Synthesis and fungicidal activity of novel 2,5-disubstituted-1,3,4- thiadiazole derivatives containing 5-phenyl-2-furan

A series of 2,5-disubstituted-1,3,4-thiadiazoles were synthesized using Lawesson’s reagent by an efficient approach under microwave irradiation in good yields. Their structures were characterized by MS, IR, 1H NMR, 13C NMR, and elemental analysis. Their in vitro and in vivo fungicidal activities revealed that the title compounds exhibited considerable activity against five selected fungi, especially to Phytophthora infestans. In order to illustrate the mechanism of title compounds against P. infestans, scanning electron micrographs (SEM) and transmission electron micrographs (TEM) were applied. The morphological and ultrastructural studies demonstrated that compound I18 led to swelling of hyphae, thickening and proliferating multilayer cell walls, excessive septation and accumulation of dense bodies. The bioassay results indicated compound I18 might act on cell wall biosynthesis, and blocked the nutrition transportation and led to cells senescence and death. Meanwhile, compound I18 had broad fungicidal activity against other twenty different kinds of fungi. These results suggested that title compounds were eligible to be development candidates and compound I18 as a promising lead compound was worthy to be further discovery, especially against P. infestans.

In continuation of our research on the synthesis of biological heterocyclic compounds, a series of novel 1,3,4-thiadiazole derivatives containing 5-phenyl-2-furan moiety was synthesized by an efficient approach under microwave irradiation (Fig. 1). Their fungicidal activity was evaluated.

Table 1. Comparison Between Conventional Heating Method and Microwave Assisted Method for Synthesis of Title Compounds in Terms of Time and Yield.
compound I18, the growth of mycelium was seriously inhibited. The rim of the colony was changed to be irregular and concave-convex, and was not as smooth as that of blank control. Furthermore, high concentration of compound I18 made this abnormal appearance much clear.
Scanning electron micrographs (SEM) images of P. infestans treated with compound I18 demonstrated the effects on the morphology of the hyphae (Fig. 3). SEM images revealed that the mycelium grew freshly and normally (the diameter was about 2.03 μ m) in the culture media of blank control with low density and fine structure (Fig. 3A,C). However, in culture media with compound I18 of 50 μ g mL −1 , mycelium grew abnormally with relatively high density of colony and some mycelia were entangled with each other. Some parts of the mycelium swelled (the diameter was about 4.14 μ m) and distorted to form the "beaded" morphology on the tip, and others ruptured to produce shriveled and empty mycelia (Fig. 3B,E).
P. infestans mycelial tip (5 mm) from the rim of an actively growing colony on PDA medium was investigated by TEM (Fig. 4). The blank control mycelia of P. infestans grown in the absence of compound I18 demonstrated some cytological and ultrastructural features, which were typical vegetative hyphae of the genus 42,43 (Fig. 4A,D). There were normal cell wall deposition and undulated plasmalemma, and cytoplasm containing vacuole and mitochondria were observed.
In the presence of compound I18 at 50 μ g mL −1 , extensive cell wall thickening was the most conspicuous ultrastructural variation observed in hyphae (Fig. 4C,D). The number of vacuoles increased and vacuoles were distorted and disrupted under treatment with compound I18 (3D). Vacuoles play an important role in mycelial growth, meanwhile with the function of maintaining fungal turgor pressure. The phenomena caused by compound I18 were roughly the same as those caused by pyrimorph and dimethomorph, which had an effect on the biosynthesis of cell walls [43][44][45] . Hence, compound I18 may retard fungal growth by acting on cell wall synthesis. The "beaded" hyphae were separated by the cell walls and false septa (Fig. 4C,H), which blocked the nutrition transportation and led to cells senescence and death. It revealed that the multilayer cell walls were formed and there was cytoplasmic substance in the interlayer of the proliferative cell walls (Fig. 4D-F). Gradually the cell walls exfoliated obviously and the cytoplasmic substance osmosis in the interlayer increased. Finally the cell wall ruptured and the cytoplasmic substance outflowed (Fig. 4F). That was the reason of producing shriveled and empty mycelia observed in SEM (Fig. 3B,E). Mitochondria and cell nuclear had the same appearance as blank control hyphae.

Conclusions
In summary, we synthesized a series of 2-substituted phenyl-5-(5′ -substituted phenyl-2′ -furoyl)-1,3, 4-thiadiazoles using Lawesson's reagent by an efficient approach under microwave irradiation in good yields. The title compounds displayed significant fungicidal activity against various fungi, especially exhibited excellent fungicidal activity against P. infestans. Moreover, it was speculated that compound I18 might act on the synthesis of cell walls from morphological and ultrastructural studies by SEM and TEM, which also revealed that compound I18 could block the nutrition transportation and led to cells senescence and death. These results suggested that title compounds were eligible to be development candidates and compound I18 as a promising lead compound was worthy to be further discovery, especially against P. infestans. However, further research should also be undergoing to confirm the specific mode of action of the title compounds.

Materials and Methods
Instruments. All   while tetramethylsilane was used as an internal standard. Analytical thin-layer chromatography was carried out on silica gel 60 F254 plates, and spots were visualized with ultraviolet light. Mass spectra were measured on a Bruker APEX IV spectrometer (Bruker, Fallanden, Switzerland). Elemental analyses were performed on a Vario EL elemental analyzer. The microwave-assisted reaction was carried out with a CEM Microwave synthesizer (CEM Discover S-Class).
General procedure for the synthesis of title compounds. A mixture of diacylhydrazines 3 (1 mmol) and Lawesson's reagent (1.5 mmol) in toluene (10 mL) was refluxed for 5-7 h. After completion of the reaction as monitored by TLC, the reaction was quenched by addition of the saturated NaHCO 3 solution. The organic layer was washed with brine, dried over MgSO 4 and concentrated in vacuo. The residue was recrystalized from ethanol to afford pure products I.
General procedure for the synthesis of title compounds by microwave radiation. All reactions were carried out in a pressure tube, sealed with a Teflon septum. The mixture of the diacylhydraziness 3 (1 mmol) and Lawesson's reagent (1.5 mmol) in toluene (20 mL) was taken in the pressure tube. The pressure tube was introduced to the centre of a CEM Discover microwave oven and irradiated for 15 min at 150 W (reaction temperature was set to 110 °C). After completion of the reaction, the reaction mixture was allowed to cool, and then, it was poured slowly with stirring into the saturated NaHCO 3 solution. The organic layer was washed with brine, dried over MgSO 4 and concentrated in vacuo. The residue was recrystalized from ethanol to afford pure products I.         13 13 13 13 13 13 13 13

Bioassays. In vitro fungicidal activity. In vitro fungicidal activity of the title compounds against
Phytophthora infestans, Valsa mali, Phomopsis aspamgi, Cladosporium fulvum Cke., Alternaria tenuis Nees and other twenty panthogenic fungi listed in Table 4 were evaluated using mycelium growth rate test 22,30 . The tested compounds were dissolved in DMSO (dimethyl sulfoxide) and mixed with sterile molten potato dextrose agar to a final concentration of 50 μ g mL −1 . Commercial fungicides pyrimorph and hymexazol were used as controls against the above mentioned fungal pathogens under the same conditions. Three replicates were performed. The relative inhibitory rate of title compounds compared to blank control was calculated via the following equation (1): In which, I stands for the rate of inhibition (%), C is the diameter of mycelia in the blank control test (in mm), and T is the diameter of mycelia in the presence of tested compounds (in mm). The EC50 values of title compounds were evaluated using logit analysis ( Table 2). EC50 results were analyzed using the statistical data processing system (DPS, 10.15, Zhejiang, China).
In vivo antifungal activity. Using the pot culture test 22,28 , in vivo antifungal activities of the title compounds against P. infestans, V. mali, P. aspamgi and C. fulvum Cke. were evaluated in greenhouse along with commercial fungicides pyrimorph and hymexazol as controls.
The culture plates were cultivated at 24 ± 1 °C. Soaking cucumber seeds in water for 2 h at 50 o C, and then, keeping the seeds moist for 24 h at 28 o C in an incubator. When the radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite and peat. Cucumber plants used for inoculations were at the stage of two seed leaves.
Tested compounds were confected to 2.5% EC (emulsifiable cocentration) formulations, in which pesticide emulsifier 600 (2.125%) and pesticide emulsifier 500 (0.375%) were the additives, DMSO (0.1%) was the solvent, and xylene was the co-solvent. The formulation was diluted to 500 μ g mL −1 with water. The pathogenic fungi were inoculated on the surface of seed leaves and then the solution of title compounds was sprayed with a hand sprayer, respectively. Three replicates for each treatment were applied. After inoculation, the plants were maintained at 24 ± 1 o C and above 80% relative humidity.
When the untreated cucumber plant (blank control) fully developed symptoms, the fungicidal activity was assessed. The area of inoculated leaves covered by disease symptoms was evaluated and compared to that of untreated ones to determine the average disease index. The relative control efficacy of compounds compared to the blank assay was calculated via the following equation (2): where I is relative control efficacy, CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing. The results are shown in Table 3.
Electron microscopy 42,43 . Scanning electron microscopy (SEM). Mycelial tip (5 mm) of P. capsici from an actively growing colony on PDA medium, which were treated by I18 at 50 μ g mL −1 , were cut from the edge of the colony after cultured for 72 h. The tips were treated with 4% glutaraldehyde at 4 o C, and then, rinsed with 0.1 M phosphate buffer (pH 7.3), and fixed with 1% w/v osmium tetraoxide solution. The mycelial tips were dehydrated using a series of acetone solutions in the order of concentration 30, 50, 70, 80, and 90% anhydrous acetone, after rinsed with 0.1 M phosphate buffer three times. After completing the processes of drying at critical point, mounting, and gold spraying, the mycelial tip was examined by a scanning electron microscope (S-3400N, Hitachi, Nissei Sanyo, Japan) with an accelerating voltage of 18-20 kV.
Transmission electron microscopy (TEM). The mycelial tip was prepared according to the same method mentioned above. After dehydrating and embedding in Epon 112, thin sections were cut and double-stained with uranyl acetate and lead citrate. The grids were examined with a JEOL-1230 (JEOL, Tokyo, Japan) transmission electron microscope.