Design, Synthesis and Insecticidal Activity of Novel Phenylurea Derivatives

A series of novel phenylurea derivatives were designed and synthesized according to the method of active groups linkage and the principle of aromatic groups bioisosterism in this study. The structures of the novel phenylurea derivatives were confirmed based on ESI-MS, IR and 1H-NMR spectral data. All of the compounds were evaluated for the insecticidal activity against the third instars larvae of Spodoptera exigua Hiibner, Plutella xyllostella Linnaeus, Helicoverpa armigera Hubner and Pieris rapae Linne respectively, at the concentration of 10 mg/L. The results showed that all of the derivatives displayed strong insecticidal activity. Most of the compounds presented higher insecticidal activity against S. exigua than the reference compounds tebufenozide, chlorbenzuron and metaflumizone. Among the synthesized compounds, 3b, 3d, 3f, 4b and 4g displayed broad spectrum insecticidal activity.


Introduction
Benzoylphenyl ureas (BPUs), acting as insect growth regulators (IGRs) and affecting the larval stages of most insects by blocking or inhibiting the synthesis of chitin, have been rapidly developed since the first benzoylphenylurea, diflubenzuron, was commercialized in 1972 [1,2]. BPUs have OPEN ACCESS attracted considerable attention for decades for their unique mode of action coupled with a high degree of activity on target pests and low toxicity to non-target organisms [3,4]. More than 20 BPUs have been developed as IGRs, such as chlorfluazuron, flufenoxuron, triflumuron and chlorbenzuron [5].
Diacylhydrazines have been another of the most important classes of insect growth regulators since the discovery of the N-tert-butyl-N,N'-diacylhydrazines in the mid-1980s by Rohm and Haas Co. [6,7]. They affect the ecdysone receptor and lead to lethal premature molting. For their unique mode of action with high insecticidal selectivity, lower toxicity to vertebrates and simple structure, diacylhydrazines have attracted considerable attention [8,9]. Among nonsteroidal ecdysone agonists, N-tert-butyl-N'-(4-ethylbenzoyl)-3,5-dimethylbenzoylhydrazine (tebufenozide) was the first introduced to market [10,11]. At present, a series of diacylhydrazines, such as methoxyfenozide, halofenozide and chromafenozide have already been widely applied as pesticides in the agrochemical field.
Metaflumizone, which was discovered by Nihon Nohyaku in the early 1990s, is a novel sodium channel blocker insecticide. It belongs to the new class of semicarbazone insecticides and its structure contains an acylhydrazone moiety. It provides good control of most of the economically important lepidopterous pests and other orders pests including Siphonaptera, Diptera, Hymenoptera and Coleoptera. Meanwhile, it presents low risk to non-target organisms as well as humans and the environment. Moreover, insect strains which were resistant to the organophosphates, imidacloprid and carbamates have not showed cross-resistance to metaflumizone. Therefore, metaflumizone has a great potential in Integrated Pest Management(IPM) strategy and resistance management [12][13][14].  An abundance of insects belonging to the order Lepidoptera, such as the diamondback moth (Helicoverpa armigera Hubner), beet armyworm (Spodoptera exigua Hiibner), cabbage caterpillar (Pieris rapae Linne) and cotton bollworm (Plutella xyllostella Linnaeus) etc., are among the most damaging pests for crops all over the world. Because they cause enormous agricultural production losses, various insecticides have been used to control Lepidoptera pests. These insecticides brought great benefits, but they have caused negative effects such as toxicity to non-target organisms, including mammals, and environmental pollution. Moreover insecticide resistance has strengthened yearly [15]. Therefore, researchers have to develop novel, low toxicity, highly efficient, friendly environmental insecticides.
In view on the facts above, we sought to incorporate a phenylurea unit with an aromatic diacylhydrazine and acylhydrazone moiety, respectively, according to the method of active groups linkage and the principle of aromatic groups bioisosterism (Figure 1), and eighteen such phenylurea derivatives were then designed and synthesized (Scheme 1). Moreover the new compounds' insecticidal activities against the third instars larvae of diamondback moth, beet armyworm, cabbage caterpillar and cotton bollworm, were evaluated. We expected that by this approach the combination of the critical components could further strengthen the biological activity of phenylurea derivatives.

Insecticidal Activities
As displayed in Table 1, all of the phenylurea derivatives 3a-3i, 4a-4i indicated strong insecticidal activities against the third instars larval of beet armyworm at the concentration of 10 mg/L. Most of the synthesized compounds showed higher insecticidal activity than the references chlorbenzuron, tebufenozide and metaflumizone at the 72 h time point. Among the synthesized compounds, the mortality of the compounds 3g, 3i, 4d and 4e exceeded 90% at 72 h. Moreover the mortality of compound 4g reached 100%. Meanwhile the data of Table 1 shows that the mortality of the compounds indicated a positive correlation with administration time. The data in Table 2 showed the mortality of the phenylurea derivatives 3a-3i, 4a-4i against the third instars larval of diamondback moth (P. xyllostella), cotton bollworm (H. armigera) and cabbage worm (P. rapae) at the concentration of 10 mg/L after 72 h. The data presented that all the phenylurea derivatives showed strong insecticidal activity against diamondback moth, cotton bollworm and cabbage worm. Among the synthesized compounds, 3b, 3d, 3f, 4b and 4g displayed broad spectrum insecticidal activity.

General Procedure for the Preparation of 1a-1m
3-Aminobenzoic acid ethyl ester (50 mmol) was dissolved in toluene (30 mL).With ice-water bath cooling, a substituted phenyl isocyanate (Ar-NCO) (50 mmol) dissolved in toluene (30 mL), was added dropwise. With the water bath, the mixture was reacted at a temperature of 25 °C, for 5 h, and then heated to reflux for 2 h. After cooling to room temperature, the reaction mixture was filtered under vacuum and dried to produce ethyl 3-(3-substituted phenylureido)benzoates 1a-1m, in yields ranging from 85% to 95%.

Bioassay for Insecticidal Activities
Wheat leaf discs (0.5 cm × 0.5 cm) were treated with 5 μL solutions made from 5 mg of test sample dissolved in 500 mL of acetone. Tebufenozide, chlorbenzuron and metaflumizone were used as positive controls and acetone was used as negative control. The third instars larvae of beet armyworm were fed with the discs. Cohorts of 24 beet armyworms were treated each time and bioassays were replicated three times. After 24 h, 48 h and 72 h, the numbers of knocked down larvae (symptoms: the larvae were narcotized, the bodies were very soft and immobilized, and response disappeared completely) were recorded respectively [16,17].
Bioassay for insecticidal activities against cotton bollworm, diamondback moth and cabbage worm was performed according to the above method. The third instars larvae of cotton bollworm, diamondback moth and cabbage worm were fed with the discs instead, respectively. The numbers of knocked down larvae after 72 h were recorded. The experiments showed that the mortality of acetone as negative controls was 0%.

Conclusions
In conclusion, eighteen phenylurea derivatives 3a-3i, 4a-4i have been designed and synthesized according to the method of active groups linkage and the principle of aromatic groups bioisosterism. The insecticidal activities of these novel phenylurea derivatives against the third instars larvae of S. exigua, H. armigera, P. xyllostella and P. rapae were evaluated, at a concentration of 10 mg/L. The bioassay results showed that all of the synthesized compounds displayed high insecticidal activity. Most of them presented higher insecticidal activity against S. exigua than the reference compounds tebufenozide, chlorbenzuron and metaflumizone. Among the synthesized compounds, 3b, 3d, 3f, 4b and 4g displayed broad spectrum insecticidal activity. The results above have encouraged us to further explore novel phenylurea derivatives as insecticidal agents and this will be reported in future work.