Background

The phytopathogenic fungus such as Gibberella zeae, which is also known as anamorph Fusarium graminearum, poses serious threats to agriculture. It is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world [1]. The high incidence of plant mortality and the lack of effective control methods make the pathogen are responsible for billions of dollars in economic losses worldwide each year [2]. In recent years, because of continued moist weather during the crop growing season and the failure of chemical control in some areas due to fungicides resistance in the pathogen population, Fusarium head blight (FHB) have been endemic in the wheat-producing areas of many province of China, which have caused an estimated 30 to 50% of reduction and even completely failure of harvests in many wheat-producing areas. Therefore, the design of new compounds to deal with G. zeae has become one of the most important areas for fungicide research today.

Pyrazole amide derivatives play an important role in development of medicine and pesticide due to their broad spectrum of biological activity [3–8]. Considerable attention for the study of synthesis and biological activity of pyrazole amide derivatives has been paid in recent years [9]. Currently, some pyrazole amide derivatives have been developed and commercialized as pesticide. As shown in Figure 1, furametpyr, penthiopyrad, tolfenpyrad, and chlorantraniliprole are known for their ability to protect certain plants from severe diseases and pests. In the past few years, hydrazone derivatives has been also attracted more and more attention due to their particular physical, chemical, and biological activities [10]. Hydrazone, a class of important substructure, can be found in numerous pharmaceutically active compounds [10, 11], and have been demonstrated to bear important biological activities (such as antibacterial activity [12, 13], analgesic activity [14], antinociceptive activity [15], insecticidal activity [16, 17], antimalarial activity [18], and antimicrobial activity [19, 20]). Some of the compounds containing hydrazone substructure have been commercialized as pesticides (such as benquinox, diflufenzopyr, and ferimzone) (Figure 1). In our recent publications [21, 22], several hydrazone derivatives have been synthesized and tested for their insecticidal activity and antibacterial activity, some of the hydrazone derivatives exhibited notable insecticidal activity against Plutella xylostella Helicoverpa armigera Culex pipiens pallens Laphygma exigua Spodoptera litura Nilaparvata lugens and Rhopalosiphum maidis[21], and some of the hydrazone derivatives containing a pyridine moiety possessed good antibacterial activity against Ralstonia Solanacearum[22].

Figure 1
figure 1

The commercialised pesticide of pyrazole amide and hydrazone derivatives.

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Keeping this in view, in an effort to discover new molecules against G. zeae, F. oxysporum and C. mandshurica, we sought to combine the sub-structural units of pyrazole amide and hydrazone together to design and synthesize novel pyrazole amide derivatives containing a hydrazone substructure. Thus, 11 novel pyrazolecarboxamide derivatives were synthesized using 3-dimethyl-1 H-pyrazol-5(4 H)-one and 2-amino-5-chloro-3-methylbenzoic acid as starting materials. All synthesized compounds were unequivocally characterized by IR, NMR and elemental analysis. The fungicidal activity on G. zeae, F. oxysporium and C. mandshurica were evaluated, the results showed that most of the synthesized compounds exhibit favorable antifungal activity against G. zeae and a certain antifungal activity against F. oxysporum and C. mandshurica, of which, compounds 7g and 7i display good activities at 50 mg/L. To the best of our knowledge, this is the first report on the antifungal activity of hydrazone derivatives containing a pyrazole moiety.

Results and discussion

Synthesis

The synthetic route to the title compounds is outlined in Scheme 1 [see Additional file 1. Intermediates 4 were prepared using 1,3-dimethyl-1 H-pyrazol-5(4 H)-one as starting materials. 1,3-dimethyl-1 H-pyrazol-5(4 H)-one was firstly subjected to Vilsmeier-Haack chloroformylation using N,N-dimethylformamide (DMF) and phosphorus oxychloride (POCl3) to yield 5-chloro-1,3-dimethyl-1 H-pyrazole-4-carbaldehyde 1[9], which was further oxidized by potassium permanganate and following chlorinated with thionyl chloride (SOCl2) to provide the intermediates 3, then intermediates 4 were prepared by treating 5-chloro-1,3-dimethyl-1 H-pyrazole-4-carbonyl chloride with 2-amino-3-methylbenzoic acid or 5-chloro-2-amino-3-methylbenzoic acid in CH2Cl2 in present of triethylamine in good yields, 2-(5-chloro-1,3-dimethyl-1 H-pyrazol-4-yl)-8-methyl-4 H-benzod[1, 3]oxazin-4-one 5 can be easily synthesized by reaction of acetic anhydride with 4 in excellent yield [21], however, it also can be prepared in a single step by the reaction of 3 with substituted 2-amino-3-methylbenzoic acid as describing in the literature [21, 22]. Finally, compounds 6 were conveniently obtained with excellent yield (>90%) by treatment of 5 with 80% hydrazine hydrate, subsequent treatment of 6 with different ketones and aldehydes (or hemiacetals) in ethanol at room temperature afforded the desired compounds (7a to 7s) with excellent yields. The structures of all new compounds were confirmed by their spectra (IR, 1 H NMR, 13 C NMR) and elemental analytical data. Additional file shows the structures, yields and elemental analysis data for title compounds in more detail [see Additional file 2. Moreover, hydrazone derivatives have two configurations due to the existence of double bond (C = N), for the title compounds, E and Z configuration can be observed in the 1 H-NMR spectra, and the E isomer was found predominantly, and the ratio between E and Z configuration can be calculated based on the integral area of E and Z isomers in 1 H-NMR spectra. Take compound 7k as an example, the E isomer of -CONHN proton can be found at δ 9.59, and the proton of Z isomer was appeared at δ 9.08, and the ratio of E isomer and Z isomer for 7k is 3.43, approximately.

Scheme 1
scheme 1

Synthetic route to target compounds 7a-7s.

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Biological activity and structure-activity relationship

All of the synthesized compounds 7a-7s was evaluated in vitro against three pathogenic fungi, G. zeae F. oxysporium, and C. mandshurica using the poison plate technique [23]. Carbendazim, one of the commercial fungicides for controlling G. zeae F. oxysporium, and C. mandshurica was used as positive control. The results provided in Table 1 indicated that most of the prepared compounds had weak to good antifungal activity against the tested fungi at 50 mg/L. Compounds 7a, 7f, 7g, 7h, 7i, 7j, 7l and 7q showed 40.82%, 47.78%, 50.32%, 40.82%, 49.05%, 48.73%, 40.19% and 45.89% activity against G. zeae, respectively. However, most of the synthesized compounds displayed lower activities against F. oxysporium and C. mandshurica, only compounds 7d and 7i showed 33.23%, 35.76% activity against F. oxysporum at 50 mg/L, respectively; compounds 7i possessed 37.25% activity against C. mandshurica at 50 mg/L. Structure activity relationship (SAR) based on activity against G. zeae showed that the compound containing two substructures of 5-chloro-1,3-dimethyl-1 H-pyrazole (compound 7g) is proven to be more active than other compounds; and the compound containing a furan moiety (7i) also display good activity, which is very close to that of compound 7g. We can conclude that changing the substituent on benzene could lead to a remarkable change in activity, for instance, the compounds without any substituent on benzene (7f, 7l) displayed much higher activities than the compounds with chorine at the 4-position of benzene (7m, 7r). Furthermore, the compounds with the same substituent but at different position on phenyl ring exhibited different activity. For instance, the activity of the compound 7 l which with 4-chloro substituent on phenyl ring was 40.19%, but the activity of the compound 7r which with 3-chloro substituent on phenyl ring was 6.65%; Moreover, different type of substituent on the phenyl ring (in R2 group) also affect the activity of the compounds, such as the compound 7q with methyl substituting at 4-position on phenyl ring possessed good activity on G. zeae, and the compound 7p with fluorine substituting at 2-position on phenyl ring displayed moderate activity, while the compound 7r with chlorine substituting at 3-position on phenyl ring demonstrate weak activity against G. zeae.

Table 1 Antifungal activity of the title compounds 7a-7s at 50 mg/L
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Experimental

Chemistry

Unless otherwise stated, all the reagents and reactants were purchased from commercial suppliers; melting points were uncorrected and determined on a XT-4 binocular microscope (Beijing Tech Instrument Co., China). The 1 H-NMR and 13 C-NMR spectra were recorded on a JEOL ECX 500 NMR spectrometer (JEOL Ltd., Japan) at room temperature operating at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR by using CDCl3 or DMSO as solvents and TMS as an internal standard; infrared spectra were recorded in KBr on a IR Pristige-21 spectrometer (Shimadzu corporation, Japan); elemental analysis was performed on an Elemen tal Vario-III CHN analyzer (Elementar, German). The course of the reactions was monitored by TLC; analytical TLC was performed on silica gel GF 254. Intermediates 1, 2, 3, and 4 were prepared according to the reported methods [9] and used without further purifications, the process for preparing of them can be found in Additional file 3.

Antifungal biological assay

All the compounds 7a-7 s were tested for in vitro antifungal activity using the poison plate technique [23]. The antifungal activity was evaluated against three pathogenic fungi, G. zeae F. oxysporium, and C. mandshurica, The results of preliminary bioassays were compared with the experimental data of a commercial agricultural fungicide, Carbendazim. Compounds were dissolved in dimethyl sulfoxide (1 mL) before mixing with potato dextrose agar (PDA, 90 mL). The compounds were tested at a concentration of 50 mg/L. All fungi were incubated in PDA at 27 ± 1°C for 4 days to get new mycelium for antifungal assay. Then mycelia dishes of approximately 4 mm diameter were cut from culture medium, and one of them was picked up with a sterilized inoculation needle and inoculated in the center of PDA plate aseptically. The inoculated plates were incubated at 27 ± 1°C for 5 days. DMSO in sterile distilled water served as negative control, while carbendazim acted as positive control. For each treatment, three replicates were conducted. The radial growth of the fungal colonies was measured and the data were statistically analyzed. The inhibitory effects of the test compounds in vitro on these fungi were calculated by the formula:

I (%)=[(C-T)/(C-0.4)]×100

Where C represents the diameter of fungi growth on untreated PDA, and T represents the diameter of fungi on treated PDA while I mean the inhibition rate.

Conclusion

In summary, a novel series of pyrazole amide derivatives bearing hydrazone moieties were synthesized. The synthesized compounds were characterized by spectral data (1 H NMR, 13 C NMR, IR) and elemental analysis. All of the compounds were subjected to fungicidal activities in vitro against G. zeae, F. oxysporium and C. mandshurica. The results indicated that the synthesized compounds possessed weak to good antifungal activities against the tested fungi, among them, compounds 7f, 7g, 7i, 7j displayed good antifungal activities against G. zeae; 7e and 7i display moderate activities against F. oxysporum and C. mandshurica respectively. Further studies are currently underway to optimize to enhance the antifungal activity of the pyrazole amide derivatives bearing hydrazone substructure.