Synthesis and bioactivities of novel trifluoromethylated pyrazole oxime ether derivatives containing a pyridyl moiety

A variety of novel 3-trifluoromethyl substituted pyrazole oxime ether derivatives containing a pyridyl moiety were designed and synthesized in satisfactory yields. Their structures were confirmed by 1 H NMR, 13 C NMR, and elemental analysis. The preliminary bioassays indicated that some of the title compounds showed potential insecticidal activities against Aphis craccivora , and some compounds also displayed plant growth regulatory activities


Introduction
The pyridyl ring is a prominent heterocyclic scaffold in lots of bioactive molecules.Numerous pyridine-based compounds have been reported to display versatile bioactivity, such as insecticidal, fungicidal, plant growth regulatory, anticancer, and antibacterial activity. 1,2Practical applications of pyridine derivatives in medicinal and pesticidal chemistry have also been achieved during the past two decades.For example, imidacloprid, acetamiprid, and thiacloprid (Figure 1), a class of neonicotinoid insecticides acting on the insect nicotinic acetylcholine receptors (nAChR), are currently used to control various insects such as aphids, whiteflies, and thrips on many crops due to their excellent insecticidal activities and good systemic properties. 3However, several aphid species have developed certain levels of neonicotinoids resistance after frequent field applications. 4Aphis craccivora belonging to pea aphids affects many crops of agricultural importance in Asia.To control Aphis craccivora more effectively, chemists devoted themselves to the development of neonicotinoids with new chemical structures and high insecticidal activities. 5n the other hand, substituted pyrazoles constitute an important class of compounds in the field of agricultural, and medicinal chemistry because of their broad spectrum biological activities. 6They are widely used as fungicide, 7 insecticide, 8 herbicide, 9 and antitumor agent. 10As known, ethiprole and fipronil (Figure 1) are significant agricultural insecticides with extensive use for the effective control of the insects on corn and soybean, as well as stored grain insect pests. 11idacloprid Acetamiprid Thiacloprid R= Et, Ethiprole R= CF 3 , Fipronil Recently, oxime ether derivatives have drawn much attention in medicinal research due to their significant bioactivity. 12Interestingly, Sun and co-workers found that some oxime ether compounds exhibited not only excellent insecticidal activities but have also good plant growth regulatory activities. 13In addition, many investigations have indicated that introducing a CF 3 group into heterocyclic molecules mostly results in the improvement of physical, chemical and biological properties. 14Therefore, trifluoromethylated heterocycles became a focus of chemical research.Encouraged by these observations, we anticipated that introduction of the important pyridyl moiety and the CF 3 group to pyrazole oxime ether molecules might generate a new group of biologically active compounds.Herein, we report the synthesis and bioactivity of some novel trifluoromethylated pyrazole oxime ether derivatives containing a pyridyl moiety.

Biological activities
The insecticidal activities of the target compounds 9a-9n, 12a-12d against pea aphids (Aphis craccivora) were investigated using a known procedure. 22As indicated in Table 3, some of the title compounds showed good to excellent insecticidal activities against A. craccivora at the dosage of 0.5 mg/mL.When the mortality of insects was recorded at 24 h, compounds 9b, 9f, 9g, 9j, 9k, 9l, 9m, 9n, and 12b displayed excellent insecticidal activities against A. craccivora with the values of 92.2, 90.1, 90.4,91.3, 90.2, 91.5, 90.8, 90.1, and 90.3%, respectively, while the alkylthio derivatives 12c and 12d showed very low insecticidal activities against A. craccivora.When the mortality of insects was recorded at 48 h, compounds 9a, 9b, 9f, 9g, 9j, 9k, 9l, 9m, 9n, and 12b exhibited around 90% inhibitory rate against A. craccivora.From the data listed in Table 3, we can find that among the chlorinated phenoxy derivatives, 4-substituted analogue 9j had more potency against A. craccivora than the corresponding 2-and 3-substituted analogues (9h and 9i); when the mortality of insects was recorded at 48 h, compounds 9h, 9i, and 9j displayed 72.4,75.7, and 94.1% insecticidal activities against A. craccivora, respectively.At the same time, compound 12b containing a 4-chlorophenylthio moiety exhibited insecticidal activity similar to that of the phenoxy analogue 9j.In addition, Table 4 showed the results of further toxicity assay about the typical candidates 9f, 12b, and the positive control nicotine sulfate against A. craccivora.The values of LC 50 listed in Table 4 indicated that compounds 9f and 12b had slightly less potency against A. craccivora than the control nicotine sulfate.All the above results implied that introduction of the important pyridyl moiety and the CF 3 group to pyrazole oxime ether molecules could afford some new compounds possessing potential insecticidal activities against A. craccivora.Further structural optimization and study of insecticidal activities are under way.
The plant growth regulatory activities of the title compounds 9a-9n, 12a-12d were evaluated by the cucumber cotyledon test at the concentration of 10 µg/mL according to a reported procedure. 23s shown in Table 3, some of the target compounds exhibited promising promotion effects on the radicle growth of cucumber cotyledons.Among these compounds, the promotional values of compounds 9a, 9e, 9f, 9j, 9k, 9l, 9m, and 12b were 83.3, 83.6, 80.1, 98.3, 92.5, 98.0, 82.3, and 98.7%, respectively.From the data listed in Table 3, we found that in the case of chlorinated phenoxy derivatives, the 4-substituted analogue 9j exhibited a much higher promotional activity than did the corresponding 2-and 3-substituted analogues (9h and 9i).In addition, the phenylthio derivative 12b displayed promotional effect comparable to that of corresponding phenoxy analogue 9j.It is interesting to note that the plant growth regulatory activities of compounds 9j, 9k, 9l, and 12b are in accordance with their insecticidal activities to a certain extent.
In summary, a variety of novel 3-trifluoromethyl substituted pyrazole oxime ether derivatives containing a pyridyl moiety were synthesized in satisfactory yields.All the target compounds were soluble in most organic solvents, which allowed an easy use.The preliminary bioassays indicated that some of the designed compounds exhibited significant insecticidal activities against Aphis craccivora, and some compounds showed promising plant growth regulatory activities.

Experimental Section
General Procedures.Melting points were determined on an X-4 binocular microscope melting point apparatus (Beijing Tech Instrument Co., Beijing, China) and were uncorrected. 1H NMR and 13 C NMR spectra were obtained on a Bruker AC-P 300 spectrometer (300 MHz, 1 H; 75 MHz, 13 C) in CDCl 3 with tetramethylsilane as the internal standard.Chemical shift values (δ) were given in ppm.Elemental analyses were determined on a Yanaco CHN Corder MT-3 elemental analyzer.All reagents were of analytical reagent grade or were chemically pure.All solvents were dried by standard methods and distilled prior to use.
General procedure for the preparation of aldehydes (10a-10c) 18 To powdered potassium hydroxide (6.5 mmol) was added water (2 mL), the mixture was stirred for 10 min at room temperature, and then a solution of substituted benzenethiol or t-BuSH (5.5 mmol) in DMF (20 mL) was added thereto.The resulting solution was heated to 55 °C and stirred for 30 min.Followed by addition of compound 6 (5 mmol), the reaction mixture was stirred at 110 °C for 6 h, cooled, and poured into ice-water (30 mL).The precipitate was filtered to give a brown solid, which was washed with water and petroleum ether.The solid was dried under vacuum at 30 °C for 1 h, affording the corresponding products, which were used without further purification except 10a.

General procedure for the preparation of the title compounds (9a-9n, 12a-12d)
To a mixture of 2-chloro-5-chloromethylpyridine 4 (5 mmol), oximes 8a-8n or 11a-11d (4 mmol), and DMF (30 mL), potassium carbonate (10 mmol) was added thereto.The reaction mixture was heated gradually to 90 °C and stirred for 10-16 h.The resulting mixture was cooled, diluted with ice water (150 mL), and extracted with dichloromethane (4 × 50 mL).The organic layer was washed with saturated brine (2 × 20 mL), and then dried over anhydrous sodium sulfate.The solvent was removed under reduced pressure, and the crude residue was purified by column chromatography on silica gel with the solvent system of ethyl acetate and petroleum ether (60-90 °C) to afford the target compounds 9a-9n, 12a-12d.

Biological assays
Bioassay of insecticidal activities.The insects, pea aphids (Aphis craccivora), were reared in the laboratory.The insecticidal activities of compounds 9a-9n, 12a-12d against pea aphids (Aphis craccivora) were tested according to a reported procedure. 22The insects were placed in a room maintained at 26 °C, 60% relative humidity, and a 14 h photoperiod.Stock solutions of each test sample was prepared in acetone at a concentration of 0.5 mg/mL, and then diluted to the required concentration with water containing TW-20.Tender shoots of soybean with 60 insects were dipped in the diluted solutions of the chemicals for 5 s, then the superfluous liquor was removed, and they were kept in the conditioned room for normal cultivation.The mortality was evaluated by the number or size of live larvae in the treated bottles relative to that in the untreated controls after 24 h and 48 h, respectively.Controls were performed under the same conditions.Each treatment was performed three times.The data were subjected to probit analysis.Bioassay of plant growth regulatory activities.The plant growth regulatory activities of compounds 9a-9n, 12a-12d were evaluated by means of cucumber cotyledon test according to a reported procedure. 23The cucumber seeds (JINKE, No.4) were supplied by the Biological Assay Center, Nankai University, China.These seeds were incubated at 24 °C in a darkroom for 3 days, and 10 pieces of cotyledons of the same size were selected.The test samples were dissolved in DMF at a concentration of 10 µg/mL.A sample solution (0.3 mL) was sprayed over a filter paper (6 cm diameter), and solvent was volatilized to dryness on air.The filter paper thus prepared was placed into an incubation vessel (6 cm diameter) and soaked with distilled water (3 mL).Finally, 10 pieces of cotyledons were added.These cotyledons were incubated at 24 °C in a darkroom for 3 days.The rhizogenesis numbers of every 10 pieces of hypocotyls were measured.In contrast, the distilled water was used as a control.Each test was performed in triplicate.Statistical Analysis.The results were expressed as means ± standard deviation (SD) of three parallel experiments.Data were analyzed by Student's t-test.The LC 50 (median lethal concentration) was analyzed using probit analysis performed with the statistical software SAS.

Table 1 .
The effects of bases and solvents on the synthesis of compound 9a

Table 2 .
Reaction time and yields of

Table 3 .
Insecticidal activities against A. craccivora and plant growth regulatory activities of the title compounds 9a

-9n, 12a-12d
a Each value represents the mean ± SD of three experiments.

Table 4 .
Toxicities against Aphis craccivora of 9f, 12b, and Nicotine sulfate a a The mortality was recorded at 48 h.b LC 50 refers to median lethal concentration.cr refers to correlative coefficient.d A refers to Nicotine sulfate.