Discovery of Novel N ‐ Pyridylpyrazole Thiazole Derivatives as Insecticide Leads

: To develop effective insecticides against Lepidoptera pests, 25 novel N ‐ pyridylpyrazole derivatives containing thiazole moiety were designed and synthesized based on the intermediate derivatization method (IDM). The insecticidal activities of these target compounds against Plutella xylostella ( P. xylostella ), Spodoptera exigua ( S. exigua ), and Spodoptera frugiperda ( S. frugiperda ) were evaluated. Bioassays indicated that compound 7g − 7j exhibited good insecticidal activities. Com ‐ pound 7g showed especially excellent insecticidal activities against P. xylostella , S. exigua , and S. frugiperda with LC 50 values of 5.32 mg/L, 6.75 mg/L, and 7.64 mg/L, respectively, which were ade ‐ quate for that of commercial insecticide indoxacarb. A preliminary structure ‐ activity relationship analysis showed that the insecticidal activities of thiazole amides were better than that of thiazole esters, and the amides with electron ‐ withdrawing groups on the benzene ring were better than the ones with electron ‐ donating groups. This work provides important information for designing novel N ‐ pyridylpyrazole thiazole candidate compounds and suggests that the 7g is a promising insecti ‐ cide lead for further studies.


Introduction
As the world's population continues to grow, food shortages will become increasingly prominent in the future, especially in underdeveloped or remote areas. Agricultural pests are the main threat to crop production: approximately 20~40 percent of crop losses each year are caused by plant diseases and insect pests [1,2]. Lepidoptera pests, such as Plutella xylostella (P. xylostella) and Spodoptera frugiperda (S. frugiperda) have a short reproductive cycle, high reproductive rate, and serious overlap of generations, which bring a great burden to agricultural producers [3,4]. Many efforts have been devoted to the development of effective control methods against these pests, such as agricultural, physical, biological, and chemical controls. Among them, chemical control with insecticides remains the major method due to its high efficacy and economic efficiency. However, the long-term and unreasonable use of chemical insecticides has brought up problems such as environmental issues and resistance to pests [5,6]. Therefore, it is necessary to discover insecticidal structures with novel scaffolds that should be environmentally safe, with less impact on non-target species and human health, which is a hot spot in current pesticide research [7,8].
Traditionally, it takes about ten years and over USD 256 million to develop a new agrochemical [9]. Several practical strategies have been proposed to enhance the probability of success, such as modification of natural compounds [10], scaffold-hopping [11], "Me Too" chemistry [12], and IDM [13]. In particular, the IDM emphasizing the Thiazole is an important heterocyclic structure widely applied in commercial pesticides, such as insecticides (e.g., thiamethoxam and clothianidin, nicotinic acetylcholine receptor competitive regulator), nematicide (e.g., fluensulfone, metabolic inhibitors of energy storage processes), and fungicides (e.g., thifluzamide, thiabendazole, and ethaboxam) (Figure 1). In our previous work, a series of thiazolyl substituted sulfonamides were found to exhibit good insecticidal or fungicidal activities [26]. These examples indicate that thiazole is an effective scaffold for designing a novel lead compound in the drug discovery process. Although thiazole scaffolds have been widely used in the field of pesticides, they are only some fungicides and commercial pesticides for controlling piercing-sucking pests, but fewer pesticides containing thiazole scaffolds for controlling chewing pests have been marketed [27][28][29]. Therefore, introducing a thiazole ring into insecticide fragments to control chewing pests is an interesting topic. In addition, amide and ester structures are widely used in the development of pesticides, because the introduction of amide and ester groups can enhance the interaction between drug molecules and receptors, leading to chemical molecules exhibiting significant insecticidal, nematocidal, and other bioactivities [30][31][32].
To obtain novel N-pyridylpyrazole thiazole derivatives with potential insecticidal activity, we sought to retain the substructure of N-pyridylpyrazole, and increase the amide bridge by introducing a thiazole moiety based on the IDM (Figure 2). A series of N-pyridylpyrazole derivatives containing thiazoles were designed and synthesized.
Structures of all synthesized compounds were characterized by 1 H NMR and 13 C NMR. Insecticidal activities of the target compounds were evaluated against P. xylostella, S. exigua, and S. frugiperda, and preliminary structure-activity relationship (SAR) was discussed.
General synthesis procedures of compounds 7a−7m. Oxalyl chloride (169 μL, 2.0 mmol) was added to a mixture of intermediate 5 (0.38 g, 1.0 mmol) in DCM (3 mL), followed by a drop of dimethyl formamide at room temperature. The mixture was stirred for 2 h and evaporated to afford the crude product, which was then dissolved in DCM (2 mL). The above acyl chloride dropwise was added to an ice-cooled solution of amines (1.2 mmol) and triethylamine (TEA, 2.0 mmol) in DCM (2 mL). The solvent was removed to afford the crude product. The mixture was diluted with DCM, washed with 1 mol/L hydrochloric acid, saturated NaHCO3, and brine, dried over anhydrous Na2SO4, concentrated in vacuo, and purified through column chromatography to obtain product 7a−7m.

Biological Materials and Methods
2.3.1. Bioassays with P. xylostella and S. exigua P. xylostella and S. exigua were collected from the laboratory of South China Agricultural University, Guangzhou, China (23°8′ N, 113°17′ E). The insecticidal activities were evaluated based on the literature reported [8,25]. Treatment with distilled water (containing 0.1% Tween-80 and 0.01% DMSO) was used as a negative control, and chlorantraniliprole and indoxacarb were selected as a positive control. First, all the tested compounds were dissolved in DMSO (dimethyl sulfoxide) and diluted with water (containing 0.1% Tween 80). Then, the cabbage leaf disks (diameter: 1.8 cm) were dipped in the tested solution for 30 s and dried naturally. Finally, the treated cabbages leaf disks were placed in Petri dishes (diameter: 9 cm), and ten third-instar larvae of P. xylostella or second-instar of S. exigua raised in the laboratory were transferred to each petri dish. Three replicates for each treatment concentration were conducted. Mortalities (%) were determined at 48 h after treatment. SPSS 21.0 software was used for one-way analysis of variance, Duncan's method was used for multiple comparisons, and the LC50 values were calculated using Finney's Probit analysis (Probit module in SPSS 21.0 Software). The corrected mortality (%) was obtained as follows: Corrected mortality (%) = (mortality in treatment − mortality in negative control)/(1mortality in negative control) × 100

Bioassays with S. frugiperda
S. frugiperda was collected from the laboratory of South China Agricultural University, Guangzhou, China (23°8′ N, 113°17′ E). Similarly, treatment with distilled water was used as a negative control, and indoxacarb and chlorantraniliprole were selected as a positive control. First, all tested compounds were dissolved in DMSO and diluted to 100 mg/L with distilled water (containing 0.1% Tween 80). Then, the young leaves of maize were cut into 5 cm lengths, dipped in the solution of the test compound for 10 s, and dried naturally. Finally, the treated maize leaves were placed in Petri dishes (diameter: 9 cm), and 10 s-instar larvae of S. frugiperda raised in the laboratory were placed in each petri dish [33]. Three replicates for each treatment concentration were conducted. Mortalities were determined 48 h after treatment.

Chemistry
The synthetic routes of the target compounds are illustrated in Scheme 1. 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid 1 was used as the starting material, and we attempted to treat 1 with ammonia in the presence of the combination of EDCI and HOBt [34] to prepare primary amide intermediate 2. Subsequently, product 3 was obtained using Lawesson's Reagent via an oxygen-sulfur exchange reaction. Then, the thioamide product 3 and ethyl bromopyruvate through the ring-closing reaction introduced the thiazole ring into the N-pyridylpyrazole scaffold to obtain the thiazole ester compound 4 in 82% yield. Compound 4 was hydrolyzed with 1 mol/L NaOH solution, and acidified with a 1 mol/L hydrochloric acid solution to afford intermediate 5, and 71.14% overall yield for the four-step sequence. Furthermore, the corresponding compounds 6a−6j were prepared via synthetic route A, and treated compound 5 with alcohols using DCC/DMAP as a catalyst [15]. Among them, different types of aliphatic alcohols bearing functional groups such as alkyl, alkynyl, trifluoromethyl, and trichloromethyl groups gave the corresponding products 6a−6g in good yields. In addition, this strategy was also compatible with different types of benzyl alcohol, delivering the desired products 6h and 6i in good yields. On the other hand, compound 7a−7m was prepared following another synthetic route B described in the preparation of compounds 5 with amines using a combination of oxalyl chloride/triethyl amine [35], affording the corresponding product yields of 63−98%. Interestingly, this protocol was also compatible with different types of acid amides-free N-H bonds (7g, 7i, and 7j). Finally, the primary amide compound 7a was further dehydrated to obtain the thiazole nitrile compound 8.

Insecticidal Activities
It is well-known that N-pyridylpyrazole derivatives possess extraordinary insecticidal activity against many insect species [12,36,37]. Hence, to screen for potential insecticide candidates, preliminary bioassays on the insecticidal activities of 25 title compounds, and positive control (indoxacarb and chlorantraniliprole) were conducted against three Lepidoptera pests (P. xylostella, S. exigua, and S. frugiperda) according to previously reported procedure [26,38]. As listed in Table 1, the preliminary screen results indicated that most of the tested compounds displayed a good insecticidal effect toward the third-instar P. xylostella larvae with mortality rates above 50% at 100 mg/L. In particular, compound 7g−7j showed mortality rates close to 100%, which is adequate for commercial insecticide indoxacarb. The LC50 values of 7g−7j against the third-instar P. xylostella larvae were further determined ( Table 2) Results also showed that the tested compounds have similar insecticidal activities against S. exigua and S. frugiperda. In short, compound 7g exhibited excellent insecticidal activity with an LC50 value of 6.75 mg/L and 7.64 mg/L, which was close to that of commercial insecticide indoxacarb. Consequently, compound 7g is a potential new lead for the development of insecticides to control Lepidoptera pests. 100.00 ± 0.00 l 100.00 ± 0.00 k 100.00 ± 0.00 j 7k 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 7l 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 7m 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 8 60. 100.00 ± 0.00 l 100.00 ± 0.00 k 100.00 ± 0.00 j a Corrected mortality rate (mean ± SE, %), SE (Standard Error), Means within a column followed by a different lowercase letter represent a significant difference (p < 0.05) b IDC = indoxacarb; c CHL = chlorantraniliprole.

Conclusions
In conclusion, the IDM strategy could be a feasible tool for the development of pesticides. In this study, a library of novel N-pyridylpyrazole derivatives containing thiazole moiety was obtained based on highly efficient ring-closing reactions, and their insecticidal activities were investigated against insect pests (P. xylostella, S. exigua, and S. frugiperda). Bioassay results showed that these compounds exhibited good insecticidal activity against three Lepidoptera pests. In particular, compound 7g exhibited adequate insecticidal activities against P. xylostella (LC50 = 5.32 mg/L), S. exigua (LC50 = 6.75 mg/L), and S. frugiperda (LC50 = 7.64 mg/L) compared to indoxacarb, making it a promising insecticide candidate. To the best of our knowledge, N-pyridylpyrazole containing thiazole scaffold is a novel lead to obtaining more potent analogs through structural modification and optimization.