Synthesis and Antifungal Activity of N-(Substituted pyridinyl)-1-methyl(phenyl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide Derivatives

A series of N-(substituted pyridinyl)-1-methyl(phenyl)-3-trifluoromethyl-1H-pyrazole-4-carboxamide derivatives were synthesized. All target compounds were characterized by spectral data (1H-NMR, 13C-NMR, IR, MS) and elemental analysis and were bioassayed in vitro against three kinds of phytopathogenic fungi (Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica). The results showed that some of the synthesized N-(substituted pyridinyl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamides exhibited moderate antifungal activities, among which compounds 6a, 6b and 6c displayed more than 50% inhibition activities against G. zeae at 100 µg/mL, which was better than that of the commercial fungicides carboxin and boscalid.


Introduction
The mitochondria are membrane enclosed organelles found in most eukaryotic cells [1], which are usually described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy [2], and they also play an OPEN ACCESS important role in plant protection against many phytopathogenic fungi. Carboxanilides are traditional active compounds which have been applied in agro-chemistry [3][4][5], and different kinds of commercial products based on the carboxanilide structure have been developed in recent years. Early carboxanilide fungicides such as carboxin have special activity against basidiomycetes, but limited activity towards other plant pathogens [6,7]. The mode of action for these commercial fungicides revealed that they can specifically bind to the ubiquinone-binding site (Q-site) of the mitochondrial complex II, interrupting electron transport in the mitochondrial respiratory chain, so the fungi can't produce vital energy to form ATP [8][9][10][11].
The results of structure-activity relationships (SAR) research revealed that highly active novel carboxanilide derivatives containing two heterocyclic rings and alkyl substitution at the amino heterocyclic ring such as furametpyr and penthiopyrad ( Figure 1) show an expanded antifungal spectrum [5,12]. Both of these commercial carboxamide fungicides have remarkable activity, not only against basidiomycetes, but also the ascomycetes (such as gray mold, powdery mildew, and apple scab) [7,12,13]. Penthiopyrad is one novel carboxamide fungicide, whose mode of fungicidal action indicates that it inhibits succinate dehydrogenase and this results in the inhibition of the citric acid cycle and mitochondrial electron transport pathways. The pyridine moiety is a traditional group with broad activity which is often used in the molecular design of agrochemicals. Based on the molecular structure of penthiopyrad, a new series of pyrazole carboxamide compounds containing an active 3-trifluoromethyl-1H-pyrazole-4-carboxamide skeleton have been designed by introducing active pyridine rings into the amino part of pyrazole carboxamide. In this work 12 novel N-(substituted pyridinyl)-1-methyl-3-trifluoromethylpyrazole carboxamide analogues and 11 novel N-(substituted pyridinyl)-1-phenyl-3-trifluoromethylpyrazole carboxamide analogues were synthesized (Scheme 1). The antifungal activity against Gibberella zeae (G. zeae), Fusarium oxysporium (F. oxysporium), Cytospora mandshurica (C. mandshurica) were then evaluated.
Then intermediates 3 and 7 reacted with lithium hydroxide in THF to afford pyrazole acids 4 and 8, respectively. Subsequently 4 and 8 were refluxed in SOCl 2 to give pyrazole acid chlorides 5 and 9. Compounds 6a-l and 10a-k were then obtained by reaction of 5 or 9 with different substituted pyridylamines, respectively [17,18]. The 1 H-NMR, 13 C-NMR, IR and MS data and elemental analysis for the synthesized new compounds were consistent with assigned structures.

Antifungal Activity
As indicated at Table 1, most of the synthesized compounds possessed antifungal activity to a certain extent, and some of the synthesized compounds exhibited moderately antifungal activity, such as 6a, 6b and 6c that displayed more than 50% inhibition activity against G. zeae at 100 µg/mL, which were better than that of the commercial fungicides carboxin and boscalid. The inhibition rates of compound 6a against G. zeae, F. oxysporum and C. mandshurica were 73.2%, 53.5%, 48.7% respectively, better than that of the other compounds.

Toxicity of Target Compound 6b on Three Kinds of Pathogenic Fungi
The EC 50 values of compound 6b against three plant pathogens (G. zeae, F. oxysporum and C. mandshurica) were 81.3 µg/mL, 97.8 µg/mL, 176.5 µg/mL, respectively ( Table 2). The preliminary structure-activity relationship analysis indicated that the antifungal activity of the synthesized compounds was obviously decreased when the methyl group at N-1 of the pyrazole ring was replaced by a phenyl group.

Chemistry
All the melting points were determined with a XT-4 binocular microscope melting point apparatus (Beijing Tech Instrument Co., Beijing, China), while the thermometer was uncorrected. 1 H-NMR and 13 C-NMR were recorded with a JEOL-ECX500 NMR spectrometer operating at 500 MHz and 125 MHz respectively, and tetramethylsilane (TMS) was used as the internal standard. Mass spectral studies were conducted on an Agilent 5973 organic mass spectrometer. Elemental analysis was performed on an Elementar Vario-III CHN analyzer (Hanau, Germany). Analytical thin-layer chromatography (TLC) was performed on silica gel GF254. Column chromatographic purification was carried out using silica gel, the spots were visualized with ultraviolet (UV) light. All reagents were of analytical reagent grade or chemically pure. All solvents were dried, deoxygenated, and redistilled before use.

Preparation of the Target Compounds 6 and 10
To a solution of 2-amino-5-(trifluoromethyl) pyridine (163 mg, 1 mmol) and 80% sodium hydride (45 mg, 1.5 mmol) in anhydrous THF (10 mL), compound 5 (214 mg, 1 mmol) was added slowly, while the temperature was controlled under 10 °C, then reacted at ambient temperature for 10 h. The solvent was distilled off, and the residue was dissolved in ethyl acetate, washed with water and brine, dried over by anhydrous sodium sulfate. After the solvent was removed under reduced pressure, the residue was purified by column chromatography on silica gel (ethyl acetate/petroleum ether 1:15) to afford the target product, N-(5-(trifluoromethyl)-pyridin-2-yl)-1-methyl-3-(trifluoro-methyl)-1H-pyrazole-4carboxamide (6a). The following compounds 6b-l and 10a-k was prepared from the corresponding starting materials in a similar manner to that described for 6a. The physical and spectral data for compounds 6b-l and 10a-k are listed below.

Antifungal Bioassays
The fungicidal activity of the target compounds 6a-l and 10a-k were tested in vitro against G. zeae, F. oxysporium, and C. mandshurica, and their relative inhibitory ratio (%) was determined using the mycelium growth rate method [19,20]. Carboxin and boscalid were used as controls. After the mycelia grew completely, the diameters of the mycelia were measured, and the inhibition rate was calculated according to the formula: where I is the inhibition rate, D 1 is the average diameter of mycelia in the blank test, and D 2 is the average diameter of mycelia in the presence of those compounds. The inhibition ratios of those compounds at the dose 100 μg/mL have been determined, and the experimental results are summarized in Table 1.
Compound 6b has been tested against three pathogenic fungi (Gibberella zeae, F. oxysporum, C. mandshurica,) at concentrations of 100, 50, 25, 12.5, 6.25, 0 μg/mL. The EC 50 (effective dose for 50% inhibition μg/mL) values were estimated statistically by Probit analysis with the help of the Probit package of the SPSS software version 11.5 using a personal computer. The average EC 50 was taken from at least three separate analyses for inhibition of growth using the basic EC 50 program of SPSS version 11.5.

Conclusions
In conclusion, a novel series of pyrazolecarboxamide compounds containing an active 3-trifluoromethyl-1H-pyrazole-4-carboxamide skeleton were designed and synthesized using penthiopyrad as the lead structure. All target compounds were characterized by spectral data ( 1 H-NMR, 13 C-NMR, IR, MS) and elemental analysis. The compounds were tested in vitro for antifungal activity against three kinds of phytopathogenic fungi (Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica). The results showed that some of the synthesized N-(substituted pyridinyl)-1-methyl-3trifluoromethyl-1H-pyrazole-4-carboxamide compounds exhibited moderately antifungal activity, and compounds 6a, 6b and 6c displayed more than 50% inhibition activities against G. zeae at 100 μg/mL, which were better than that of the commercial fungicides carboxin and boscalid. The preliminary structure-activity relationship analysis indicated that the antifungal activity of the synthesized compounds was obviously decreased when a methyl at the 1-position of the pyrazole ring was substituted by a phenyl. The structures of the target products need to be optimized to enhance their antifungal activity. Future structural modification and biological evaluation to explore the full potential of this novel class of antifungal molecules are under way.