Discovery of flometoquin, a novel quinoline insecticide

Abstract

Flometoquin, 2-ethyl-3,7-dimethyl-6-[4-(trifluoromethoxy)phenoxy]quinolin-4-yl methyl carbonate, is a novel insecticide with a structurally unique phenoxy-quinoline. It was discovered in 2004 by the collaborative research of Nippon Kayaku and Meiji Seika Kaisha, Ltd. (currently, Mitsui Chemicals Crop & Life Solutions, Inc.). The compound demonstrates strong and quick insecticidal action against a variety of thrips species at the nymphal and adult stages through contact and feeding activity, which could minimize crop damage and economic loss by insect pest species. In addition, flometoquin is safe for tested non-target arthropods, which makes it suitable for controlling the insect pests mentioned above under Integrated Pest Management (IPM) programs. Here, we describe a structure–activity relationship study from lead generation to the discovery of flometoquin and its insecticidal properties, including knockdown activity and effects against non-targeted arthropods.

Introduction

Thrips, one of the most commercially important pests, are represented by western flower thrips (Frankliniella occidentalis), melon thrips (Thrips palmi), and onion thrips (Thrips tabaci).^1,2) Without adequate control, these insect pests can cause severe damage by directly feeding on crops, thereby reducing the quality and transmitting viruses causing serious damage to crops.^3,4) Thrips management is generally categorized into cultural, biological, and chemical control, but incorporating a biological control into the cropping system using pesticides has been currently recommended as the most effective strategy. But insecticides that can be used in combination with biological control are limited. Chemical control still remains a necessary tool^5,6) and new active ingredients with novel modes of action (MoA) that are safe for non-target organisms are constantly needed for controlling these species.

To address this problem, we initiated a collaborative research study between Nippon Kayaku and Meiji (currently, Mitsui Chemicals Crop & Life Solutions, Inc., referred to as “Meiji” in this paper), with the goal of creating insecticides with a novel chemical class that can act quickly and are highly effective against thrips. In our preliminary studies, we had found that cpd1 and its derivatives (Fig. 1) have some insecticidal activity against diamond backmoth (Plutella xylostella) and F. occidentalis at high doses. Although the activity levels of these derivatives were not satisfactory, they attracted our attention for their distinctive knockdown activity. In addition, insecticidal quinoline derivatives were structurally unknown and quite unique; thus, we focused on them as a promising chemical class with a novel MoA. The schematic pathway of our discovery strategy leading to the identification of flometoquin is shown in Fig. 1.

Fig. 1. Schematic pathway for the discovery strategy from lead compound to flometoquin.

First, we set cpd1 as the lead compound for structural optimization. We subsequently attempted to modify the substituents from position 2 to 5, followed by the optimization of substituents at position 4′ in a 6-phenoxy ring. In light of the selective toxicity and insecticidal activity we found, we set cpd2 as the second lead compound group for further investigation and explored various derivatives in reference to combinations R₃/R₄ and R₅. As a result of the optimization of cpd2, we found two fluoromethoxy derivatives to be highly effective against all test species, and discovered flometoquin (cpd3) through field trials. Here, we describe the discovery, chemistry, structure–activity relationship (SAR), and some biological features of flometoquin.

Materials and methods

1. Preparation of compounds

Melting points were measured with a Mettler FP80 melting point apparatus and left them as uncorrected values. Chemical structures were confirmed by ¹H-NMR spectroscopy using a JEOL JNM-Λ400 FT-NMR system at 400 MHz with tetramethylsilane as the internal standard.

1.1. Synthetic route of flometoquin and its derivatives for SARs

Derivatives for SARs were obtained via the cyclization of aniline, as shown in Fig. 2. Isomers of the 5- (cpd6) and 7-position (cpd7) on the quinoline ring were separated from the mixture (cpd5) of isomers that were simultaneously generated from m-substituted aniline (cpd4).

Fig. 2. Synthetic pathway of flometoquin at the discovery stage.

1.2. Synthesis of 2-ethyl-3,7-dimethyl-6-[4-(trifluoromethoxy)phenoxy]quinolin-4-yl methyl carbonate (flometoquin)

A solution of 3.4 g of 4-(4-trifluoromethoxyphenoxy)-3 trifluoromethyl-aniline, 3.5 g of ethyl 2-methyl-3-oxopentanoate, and 2.1 g of p-toluenesulfonic acid dissolved in 100 mL of xylene was heated under reflux for 10 hr. The reaction solution was cooled, and the precipitated crystals were then collected by filtration to give 6.0 g of a mixture of 2-ethyl-3-methyl-4-hydroxy-6-(4-trifluoromethoxyphenoxy)-5-trifluoromethyl-quinoline with 2-ethyl-3-methyl-4-hydroxy-6-(4-trifluoromethoxyphenoxy)-7-trifluoromethyl-quinoline. Next, 50 mL of dimethylacetamide was added to 6.0 g of the crystals, and then 1.7 g of 60% sodium hydride and 4.6 g of methyl chloroformate were added at 0°C. The mixture was stirred at 4 to 24°C for 1.5 hr, and 100 mL of toluene and 100 mL of distilled water were then added to the reaction solution. The organic layer was washed with water and was then concentrated under reduced pressure. The crude product was purified by column chromatography on a silica gel (BW300, manufactured by Fuji Silysia Chemical, Ltd., solvent: n-hexane/ethyl acetate) to give 0.63 g of 4-methoxycarbonyloxy 2-ethyl-3-methyl-6-(4-trifluoromethoxyphenoxy)-5 trifluoromethyl-quinoline (yield 12.9%) and 2.00 g of 4-methoxycarbonyl-2-ethyl-3-methyl-6-(4-trifluoromethoxyphenoxy)-7-trifluoromethyl-quinoline (yield 40.9%). Reaction yields were not optimized in this study; thus, the synthetic pathway for manufacturing should be quoted from our process patents.^7,8) The other derivatives were synthesized using similar methods and their melting points and ¹H-NMR spectrum data are described in Table S1.

2. Biological assay

2.1. Insect pests and non-target arthropods

We used laboratory strains of P. xylostella, common cutworm (Spodoptera litura), sweet potato whitefly (Bemisia tabaci), F. occidentalis, T. palmi, and T. tabaci. Non-target arthropods used in each test are shown in Table S2.

2.2. Insecticidal activity for SARs and biological properties of flometoquin

In the SAR study, each derivative was formulated as a 5% emulsifiable concentrate (EC) according to our previous patent,⁹⁾ and test solutions at various concentrations were prepared by diluting them with tap water. Bioassays were conducted according to our laboratory protocol (Table S3). Based on the percent knockdown and mortality (which were corrected when necessary) and using Abbott’s formula,⁹⁾ EC₉₀ values were determined 2–5 days after treatment. Living insects that dropped from leaves or were moribund were included in the number of knockdowns, while completely motionless insects were counted as dead. Corrected percentages of knockdowns and mortality were defined by the following formula:

  

  

where A is the number of survivors in the control group, B is the number of survivors in the treatment group, and C is the number of survivors (excluding knockdown survivors) in the treatment group. The biological properties of flometoquin were demonstrated as LC₅₀ values in the same protocol of SARs (refer to Table S3). In order to obtain LC₅₀ values against each insect pest, test solutions of flometoquin at four concentrations were prepared by diluting a 100 g/kg suspension concentrate (SC: FINESAVE) formulation. The dose-response data was calculated based on the corrected percent mortality and assessed by SAS-probit analysis.¹¹⁾

2.3. Knockdown assay

Similar to the method shown for determining the biological properties of flometoquin (section 2.2.), test solutions of reference compounds were also prepared with commercially available product forms of 150 g/L tolfenpyrad (Hachi-Hachi), 250 g/kg spinosad (SPINOACE), 113 g/L spinetram (DIANA), and 103 g/L cyantraniliprole (BENEVIA). The time required for lethal and knockdown effects was measured and recorded at appropriate intervals for each insect; every 30 min for F. occidentalis and T. tabaci and every 10 min for T. palmi. The experimental design and evaluation method were the same as those described in section 2.2 (refer to Table S3). All experiments were replicated at least twice.

2.4. Effect of flometoquin on non-target arthropods and microbial pesticides

In order to examine the effect of flometoquin on non-target arthropods, an appropriate method for each species, including insect dipping, contact with a dry film, or insect spraying, was employed. We followed the method described in the list regarding the toxicity of pesticides on natural enemies, published by the Japan BioControl Association (JCBA).¹²⁾ The acute oral and contact toxicity of flometoquin to adult workers of honeybees and bumblebees was evaluated by the methods of OECD 213¹³⁾ and 214.¹⁴⁾ The effect of flometoquin on microbial pesticides was examined according to a previously published paper.¹⁵⁾

Results and discussion

1. Lead optimization

1.1. Activity of derivatives substituted at positions 2 and 4 (from cpd8 to cpd11)

Summary of results for SARs are shown in Table 1. Optimal substituents at positions 2 and 4 for insecticidal activity against P. xylostella were the ethyl (cpd8) and acyloxy groups (cpd10), respectively. Bulky substituents, such as butyl groups in position 2 (cpd9) and position 4 (cpd11), resulted in the reduction or loss of insecticidal activity against P. xylostella and F. occidentalis.

Table 1. Insecticidal activities of 6-phenoxy quinoline derivatives against Plutella xylostella, Spodoptera litura, and Frankliniella occidentalis

Compound No. (1st screening)	R1	R2	R3	R4	R5	EC90 (mg a.i./L)
						P. xylosotella	S. litura	F. occidentalis
cpd1 (Lead)	Me	Me	Cl	H	4′-Cl	100	>100	>200
cpd8	Et	Me	Cl	H	4′-Cl	5–20	>100	50–200
cpd9	n-Bu	Me	Cl	H	4′-Cl	>100	>100	>200
cpd10	Me	OMe	Cl	H	4′-Cl	10–20	>100	>200
cpd11	Me	t-Bu	Cl	H	4′-Cl	>100	>100	>200
cpd12	Me	Me	CF3	H	4′-Cl	<20	100	200
cpd13	Me	Me	CN	H	4′-Cl	>100	>100	>200
cpd14	Me	Me	Me	H	4′-Cl	20–100	>100	>200
cpd15	Me	Me	Cl	H	2′-Cl	100	>100	>200
cpd16	Me	Me	Cl	H	3′-Cl	20–100	>100	>200
cpd17	Me	Me	Cl	H	4′-CN	100	20–100	>200
cpd18	Me	Me	Cl	H	4′-Me	>100	>100	N.D.
cpd19	Me	Me	Cl	H	4′-F	100	>100	>200
cpd20	Me	Me	Cl	H	4′-CF3	20–100	20–100	<50
cpd21	Me	Me	Cl	H	4′-OMe	>100	>100	>200
cpd22	Me	Me	Cl	H	4′-OCF3	<20	100	<50
cpd23	Me	Me	CF3	H	4′-OCF3	1.25–2.5	2.5–5	20–50
cpd24	Me	Me	Me	H	4′-OCF3	20–100	20–100	100–200
cpd25	Me	Me	H	Me	4′-OCF3	2.5–5	20–100	100–200
cpd26	Me	Me	Me	Me	4′-OCF3	5–20	20–100	100
cpd27	Me	Me	Cl	Cl	4′-OCF3	>100	>100	20–100

N.D.: No Data, mg a.i./L: mg active ingredient/L

1.2. Activity of derivatives substituted at position 5 (from cpd12 to cpd14)

Potent substituents at position 5 for P. xylostella were trifluoromethyl (cpd12) and methyl (cpd14), and only trifluoromethyl was effective for S. litura. In the case of cyano (cpd13), insecticidal activity against all test species disappeared. In this position, although both the electron-donating and electron-withdrawing groups were generally acceptable, with some exceptions, trifluoromethyl in particular, exhibited a wider insecticidal spectrum than any other functional group. Unfortunately, there was no promising substituent for F. occidentalis, suggesting that substituent change at extra position 5 should be needed.

1.3. Activity of derivatives substituted at position 4′ of the 6-phenoxy ring (from cpd19 to cpd22)

We subsequently checked the best position of chlorine in the 6-phenoxy ring and found that the insecticidal activity of these derivatives had similar levels (cpd1, 15, and 16) for P. xylostella, and although cpd16 was slightly better than any other derivatives, it was not satisfactory. Therefore, we explored other functional groups at position 4′. To our surprise, trifluoromethyl (cpd20) and trifluoromethoxy (cpd22) were found to be remarkably active against all test species, including F. occidentalis, as compared with other substituents. In the derivatives from cpd17 to 22, activities against P. xylostella were in the following order: trifluoromethoxy (cpd22)>trifluoromethyl (cpd20)>fluoro (cpd19)>cyano (cpd17) and methyl (cpd18)>methoxy (cpd21), indicating that activity increased as the electron withdrawing groups at position 4′ of the 6-phenoxy ring strengthened. Especially, trifluoromethoxy (cpd22) and trifluoromethyl (cpd20) were also revealed to be effective against F. occidentalis.

1.4. Activity of derivatives substituted at positions 5 and 7 (from cpd23 to cpd27)

Substituent changes at positions 5 and 4′ of the 6-phenoxy ring were found to be critical key structures for activity; thus, we set trifluoromethoxy at position 4′ and investigated the combinational effects of positions 5 to 7 in this section. We found that cpd23 was better than the other compounds from cpd24 to 27, and totally demonstrated greater activity than any of them. However, 5-trifluoromethyl quinoline derivatives demonstrated high mammalian acute toxicity and phytotoxicity. Fortunately, tested derivatives that had 7-methyl (cpd25) or 5,7-dimethyl (cpd26) groups exhibited moderate activity against all test species and lower toxicity on crops and mammals than 5-triflumethyl quinoline, including cpd23. For our goal of discovering a selective candidate between insect pests and others, we focused on these two compounds and conducted structural optimization regarding the combination of substituents at positions 5 to 7 and 1′ to 6′ by setting ethyl at position 2 and acyloxy at position 4 as the second lead compound group. The results are described in the next section.

1.5. Activity of the second lead compound group and the discovery of flometoquin (from cpd28 to cpd32 in addition to cpd3)

The second lead compound group (cpd2) was highly active against not only F. occidentalis but also B. tabaci and T. palmi, which were all highly resistant to conventional insecticides around the world and are commercially important pests. Thus, we focused on these pests and structurally optimized them. We synthesized a number of compounds with substituents at positions 5–7 and the 6-phenoxy group. Representative compounds and their activities are shown in Table 2. They exhibited 5- to 10-fold higher activity against target pests than the first-generation derivatives shown in Table 1 did, and their optimal chemical structure differed depending on the pest. However, in the laboratory, cpd32 and cpd3 showed the best activity against any of the three pests, F. occidentalis, B. tabaci, and T. palmi. We subsequently conducted many studies—including field trials—to finally obtain flometoquin (cpd3).

Table 2. Insecticidal activities of 6-phenoxy quinoline derivatives against Frankliniella occidentalis, Bemisia tabaci, and Thrips palmi

Compound No. (2nd screening)	R3	R4	R5	EC90 (mg a.i./L)
				F. occidentalis	B. tabaci	T. palmi
cpd28	Me	Me	2′-Cl,4′-OCF3	5–20	1.25–2.5	20
cpd29	Me	Me	4′-OCF2CF2H	100–200	5–20	20–100
cpd30	Me	Me	4′-OCF3	5–20	<2.5	5–20
cpd31	H	Me	2′-Cl,4′-OCF3	5–20	<2.5	<5
cpd32	H	Me	4′-OCF2CF2H	5	<2.5	<5
cpd3 (Flometoquin)	H	Me	4′-OCF3	5	2.5–5	5–20

mg a.i./L: mg active ingredient/L

2. Biological features of flometoquin

2.1. Biological activity against each test species

The LC₅₀ values of flometoquin against each test species are shown in Table 3. They demonstrate that flometoquin was highly effective against third-instar larvae of P. xylostella, first-instar nymphs and adults of B. tabaci, T. tabaci and F. occidentalis, and all stages of T. palmi.

Table 3. LC₅₀ values of flometoquin against Thrips palmi, Thrips tabaci, Frankliniella occidentalis, Bemisia tabaci and Plutella xylostella

Test pest species	Test method	Stage	LC50 (mg a.i./L)	95%CL
Thrips palmi	Foliar spraying	1st nymph	1.35	1.08–1.68
		2nd nymph	1.16	0.82–1.49
		Adult	1.40	1.87–3.00
Thrips tabaci	Leaf dipping	1st nymph	0.40	0.29–0.61
		Adult	0.49	0.38–0.73
Frankliniella occidentalis	Leaf dipping	1st nymph	0.47	0.36–0.60
		Adult	0.50	0.42–0.59
Bemisia tabaci	Foliar spraying	1st nymph	0.85	0.71–1.03
		Adult	0.79	0.73–0.87
Plutella xylostella	Leaf dipping	3rd larvae	1.47	0.59–2.38

mg a.i./L: mg active ingredient/L

2.2. Knockdown effect against three representative thrips species

We assessed the quick knockdown effects against three pest thrips species: F. occidentalis, T. palmi, and T. tabaci. As shown in Fig. 3, in each test, flometoquin had a fairly quick-acting effect as compared with the two commercial standards, which are conventional insecticides that reduce tospovirus transmission by thrips.^16–19) When applied to the first instar nymph and adults of F. occidentalis, tolfenpyrad exhibited a quick knockdown effect at the same level as flometoquin, but its nymph and adult mortality rates were 30% and 7%, respectively, which were remarkably less than those of flometoquin (100% and 81%) after 180 min of treatment. Spinosad demonstrated high knockdown activity against adults, but the speed of knockdown was drastically slower, and the activity level after 30 min (26%) was much less than that of flometoquin (100%) (Fig. 3A and B). When used on adults of T. palmi, the activity levels of flometoquin for lethal effect (10 min after treatment) and knockdown effect (90 min after treatment) were 57% and 46%, respectively, which indicate more potency than those of spinetram and cyantraniliprole (Fig. 3C). In T. tabaci adults, flometoquin exhibited a complete knockdown effect within 60 min, although no insecticide tested, including spinetram and cyantraniliprole, had any lethal action within 240 min (Fig. 3D). A previous study reported that only later instar nymph and adult thrips infesting virus-infected host plants after a latent period can transmit the virus.²⁰⁾ Therefore, flometoquin, available for controlling both nymphal and adult thrips with a quicker action than tested commercial standards including spinetram and cyantraniliprole, might contribute to reducing the transmission of viruses such as Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV). Further evidence with a detailed study focusing on thrips vector control through application of flometoquin will be reported in another paper.

Fig. 3. Knockdown and lethal activity against first nymphs (A) and adults (B) of Frankliniella occidentalis and adult Thrips palmi (C) and Thrips tabaci (D). Light blue bars indicate the percentage of knockdown, and deep blue bars indicate the percentage of mortality.

3. Compatibility with integrated pest management (IPM)

The acute toxicity evaluation of flometoquin on several species of non-target arthropods and microbial pesticides is shown in Table 4, which demonstrates no adverse effects in the tests. Flometoquin was inactive against honeybees and bumblebees at the adult stage with EC₅₀ values of more than 100 µg/bee. Natural enemies were also tested in each experiment at registered application doses. The direct treatment of flometoquin on adults and eggs in each of the five predatory mite species had no adverse effect, suggesting that it would be compatible with the key natural enemies of arthropod pests in both open fields and greenhouses. Additionally, flometoquin did not exhibit any inhibitory action against the efficacy of the microbial pesticides listed. These results indicate that flometoquin would be compatible with the IPM programs.

Table 4. Compatibility of flometoquin with pollinators, natural enemies, and microbial pesticides

Category	Scientific name	Stage	Evaluation method	EC30 or *EC50 (mg a.i./L or *µg/bee)
Predatory mite	Amblyserius cucumeris	Egg & Adult	Direct spray	>100
	Phytoseiulus persimilis
	Neoseiulus californicus
	Amblyserius swirskii	Egg & Adult	Direct spray	>200
	Amblydromalus limonicus
Pollinator	Apis mellifera	Adult	Oral application	*>10
	Bombus ignites		Topical application
	Bombus terrestris	Beehives and Adults	Set beehives after foliar spraying in greenhouse	>100
	Lucilia sericata	Adult	Direct spray	>200
Parasite wasp	Aphidius colemani	Pupa	Insect dipping	>100
	Encarsia formosa	Pupa	Insect dipping	>100
		Adult	Spraying on food	>100
	Eretmocerus eremicus	Pupa	Insect dipping	>100
		Adult	Spraying on food	>100
	Diglyphus isaea	Adult	Dry film	>50
Predatory bug	Nesidiocoris tenuis	Nymph	Direct spray	>100
		Adult	Insect dipping	>100
Green lacewing	Chrysoperla carnea	Larva	Spraying on food	>100
	Chrysopa formosa	Adult	Spraying on food	>100
Microbial pesticide	Verticillium lecani	Culture	Disk diffusion test	>100
	Beauveria bassiana
	Erwinia carotovora
	Bacillus subtilis

mg a.i./L: mg active ingredient/L

Conclusion

Flometoquin was discovered as a novel class of insecticide with a structurally unique 6-phenoxy quinoline. It showed insecticidal activity against thrips, whiteflies, and diamondback moth. In particular, flometoquin demonstrated excellent insecticidal activity against a variety of species with knockdown effects considerably faster than those of available commercial standards. This suggests that flometoquin can potentially reduce virus transmission by thrips. Furthermore, it was very safe for natural enemies and, therefore, is suitable for IPM. We strongly believe that these properties of flometoquin will greatly assist domestic and global farmers struggling with resistance in thrips control.

Acknowledgements

Part of this work was conducted by the Eco-Science Corporation (Nagano, Japan) and the Japan Plant Protection Association (JPPA) (Tokyo, Japan), who greatly contributed to the toxicity evaluation relevant to non-target arthropods. TM devoted all of his energy to discovering and developing flometoquin during his lifetime. Finally, we would like to thank all of the people who were involved in the research and development of flometoquin.

Conflict of interests

The authors declare no conflicts of interest associated with this manuscript.

Electronic supplementary materials

The online version of this article contains supplementary materials (Tables S1–S3), which are available at https://www.jstage.jst.go.jp/browse/jpestics/.

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