Synthesis and Bioactivities Study of Novel Pyridylpyrazol Amide Derivatives Containing Pyrimidine Motifs

In this study, thirteen new pyridylpyrazolamide derivatives containing pyrimidine motifs were synthesized via six-step reactions. Bioassay results showed that some of the synthesized compounds revealed good antifungal properties against Sclerotinia sclerotiorum, Phytophthora infestans, Thanatephorus cucumeris, Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica, Botryosphaeria dothidea, and Phompsis sp. at 50 μg/mL, which were similar to those of Kresoxim-methyl or Pyrimethanil. Meanwhile, bioassay results indicated that the synthesized compounds showed a certain insecticidal activity against Spodoptera litura, Mythimna separata, Pyrausta nubilalis, Tetranychus urticae, Rhopalosiphum maidis, and Nilaparvata lugens at 200 μg/mL, which was lower than that of Chlorantraniliprole. To the best of our knowledge, this study is the first report on the antifungal and insecticidal activities of pyridylpyrazol amide derivatives containing a pyrimidine moiety.


INTRODUCTION
Plant fungal and insect diseases have posed serious threats to crops in the world and caused a severe loss throughout the world (Strange and Scott, 2005;Yang et al., 2015). Nowadays, some of the available traditional fungicides and insecticides, such as Kresoxim-methyl, Pyrimethanil, Chlorantraniliprole, etc., are widely used to prevent plant harmful fungal and insect diseases. However, prolonged use of traditional pesticides can not only lead to drug resistance, but also have a harmful influence on the safety of the plants and the environment. Therefore, the development of novel and promising fungicides and insecticides is still an urgent task.
To develop effective pesticide agents, we aim to introduce the pyrimidine ring to the pyridiylpyrazol amide skeleton to design a series of novel pyridiylpyrazol amide derivatives containing a pyrimidine moiety (Figure 3). As far as we know, it is the first report on the antifungal and insecticidal activities of pyridylpyrazol amide derivatives containing a pyrimidine moiety.

General Information
JEOL-ECX 500 NMR spectrometer (JEOL, Tokyo, Japan) was used to analyse the NMR spectral ( 1 H NMR and 13 C NMR) at room temperature using TMS as an internal standard and DMSO-d 6 as the solvent. Elemental analysis was performed on the Elementar Vario-III CHN analyser (Elementar, Hanau, Germany). Mass spectral were conducted on the Agilent 5973 organic mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Melting points were determined on the XT-4 binocular microscope (Beijing Tech Instrument Co., China). All commercial reagents and solvents were used as they did not require any purification before use.

Synthesis
Preparation Procedure of the Key Intermediate 5 The synthetic procedure for the key intermediate 5 is shown in Scheme 1. To a mixture of 2,3-dichloropyridine (0.1 mol) dissolved in anhydrous ethanol (120 mL), 80% hydrazine hydrate (80 mL) was added dropwise and reacted under reflux. Upon completion of the reaction, the reaction solution was cooled to room temperature and the solvent was removed under reduced pressure. The residue was washed with water and recrystallized with ethanol to gain intermediate 1. Intermediate 1 (90 mmol) and diethyl maleate (90 mmol) were added to the mixture of sodium ethoxide (90 mmol) and ethanol (100 mL), then a moderate amount of glacial acetic acid was added when the temperature was below 60 • C. Upon completion of the reaction, the reaction solution was poured into 100 mL distilled water to precipitate the solid, then the solid was recrystallized with ethanol to obtain intermediate 2. Then, a mixture of intermediate 2 (45 mmol), phosphorus oxychloride (POCl 3 , 50 mmol) or phosphorus oxybromide (POBr 3 , 50 mmol), and acetonitrile (CH 3 CN, 50 mL) was reacted under reflux. After ending the reaction, the reaction mixture was poured into 30 mL distilled water, extracted with dichloromethane (CH 2 Cl 2 ), and dried with anhydrous sodium sulfate (Na 2 SO 4 ) to give intermediate 3 (Wang B. L. et al., 2018). After that, a mixture of intermediate 3 (40 mmol), potassium persulfate (K 2 S 2 O 8 , 44 mmol), and CH 3 CN (50 mL) reacted under reflux. Upon completion of the reaction, the reaction mixture was poured into 100 mL of distilled water. The residue was washed with water and recrystallized with ethanol to obtain intermediate 4 (Wang B. L. et al., 2018). Finally, sodium hydroxide (NaOH, 30 mmol) dissolved in 10 mL of water was added to the mixture of intermediate 4 and methanol (20 mL) and reacted under reflux. When the reaction was completed, the reaction mixture was poured into 100 mL of distilled water and acidified the mixture to pH 5-6 using concentrated hydrochloric acid. The key intermediate 5 was attained after recrystallization with ethanol. 1 H NMR spectral data for intermediates 1-5 are reported in the Supplementary Data.

Antifungal Biological Assay
The antifungal activities of the title compounds 6a-6m against Sclerotinia sclerotiorum (S. sclerotiorum), Phytophthora infestans (P. infestans), Thanatephorus cucumeris (T. cucumeris), Gibberella zeae (G. zeae), Fusarium oxysporum (F. oxysporum), Cytospora mandshurica (C. mandshurica), Botryosphaeria dothidea (B. dothidea), and Phompsis sp. were evaluated at the concentration of 50 µg/mL (Min et al., 2016;Wu et al., 2019a,b). The target compounds 6a-6m (5 mg) were dissolved in dimethyl sulfoxide (1 mL) and sterile water (9 mL) before mixing with 90 mL potato dextrose agar (PDA) to generate a final concentration of 50 µg/mL. Then, 4 mm diameter of the mycelia dishes were cut from a culture medium of pathogenic fungi, then inoculated in the middle of PDA and cultivated at 27 ± 1 • C for 4-5 days. DMSO in sterile distilled water served as a negative control, while Kresoxim-methyl and Pyrimethanil acted as positive controls. For each treatment, three replicates were conducted. The radial growth of the fungal colonies was measured and the data were statistically analyzed. The inhibition rate I (%) of the test compounds against eight pathogenic fungi were calculated by the following formula, where C represents the diameter of fungi growth on untreated PDA, and T represents the diameter of fungi on treated PDA. (1)
Chlorantraniliprole was used as a control. All bioassays were performed in the laboratory at 27 ± 1 • C for 48 h. Three replicates were performed for each treatment. The percentage of mortalities for the target compounds were determined using Abbott's formula.

RESULTS AND DISCUSSION
In this study, using 2,3-dichloropyridine as the starting material, the title compounds 6a-6m were synthesized in six steps, including hydrazidation, cyclization, bromination or chlorination, oxidation, hydrolyzation, and condensation. The target compounds structures were confirmed by 1 H NMR, 13 C NMR, MS, and elemental analysis. In the 1 H NMR spectra of compound 6a, a singlet at 2.45 ppm assigned to CH 3 protons of pyrimidine-CH 3 , the doublet signal at 2.67 ppm indicated the presence of CH 3 proton in CONH-CH 3 , meanwhile, a singlet at 8.78 and 7.32 ppm indicated the presence of pyrimidine and pyrazole ring. Two proton signals of two -CONH-in amide moiety was observed at 12.33 and 8.74 ppm. The structure of 6b was also confirmed by its mass spectral data. In its mass spectrum, the molecular ion peak was noticed m/z at 450.1 ([M+H] + ) corresponding to its molecular weight. The in vitro antifungal activities at 50 µg/mL of the target compounds against eight plant fungi are listed in Table 1. Table 1 showed that, at 50 µg/mL, compounds 6a-6m indicated certain antifungal activities against S. sclerotiorum, P. infestans, T. cucumeris, G. zeae, F. oxysporum, C. mandshurica, B. dothidea, and Phompsis sp. with the inhibition rates of 6.5-54.1%, 8.7-48.6%, 3.2-54.7%, 0-54.9%, 0-66.3%, 10.7-49.4%, 30.1-85.9%, and 36.2-81.2%, respectively. Among the title compounds, compound 6i revealed good in vitro antifungal activities against P. infestans and B. dothidea, with the inhibition rates of 48.6% and 85.9%, respectively, which were equally to those of Kresoximmethyl or Pyrimethanil. Meanwhile, compound 6j revealed good in vitro antifungal activities against S. sclerotiorum, G. zeae, C. mandshurica, and B. dothidea, with the inhibition rates of 54.1, 54.9, 49.4, and 85.9%, respectively, which were similar with those of Kresoxim-methyl or Pyrimethanil.
Based on the preliminary antifungal bioassays, the EC 50 values of compounds 6i and 6j were also tested and presented in Table 2. Table 2 showed that compounds 6i and 6j showed good activities against B. dothidea, with EC 50 values of 56.4 and 65.3 µg/mL, respectively, which were similar to that of Pyrimethanil (57.6 µg/mL).

CONCLUSIONS
In summary, thirteen novel pyridylpyrazolamide derivatives containing pyrimidine motifs were synthesized, and their structures confirmed by 1 H NMR, 13 C NMR, MS and elemental analysis. Bioassay results showed that some of title compounds revealed good antifungal and insecticidal properties. The results provided strategy for leading the synthesis of novel pyridylpyrazolamide derivatives containing pyrimidine motifs.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
WW and QF contributed to the synthesis, purification, characterization of all compounds, and prepared the original manuscript. YZ and YG performed the activity research. MC and HC perfected the language and assisted with the structure elucidation and manuscript revision. GO and MY designed and supervised the research and revised the manuscript. All authors discussed, edited, and approved the final version.