Synthesis and Antifungal Activity of 5-Chloro-6-Phenyl-pyridazin-3(2H)-one Derivatives

An effective method has been developed for the preparation under mild conditions of novel pyridazine derivatives from the easily accessible starting materials mucochloric acid and benzene. All the synthesized compounds were fully characterized and some of them displayed good antifungal activities against G. zeae, F. oxysporum and C. mandshurica in preliminary antifungal activity tests.


Chemistry
3,4-Dichloro-5-phenylfuran-2(5H)-one (1) was synthesized via Friedel-Crafts reaction by employing mucochloric acid and benzene as starting materials in the presence of the Lewis acid AlCl 3 , in accordance with the known synthetic protocols described in the literature [27,28].
Preparation of the compound 5-chloro-6-phenylpyridazin-3(2H)-one (2) has been described previously [27,29], but unfortunately in our hands under the same reaction conditions the yield was much lower (46%) compared to the one reported in the literature. It was observed that the type of solvent, reaction temperature and reaction time were the most important parameters affecting the purity and yield of the final product. The best result, affording a yield of 68%, was achieved when the reaction was perfomed in the solvent DMF at 80°C for 40 min.
5-Chloro-6-phenyl-2-substituted-pyridazin-3(2H)-ones 3a-3h were then conveniently prepared in good yields by treatment of 5-chloro-6-phenylpyridazin-3(2H)-one (2) with halide (XCH 2 R, X = Cl or Br) in acetone, acetonitrile or N,N-dimethylformamide. Among these solvents, however, acetone provided the best results and the reaction could be successfully conducted at room temperature. The reaction was much faster and high yielding when X was bromine instead of chlorine. This kind of substitution at the nitrogen atom of 2 in the presence of a base was also previously investigated [28] by Estevez et al.
Compound 2 was also separately reacted (Scheme 1) with paraformaldehyde and thionyl chloride in benzene to afford 5-chloro-2-(chloromethyl)-6-phenylpyridazin-3(2H)-one (4) in a single step in 73% yield. In comparison with the two-step process as reported in the literature [30], the operation was much more convenient and the reaction time was significantly shortened.
Finally, the pyridazine derivatives 6a-6i with 1,3,4-thiadiazole or 1,3,4-oxadiazole moieties were easily obtained in 60-80% yields by the reaction of 5-chloro-2-(chloromethyl)-6-phenylpyridazin-3(2H)-one (4) with 5 as depicted in Scheme 2. As the progress of the reaction was monitored by TLC, the possibility of a side reaction through the chlorine atom at C-5 position of pyradazine ring could not be ruled out. The reaction time and temperature were critical for this reaction. In general, the products were obtained under mild conditions at 50 °C with a reaction time of 3-4 hours. The compounds 6a-6c were eventually oxidized to 7a-7c by H 2 O 2 and (NH 4 ) 6 Mo 7 O 24 as shown in Scheme 2.
The structures of the compounds 1, 2, 3a-3h, 4, 6a-6i and 7a-7c were established on the basis of their spectroscopic data. The IR spectra showed absorption bands around 3,049-3,099 cm -1 for the Ar-H stretching vibrations and near 1,662-1,678 cm -1 for the presence C=O functional groups. In the 1 H-NMR spectra of the pyradazine derivatives, the 4-H signal appeared as a singlet in 7.14-7.26 ppm range, while the Ar-H peaks of all the derivatives were observed near 6.64-8.40 ppm as a multiplet. The CH 2 peaks were observed as singlets in 5.30-6.31 ppm range.

Antifungal activity bioassay
The in vitro antifungal screening data of the pyridazine derivatives are provided in Table 1. It was observed that these synthesized compounds showed weak to good antifungal activities against the tested fungi at 50 μg/mL. Compounds 3d, 3e and 6b were shown to inhibit the growth of G. zeae at 45.1%, 43.8%, and 40.4%, respectively; compounds 3d, 3f and 7c exhibited good activities on F. oxysporum at 38.2%, 44.2% and 43.1%, respectively while compounds 3d, 3e and 3h inhibited the growth of C. mandshurica at 43.5%, 40.6% and 47.8%, respectively. These figures were slightly lower than those of hymexazol. It should be noted that compounds 3h, 7b and 7c showed good activities on G. zeae at 50.3%, 57.9% and 60.5%, respectively; compounds 3e and 3h exhibited the growth of F. oxysporum at 53.2% and 50.9% respectively and compound 7c exhibited good activity on C. mandshurica. Amongst the four compounds 3e, 3h, 7b, 7c that exhibited similar activities as that of hymexozole on their corresponding fungi, the last two showed considerable promise. Although, a definite structure activity relationship could not be established with the limited experimental data and available compounds, it appears that incorporation of oxadiazole or thiadiazole unit through thiol 5 into parent pyridazine derivative and subsequent oxidation of the resulting product to sulfone 7 might have a positive influence to enhance antifungal activity of the designed compounds.

General
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 at room temperature operating at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR, using CDCl 3 , CD 3 COCD 3 or DMSO as solvents and TMS as an internal standard; infrared spectra were recorded in KBr on a Bruker VECTOR 22 spectrometer; elemental analysis was performed on an Elemental Vario-III CHN analyzer. The course of the reactions was monitored by TLC; analytical TLC was performed on silica gel GF 254 ; column chromatographic purification was carried out using silica gel. 5-Subsititued phenyl-1,3,4-thiadiazoles (or oxadiazole)-2-thiols 5 were prepared according to the literature procedure [25][26] from substituted benzoic acid as the starting material through esterification, hydrazidation, salt formation, and cyclization. All compounds were synthesized under mild conditions with moderate yields. (1) Mucochloric acid (33.0 g) was slowly added with stirring to a mixture of benzene (160 mL) and anhydrous aluminum chloride (40.0 g). After completion of the addition, stirring was continued for 3 h at room temperature. After addition of ice (60.6 g) and conc. HC1 (128 mL), the resulting mixture was extracted with benzene (4 × 50 mL). The combined extract was dried on anhydrous Na 2 SO 4 and concentrated under vacuum to afford a crystalline solid which was filtered off and recrystallized from methanol, m.p. 75-77 °C (lit. [27], m.p. 79-81 °C); yield 60%. (2) Hydrazine hydrate (80%, 6.0 g) was slowly added to a solution of 3,4-dichloro-5-phenylfuran-2(5H)-one (5.0 g) dissolved in N,N-dimethylformamide (30 g). The resulting solution was stirred at 80 °C for 40 min; after cooling, the mixture was added to water (150 mL) to give a precipitate which was filtered off, washed with water and recrystallized from dioxane to give a yellow solid. Yield, 68%; m.p. 231-232 °C (lit. [27], m.p. 230-231°C).

Preparation of 5-chloro-6-phenyl-2-substitutedpyridazin-3(2H)-ones 3a-3h
To a well stirred solution of 5-chloro-6-phenylpyridazin-3(2H)-one (1.00 mmol) in acetone (8 mL) anhydrous potassium carbonate (0.5 g) and halide (1.00 mmol) were added. The mixture was stirred at room temperature and monitored by TLC. After completion of the reaction, the solid was filtered; the solvent was evaporated and the crude product was purified by preparative TLC with a mixture of petroleum ether and ethyl acetate (v:v = 1:1) as developing solvent to give title compounds 3a-3h.

Antifungal bioassays
The antifungal activity of all synthesized compounds was tested against F. oxysporum, G. zeae, and C. mandshurica by the poison plate technique [31]. All the compounds were dissolved in DMSO (10 mL) before mixing with Potato Dextrose Agar (PDA, 90 mL). The final concentration of the compounds in the medium was fixed at 50 μg/mL. The three kinds of fungi were incubated in PDA at 25 ± 1 °C for 5 days to get new mycelium for the antifungal assays, and then a mycelia disk of approximately 0.45 cm diameter cut from the culture medium was picked up with a sterilized inoculation needle and inoculated in the center of PDA plate. The inoculated plates were incubated at 25 ± 1 °C for 5 days. DMSO in sterilized distilled water served as control, while hymexazole was used as positive control for each treatment with three replicates being conducted for each experiment. The radial growth of the fungal colonies was measured on the sixth day and the data were statistically analyzed. The in vitro inhibiting effects of the test compounds on the fungi were calculated by the formula CV = (A − B)/A, where A represents the diameter of fungi growth on untreated PDA, B represents the diameter of fungi on treated PDA, and CV represents the rate of inhibition.

Conclusions
In the present study, a mild and effective method for the preparation of 21 novel pyridazine derivatives were undertaken by employing mucochloric acid and benzene as the starting materials. The synthesized compounds were characterized by spectral data ( 1 H-NMR, 13 C-NMR, IR) and elemental analysis. The compounds were subjected to fungicidal activities in vitro against G. zeae, F. oxysporum and C. mandshurica. The results showed that the synthesized pyridazine compounds possessed weak to good antifungal activities against the tested fungi, among which, compounds 3e, 3h, 7b, 7c displayed good antifungal activities. Further studies are currently underway to establish a definite structure activity relationship.