot be explained by the same docking pose. The docking results predicted that the A-ring moiety of 2,6-(OMe)₂-substituted IOXs is positioned around the enzyme’s active site. On the other hand, the binding poses of 2,6-F₂-substituted analogs were not uniquely determined (Supplementary Fig. S7), and docking could not explain the differences in activity due to the introduction of B-ring substituents. It is possible that the enzyme’s active site is flexible and mobile, which may have prevented the docking structure from adequately reproducing the binding of 2,6-F₂-substituted analogs.

In conclusion, 2,6-F₂ substitution at the A-ring moiety is more favorable than 2,6-(OMe)₂ for the inhibition of chitin synthesis. The substituent effect at the A-ring moiety of IOX was like that of BPU. The 2,6-F₂ analog with 4-Cl (5) at the 3-phenyl (B-ring) moiety also showed significant larvicidal activity against C. suppressalis and S. litura. By changing Cl at the B-ring of compound 5 with other substituents, some compounds showed significant larvicidal activity against S. litura. Although the introduction of bulky substituents such as CF₃ and t-Bu is detrimental in 2,6-(OMe)₂ benzoyl-type IOXs, it is favorable in 2,6-F₂ types. Even though the reason for the different structure–activity relationship for the B-ring moiety between 2,6-(OMe)₂ and 2,6-F₂ benzoyl type is unknown, QSAR study for the expanding set of compounds and other computational approaches such as docking simulation and MD will solve the molecular interaction between IOXs and CHS1 in the future.

Note

“5-Benzoylamino-3-phenylisoxazole” is used in the title as the naming of a core structure, instead of the IUPAC nomenclature “N-(3-phenylisoxazol-5-yl)benzamide”.

Electronic supplementary materials

The online version of this article contains supplementary materials (Supplemental Fig. S1–S7), which are available at http://www.jstage.jst.go.jp/browse/jpestics/.