Synthesis and Antifungal Activity of Novel Sulfone Derivatives Containing 1,3,4-Oxadiazole Moieties

A series of new sulfone compounds containing 1,3,4-oxadiazole moieties were synthesized. The structures of these compounds were confirmed by spectroscopic data (IR, 1H- and 13C-NMR) and elemental analyses. Antifungal tests indicated that all the title compounds exhibited good antifungal activities against eight kinds of plant pathogenic fungi, and some showed superiority over the commercial fungicide hymexazol. Among them, compounds 5d, 5e, 5f, and 5i showed prominent activity against B. cinerea, with determined EC50 values of 5.21 μg/mL, 8.25 µg/mL, 8.03 µg/mL, and 21.00 µg/mL, respectively. The present work demonstrates that sulfone derivatives such as 5dcontaining a 1,3,4-oxadiazole moiety can be used as possible lead compounds for the development of potential agrochemicals.


Introduction
The emergence of fungal resistance to existing fungicides has posed a serious concern for pesticide professionals during the last decade, and the desire for safer and more effective agrochemicals with reduced environmental toxicity also remains a high priority [1]. A further aim is to produce novel fungicides that do not impede the role of beneficial organisms in plant development and which do not persist in the environment and food chains [2], so the synthesis and antifungal evaluation of new compounds is greatly needed.
The SAR suggested that 2-(methylsulfonyl)-1,3,4-oxadiazole is the core ring system that affords potent antifungal activities [22,23]. Given the apparent lack of scope for changes to the core ring system, the majority of our efforts were directed towards compounds of the general structure of the title compounds in which the core ring system was kept constant and the peripheral groups were altered. As a consequence, in an attempt to increase the fitting to the pharmacophoric model, and possibly to obtain new fungicides, we report here the synthesis, characterization and antifungal activity of novel sulfone derivatives containing 1,3,4-oxadiazole moieties 5, as shown in Scheme 1.

Scheme 1.
Synthetic route to the title compounds.

Chemistry Benzohydrazide
The key intermediate 2-thiol-5-substituted-1,3,4-oxadiazole (3) was prepared by cyclization of substituted phenylhydrazide, potassium hydroxide, and carbon disulfide in ethanol under reflux conditions. The key point of this reaction is that water must be removed completely; as the presence of even a little water may cause the cyclization to fail.
Although the electron rich methylthio moiety in compounds 4 can be oxidized to a sulfone by a variety of agents such as m-CPBA [24] or H 2 O 2 catalyzed by methyltrioxorhenium [25], unfortunately, most of these reagents are not satisfactory. They are either harmful or expensive, and a simple procedure is not easily available. In this experiment, the methylthio moiety in 4 was oxidized with potassium permanganate [26] in glacial acetic acid to afford the corresponding methylsulfonyl species 5, the advantage of this oxidation is that it uses an inexpensive oxidant without a catalyst; furthermore, the reaction is quickly complete and easy to work up. The physical characteristics, IR, 1 H-NMR, 13 C-NMR and elemental analyses data for all the synthesized compounds are reported in the Experimental section.

The Antifungal Activities of Oxadiazole Methyl Sulfones
The inhibitory effects of the synthesized oxadiazole methyl sulfone compounds on phytopathogenic fungi was studied. Two fungi, F. oxysporum and C. mandshurica, representing typical fungi often occurring in the Chinese agro-ecosystem were chosen for fungicide screening using the mycelial growth rate method. The results were compared with that of the commercial agricultural fungicide hymexazol (a broad spectrum fungicide), as indicated in Table 1. As indicated in Table 1, at the concentration of 50 µg/mL, all of the tested compounds exhibited good inhibitory effects against F. oxysporum, and all of them showed superiority over the commercial fungicide hymexazol. Among them, compounds 5b, 5e and 5f almost completely inhibited the growth of F. oxysporum, with control efficacies of 97.5%, 98.8%, and 94.0%, respectively. Compound 5g showed 89.6% fungicidal activity against F. oxysporum, while the others had inhibition activities between 67.1% and 77.0%. As the results described in Table 1 indicate, all of the tested compounds possessed promising inhibitory effects against C. mandshurica. Among them, compounds 5e, 5f, 5g, and 5h almost completely inhibited the growth of C. mandshurica, with inhibition values of more than 90.0%, whereas compound 5b showed 89.7% fungicidal activity against C. mandshurica, and compounds 5a, 5c, 5d, 5i, and 5j inhibited the growth of C. mandshurica by 68.3%, 78.2%, 64.0%, 79.6%, and 64.0%, respectively.

Toxicity of Some Title Compounds on 8 Kinds of Pathogenic Fungi
We choose some representative compounds with good, ordinary antifungal activity, as indicated in the previous bioassays, to conduct further work which disclosed that some sulfone compounds containing a methyl oxadiazole showed remarkable inhibitory effect on eight kinds of plant pathogenic fungi, which represent typical fungi often occurring in the Chinese agro-ecosystem. The results are summarized in Table 2. As indicated in Table 2, all of the test compounds possessed prominent antifungal activities against eight plant pathogens (F. oxysporum, C. mandshurica, R. solani, T. cucumeris, S. sclerotiorum, C. gloeosporioides, P. infestans, B. cinerea), with EC 50 values between 5.21 µg/mL to 98.18 µg/mL. Among them, the bioassay results showed that compounds 5d, 5e, 5f, and 5i showed prominent activity against R. solani (EC 50 values of 6.43 µg/mL, 21.23 µg/mL, 16.16 µg/mL, and 20.02 µg/mL, respectively), which was superior to the activity of the commercial fungicide hymexazol (38.64 µg/mL). B. cinerea has been identified as a pathogen of more than 235 plant species, including grapes, lettuce, tomatoes, tobacco, and strawberries, producing a gray powdery mold on the infected crops. To our delight, compounds 5d, 5e, 5f, and 5i showed prominent activity against B. cinerea, the EC 50 values of 5.21 µg/mL, 8.25 µg/mL, 8.03 µg/mL, and 21.00 µg/mL, respectively, all of them being more effective than the positive control. The results also suggested that compound 5d can be used as a possible lead compound for the development of potential agrochemicals. Compounds 5d, 5e, 5f, and 5i showed ordinary activity against S. sclerotiorum, the EC 50 values of 8.49 µg/mL, 14.97 µg/mL, 17.20 µg/mL, and 14.78 µg/mL, respectively, less effective than the positive control hymexazol (7.76 µg/mL). Compounds 5d, 5e, 5f, and 5i showed weak effectivity against C. mandshurica, with EC 50 values of 79.92 µg/mL, 38.27 µg/mL, 98.18 µg/mL, and 44.50 µg/mL, respectively. Generally speaking, compounds 5d, 5e, 5f, and 5i showed weak to normal effective against F. oxysporum, T. cucumeris, S. sclerotiorum, C. gloeosporioides, and P. infestans, with EC 50 values ranging from 14.73 µg/mL to 85.41 µg/mL. As indicated in Table 1 and Table 2, with C. mandshurica as the experimental target the relationship between antifungal activity and substituent attached to the benzene ring showed that a fluorinated phenyl group, such as compound 5e (R = 2,6-2F), had better antifungal activity (97.8% fungicidal activity against C. mandshurica at 50 µg/mL, and the EC 50 was 38.27 µg/mL).
Compound 5d had prominent antifungal activities against most of the tested fungi, and showed a broad-spectrum bioactivity; the inhibition effects of compound 5d on mycelia growth in vitro at different concentrations are illustrated in Figure 1.

General
Unless otherwise indicated, all common reagents and solvents were used as obtained from commercial suppliers without further purifications. The melting points of the products were determined on a XT-4 binocular microscope (Beijing Tech Instrument Co., China) and were not corrected. The IR spectra were recorded on a Bruker VECTOR 22 spectrometer in KBr disks. 1 H-and 13 C-NMR spectra (solvent CDCl 3 ) were recorded on a JEOL-ECX 500 NMR spectrometer at room temperature using TMS as an internal standard, chemical shift values (δ) are given in parts per million. Elemental analysis was performed on an Elementar Vario-III CHN analyzer. Analytical TLC was performed on silica gel GF254. Column chromatographic purification was carried out using silica gel.

Preparation of the Intermediates 1-3
Starting from the corresponding acid, and following the reported method [27,28], intermediates 3 were synthesized in three steps involving esterification, hydrazination, and cyclization. Specifically, 2-thiol-5-substituted-1,3,4-oxadiazole intermediates 3 were prepared by the reaction of substituted phenylhydrazide intermediates 2, potassium hydroxide, and carbon disulfide in ethanol under reflux conditions. Intermediates 2 were synthesized from intermediates 1 and hydrazine hydrate in methanol under reflux condition. Intermediates 1 (substituted benzoic acid esters) were made through esterification reactions from the starting acids.

Preparation of the Intermediates 4
To a solution of 2-thiol-5-substituted-1,3,4-oxadiazole 3 (2 mmol) and sodium hydroxide (0.08 g, 2 mmol) in water (20 mL), dimethyl sulfate (0.32 g, 2.5 mmol) was added dropwise. After stirring for 3 h at room temperature (20-23 °С), for solid products, the precipitate was filtered off, washed with 5% Na 2 CO 3 solution and distilled water, dried and recrystallized from ethanol to afford the white solid products 4; for liquid products, the reaction mixture was washed with 5% Na 2 CO 3 solution, and then extracted with ethyl ether (4 × 20 mL), the ethyl ether layer was dried with anhydrous Na 2 SO 4 , after removal of the organic solvent under reduced pressure, and oily products 4 were thus obtained.

Preparation of the Title Compounds 5
A solution of 2-methylthio-5-substituted-1,3,4-oxadiazole 4 (3.83 mmol) in glacial acetic acid (10 mL) was treated dropwise at 10 °C with potassium permanganate (0.78 g, 4.98 mmol) as a 5% aqueous solution over 20 min. The reaction was allowed to proceed for an additional 20 min, 40% strength aqueous sodium hydrogen sulphite solution was subsequently added until the mixture was decolorized, and diluted with 50 mL of water. The product was filtered and recrystallised from ethanol give the title compounds 5.

Antifungal Activities Test
The antifungal activities were tested against two pathogenic fungi, Fusarium oxysporum and Cytospora mandshurica, by the poison plate technique [29]. 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 μg/mL. 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 hymexazol 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 means the inhibition rate.
Some of the title compounds were tested against eight pathogenic fungi namely F. oxysporum, C. mandshurica, Phytophthora infestans, Rhizoctonia solani, Thanatephorus cucumeris, Colletotrichum gloeosporioides, Botrytis cinerea and Sclerotinia sclerotiorum at different concentrations of 50, 25, 12.5, 6.25, 3.125, 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 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 version SPSS 11.5.

Conclusions
In summary, a series of new sulfone compounds containing 1,3,4-oxadiazole moieties and based on the lead compound Ia was designed and synthesized. The title compounds showed promising antifungal activities against some typical fungi often occurring in the Chinese agro-ecosystem, including F. oxysporum, C. mandshurica, R. solani, T. cucumeris, S. sclerotiorum, C. gloeosporioides, P. infestans, B. cinerea. Among them, some compounds showed superiority over the lead compound Ia, such as compound 5b with EC 50 values ranging from 5.21 μg/mL to 79.2 μg/mL, and some, such as compounds 5b, 5d and 5e showed superiority over the commercial fungicide hymexazol. In particular, compounds 5d, 5e, 5f, and 5i showed prominent activity against B. cinerea, with EC 50 values of 5.21 µg/mL, 8.25 µg/mL, 8.03 µg/mL, and 21.00 µg/mL, respectively. These results demonstrated that some compounds such as 5d can be used as possible lead compounds for the development of potential agrochemicals.