Synthesis and Biological Evaluation of Benzimidazole Phenylhydrazone Derivatives as Antifungal Agents against Phytopathogenic Fungi

A series of benzimidazole phenylhydrazone derivatives (6a–6ai) were synthesized and characterized by 1H-NMR, ESI-MS, and elemental analysis. The structure of 6b was further confirmed by single crystal X-ray diffraction as (E)-configuration. All the compounds were screened for antifungal activity against Rhizoctonia solani and Magnaporthe oryzae employing a mycelium growth rate method. Compound 6f exhibited significant inhibitory activity against R. solani and M. oryzae with the EC50 values of 1.20 and 1.85 μg/mL, respectively. In vivo testing demonstrated that 6f could effectively control the development of rice sheath blight (RSB) and rice blast (RB) caused by the above two phytopathogens. This work indicated that the compound 6f with a benzimidazole phenylhydrazone scaffold could be considered as a leading structure for the development of novel fungicides.


Introduction
As an important chemical scaffold in medicinal chemistry and agrochemicals [1], benzimidazole derivatives have been attracting the interest of organic chemists for their varieties of biological activities, such as antifungal [2], anticancer [3], antibacterial [4], antioxidant [5], anti-inflammatory [6], and anti-parasitic [7]. In particular, benzimidazole fungicides, with benzimidazole as the core substructure (Figure 1), have been widely used throughout the world to fight against destructive plant pathogens, such as Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Magnaporthe oryzae [8,9]. However, excessive use of benzimidazole fungicides has led to a series of negative problems, such as pesticide residues, drug resistance and serious environmental pollution [10,11]. Therefore, the development of new benzimidazole fungicides with high activity, good selectivity and eco-friendly properties is extremely urgent.
In our previous work, molecules with phenylhydrazone showed significant antifungal and antioxidant activities [12,13]. Typically, 1,2,3-triazole phenylhydrazone derivatives displayed potent antifungal activity in vitro and in vivo. Inspired by these studies, we initiated a study to synthesize benzimidazole phenylhydrazone derivatives to screen high activity antifungal compounds. Their antifungal activities against two phytopathogenic fungi (R. solani and M. oryzae) were evaluated in vitro and in vivo, and their structure-antifungal activity relationships were also discussed.

Crystal Structure of Compound 6b
Among these compounds, the crystal structure of compound 6b was determined by X-ray diffraction analysis. Figure 2 gave a perspective view of 6b with the atomic labelling system. The Xray data had been deposited at the Cambridge Crystallographic Data Centre with the CCDC number 1038194. The result demonstrated that the -C=N-NH-bond bears an (E)-configuration rather than (Z)configuration.

Synthesis of Compounds
A total of 35 target compounds were synthesized following the routes outlined in Scheme 1. The compounds 2a-2d were synthesized by the reaction of o-phenylenediamine with glycolic acid in 75%-85% yield. Then the compounds 2a-2d were further oxidized to aldehydes (3a-3d) using MnO 2 in ethyl acetate. The aldehydes were purified by filtration to remove the excess MnO 2 , then compounds 3a-3d were obtained as white crystals in 60%-76% yield. The anilines 4a-4s were diazotized by NaNO 2 , then restored by SnCl 2 to obtain substituted phenylhydrazines 5a-5s in 55%-80% yield. Finally, 3a-3d and 5a-5s were condensed to form the -C=N-NH-bond, giving benzimidazole derivatives 6a-6ai in 45%-80% yield. 1 H-NMR, MS (ESI), and elemental analysis (CHN) data of the target compounds were in fully accordance with their assigned structures. Among them, compounds 6a, 6q, 6t, 6x, and 6z had been reported previously [14,15], while the remaining 30 compounds were first reported in this study.

Crystal Structure of Compound 6b
Among these compounds, the crystal structure of compound 6b was determined by X-ray diffraction analysis. Figure 2 gave a perspective view of 6b with the atomic labelling system. The X-ray data had been deposited at the Cambridge Crystallographic Data Centre with the CCDC number 1038194. The result demonstrated that the -C=N-NH-bond bears an (E)-configuration rather than (Z)-configuration.

Antifungal Activities In Vitro
Different concentrations of compounds 6a-6ai were evaluated for their antifungal activities in vitro against R. solani and M. oryzae. The EC50 values were calculated using linear-regression analysis, with validamycin A, carbendazim and isoprothiolane as positive controls (Table 1). Figure 2. X-ray diffraction structure of 6b, thermal ellipsoids was drawn on the 35% probability.

Antifungal Activities In Vitro
Different concentrations of compounds 6a-6ai were evaluated for their antifungal activities in vitro against R. solani and M. oryzae. The EC 50 values were calculated using linear-regression analysis, with validamycin A, carbendazim and isoprothiolane as positive controls (Table 1). Table 1. Antifungal activity of compounds against two phytopathogens a .

Compound
R  The results were expressed as the mean ± SD of triplicate experiments; b Compounds that were first reported.
Based on the results of the antifungal activities shown in Table 1, 6f was selected for further antifungal activity tests in vivo.

Inhibition of 6f on the Sclerotia Germination of R. solani
As shown in Table 2, when grain dry sclerotia was inoculated, germination could be observed in the water of the blank control after a certain incubation period (about four days) at 25 • C in the dark. 6f had better controlling effects than sclerotia.

Protective Activity of 6f against Rice Sheath Blight (RSB) In Vivo
Compound 6f was selected for evaluating the protective activity against RSB caused by R. solani. As shown in Figure 3 and Table 3, four days after inoculating with R. solani, significant differences could be observed between the treated and untreated groups. Tan spots appeared on the leaf sheath of the blank control, and the scab length reached 16.4 mm. When at a concentration of 200 µg/mL, the protective effect of 6f could reach 68.9% in vivo, close to validamycin A (73.8%), which was commonly used as a fungicide against RSB. When at a concentration of 100 µg/mL, the protective effect of 6f and validamycin A reached 66.5% and 61.0% respectively. Thus, 6f showed similar activity with validamycin A against RSB in vivo.

Inhibition of 6f on the Conidium Germination of M. oryzae
As shown in Figure 4, conidia of M. oryzae were inoculated after 24 h, germination could be observed in the water of the blank control under the microscope with a germination rate of 100%, and 1% DMSO had no effect on the conidium germination. The conidia could not form the germ tube or appressorium when incubated with 6f at the concentration of 1.0 μg/mL (1% DMSO).

Protective Activity of 6f against Rice Blast (RB) In Vivo
The efficacy of the protective activity experiment is shown in Figure 5 and Table 4, fungus spores were inoculated on leaves, blast lesions could be observed after 7 days. At a concentration of 50 μg/mL, the protective effect of 6f could reach 21.6%, whereas carbendazim treatment resulted in 88.0%.

Inhibition of 6f on the Conidium Germination of M. oryzae
As shown in Figure 4, conidia of M. oryzae were inoculated after 24 h, germination could be observed in the water of the blank control under the microscope with a germination rate of 100%, and 1% DMSO had no effect on the conidium germination. The conidia could not form the germ tube or appressorium when incubated with 6f at the concentration of 1.0 µg/mL (1% DMSO).

Inhibition of 6f on the Conidium Germination of M. oryzae
As shown in Figure 4, conidia of M. oryzae were inoculated after 24 h, germination could be observed in the water of the blank control under the microscope with a germination rate of 100%, and 1% DMSO had no effect on the conidium germination. The conidia could not form the germ tube or appressorium when incubated with 6f at the concentration of 1.0 μg/mL (1% DMSO).

Protective Activity of 6f against Rice Blast (RB) In Vivo
The efficacy of the protective activity experiment is shown in Figure 5 and Table 4, fungus spores were inoculated on leaves, blast lesions could be observed after 7 days. At a concentration of 50 μg/mL, the protective effect of 6f could reach 21.6%, whereas carbendazim treatment resulted in 88.0%.

Protective Activity of 6f against Rice Blast (RB) In Vivo
The efficacy of the protective activity experiment is shown in Figure 5 and Table 4, fungus spores were inoculated on leaves, blast lesions could be observed after 7 days. At a concentration of 50 µg/mL, the protective effect of 6f could reach 21.6%, whereas carbendazim treatment resulted in 88.0%.

Reagents and Analysis
All reagents bought from Alfa Aesar (Ward Hill, MA, USA), Aladdin (Shanghai, China) or Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) were pure analytical grades and used without further treatments. Reactions were monitored by TLC using silica gel coated glass slides (silica-gel 60 GF254, Qingdao Haiyang Chemical, Qingdao, China). Melting points were measured on WRS-1B digital melting-point apparatus (SPSIC, Shanghai, China), uncorrected. 1 H-NMR spectra were recorded on a Bruker Avance III 400 NMR spectrometer (Bruker, Stuttgart, Germany). The chemical shifts (δ) are reported in ppm with reference to internal TMS, and coupling constants (J) are given in Hz. ESI-MS spectra were recorded on a Bruker UHR-TOF maxis (Bruker, Billerica, MA, USA). X-ray single crystal diffraction analysis was conducted on a Bruker D8 Venture diffractometer (Bruker, Karlsruhe, German). Elemental analyses were performed on a Elementar Vario MICRO instrument (Elementar, Langenselbold, German) and were within ±0.4% of the theoretical values.

Synthesis and Purification of Compound 2a-2d
The procedures were conducted according to the literature [16]. Glycolic acid (15.24 g, 0.2 mol) was dissolved in 40 mL HCl (20% aqueous) in the flask. When the temperature reached 65 °C, compound 1a-1d (16.27 g, 0.15 mol) was added at three times, then refluxed at 100 °C for 5 h. After

Reagents and Analysis
All reagents bought from Alfa Aesar (Ward Hill, MA, USA), Aladdin (Shanghai, China) or Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) were pure analytical grades and used without further treatments. Reactions were monitored by TLC using silica gel coated glass slides (silica-gel 60 GF254, Qingdao Haiyang Chemical, Qingdao, China). Melting points were measured on WRS-1B digital melting-point apparatus (SPSIC, Shanghai, China), uncorrected. 1 H-NMR spectra were recorded on a Bruker Avance III 400 NMR spectrometer (Bruker, Stuttgart, Germany). The chemical shifts (δ) are reported in ppm with reference to internal TMS, and coupling constants (J) are given in Hz. ESI-MS spectra were recorded on a Bruker UHR-TOF maxis (Bruker, Billerica, MA, USA). X-ray single crystal diffraction analysis was conducted on a Bruker D8 Venture diffractometer (Bruker, Karlsruhe, German). Elemental analyses were performed on a Elementar Vario MICRO instrument (Elementar, Langenselbold, German) and were within ±0.4% of the theoretical values.

Synthesis and Purification of Compound 2a-2d
The procedures were conducted according to the literature [16]. Glycolic acid (15.24 g, 0.2 mol) was dissolved in 40 mL HCl (20% aqueous) in the flask. When the temperature reached 65 • C, compound 1a-1d (16.27 g, 0.15 mol) was added at three times, then refluxed at 100 • C for 5 h.

Synthesis and Purification of Compound 5a-5s
Substituted aniline 4a-4s (50 mmol) was dissolved in the 50 mL HCl (18%, aqueous) in the ice bath. NaNO 2 (50 mmol) dissolved in 50 mL water was added dropwise. The reaction mixture was stirred for 1 h to obtain a clear solution. Then the solution of SnCl 2 (0.1 mol) in 30 mL of concentrated HCl was added dropwise at 0 • C. The mixture was stirred at room temperature for 2 h. Afterwards, the mixture was extracted with 50 mL EtOAc and the organic impurities were discarded. Then the solution was basified with NaOH (40%, aqueous) until it reached pH 7.0. The reaction mass was extracted with EtOAc three times. Finally, substituted phenylhydrzine 5a-5s was afforded after being vapored under reduced pressure (in 55%-80% yield) [12].

Protective Activity of 6f against RSB In Vivo
For evaluating the antifungal activity of 6f against RSB in vivo, rice cultivar (Shanyou 63) was sown and grown in plastic pots in the greenhouse [22]. The compound 6f at the concentration of 200 µg/mL and 100 µg/mL was sprayed on the cultivar, respectively, and inoculated with R. solani 24 h later. Validamycin A at the concentration of 200 µg/mL and 100 µg/mL was used as the positive control. All the treatments were replicated for 20 plants and incubated in the greenhouse with an average midday relative humidity of 85%. A visual disease assessment was made 7 days after inoculating with R. solani by measuring the lesion length. The protective efficacy was calculated as: Protection efficacy = ((average lesion length of control − average lesion length of treated group)/average lesion length of control) × 100%.

Inhibition of 6f on the Conidium Germination of M. oryzae
Abundant spores of M. oryzae were collected and suspended to a concentration of 5 × 10 4 spores per milliliter in sterile water for conidium germination test. A series of concentrations of tested samples (6f and control) were mixed with conidial suspension, respectively. Aliquots of 10 µL of prepared conidial suspension were placed on separate glass slides in triplicate, which were incubated in a moisture chamber at 25 • C for 24 h. Each slide was then observed under the microscope, and the appressorium formation percentage was examined [23].

Protective Activity of 6f against RB In Vivo
Compound 6f was further measured for its antifungal activity in vivo against RB on rice (Shanyou 63). Different concentrations (50 µg/mL and 100 µg/mL) of 6f (4 mL) were sprayed on two-week old seedlings of rice leaves. After 24 h, previously prepared spore suspension of M. oryzae was also sprayed with 0.2% (w/v) gelatin. Plants were kept at 25 • C with 90% humidity and in a 12/12 h light/dark cycle. The positive control is carbendazim of 25 and 50 µg/mL. Lesion formation was observed daily and the protective efficacies were calculated after 7 days [24].

Conclusions
In conclusion, a series of benzimidazole derivatives had been synthesized and investigated for the antifungal activities against R. solani and M. oryzae in vitro and in vivo. The result showed that many of the compounds had significant antifungal activities. Compound 6f exhibited most potent antifungal activity with an EC 50 value of 1.20 µg/mL against R. solani, which was better than carbendazim (1.84 µg/mL). The protective activity of 6f in vivo reached 66.5% at 100 µg/mL, whereas validamycin A was 61.0%. At the same time, 6f displayed moderate activity against M. oryzae with EC 50 value of 1.85 µg/mL, and inhibited the conidium germination at 1.0 µg/mL. The result indicated that the compound 6f with a benzimidazole phenylhydrazone scaffold could be considered as a leading structure for the development of novel fungicides.