Microwave-assisted Synthesis and Antifungal Activity of Some Novel Thioethers Containing 1,2,4-triazole Moiety

These authors contributed equally to this work. Abstract: A series of novel thioether derivatives containing 1,2,4-triazole moiety were designed and synthesized from 4-chlorophenol and ethyl 2-chloroacetate as starting materials by multi-step reactions under microwave irradiation, and their structures were characterized by 1 H-NMR, MS and elemental analysis. The antifungal activity of title compounds was determined. The results indicated that some of title compounds exhibited moderate antifungal activity.

Microwave-assisted technique is a green method in current organic synthesis [26][27][28].It is attractive offering reduced pollution, low cost and high yields.The method can often shorten the reaction time.In recent years, many references had been reported this method.For example, MW-assisted synthesis heterocycles have been conducted successfully earlier [29,30].
In view of all these facts and as continuation of our research on bioactive compounds [31][32][33], herein a series of novel thioether derivatives containing 1,2,4-triazole moiety were synthesized under microwave irradiation.The antifungal activity of title compounds was tested.

Synthesis
The ethyl 2-(4-chlorophenoxy)acetate was obtained from 4-chlorophenol and ethyl 2-chloroacetate catalyzed by KI at reflux condition.The KI was used as catalyst in order to increase the yield of ethyl 2-(4-chlorophenoxy)acetate.The ethyl 2-(4-chlorophenoxy)acetate reacted with 85% hydrazine hydrate to give 2-(4-chlorophenoxy)acetohydrazide at refluxing condition.Then the 2-(4-chlorophenoxy)acetohydrazide reacted with isothiocyanatobenzene.Then 2-(2-(4-chlorophenoxy)acetyl) -N-phenylhydrazinecarbothioamide was collected easily by filtered.The key intermediate 5-((4-chlorophenoxy)methyl)-4-phenyl-4H-1,2,4-triazole-3-thiol was cyclized under alkaline condition, such as NaOH, then the concentrate HCl was added to this solution.The key intermediatethiol is reacted with substituted benzyl chloride or alkylchloride to afford title compounds.The microwave-assisted synthesis and conventional method was also employed in this experiment.NaOH/DMF/H2O system was applied under microwave irradiation.The best reaction condition is at 90 °C for 15 min under microwave irradiation synthesis.Taking the compound 5b as example, we can see that the yield is higher than that of conventional method, also the reaction time is shorter from Table 1.

Instruments
Melting points were recorded using an X-4 apparatus and uncorrected (Beijing Tech Instrument Co., Beijing, China). 1 H-NMR spectra were performed on a Bruker AV-400 instrument (Bruker, Fallanden, Switzerland) using TMS as an internal standard and CDCl3 as the solvent.Elemental analyses were determined on a Vario EL elemental analyzer (Elementar, Hanau, Germany).All the reagents are of analytical grade or freshly prepared before use.

Synthesis
The synthetic procedure for title compound is shown in Scheme 1.  4-Chlorophenol (0.1 mol), acetone(40 mL), ethyl 2-chloroacetate (0.12 mol), K2CO3 (16.56 g, 0.12 mol) and catalytic amount of KI were added to a 100 mL three-necked flask, then the mixture was stirred at reflux.The reaction was monitored by GC until the start material 1a disappeared completely.The mixture was poured into ice water and extracted with ethyl acetate (3 × 50 mL) and then dried by anhydrous Na2SO4 and evaporated to dryness to get crude solid 1.

General Procedure for Thioether (5)
A CEM designed 10 mL pressure-rated vial was charged with DMF (5 mL), 4 (1 mmol), RCH2Cl (1.1 mmol) and NaOH (1.2 mmol).The mixture was irradiated in a CEM Discover Focused Synthesizer (150 W, 90 °C, 200 psi, 15 min).The mixture was cooled to room temperature by passing compressed air through the microwave cavity for 2 min.It was poured into cold ice (40 mL) and the formed precipitate was filtered.The crude solid was recrystallized from EtOH to give the title compounds 5a.All the other compounds are synthesized according to the procedure (Scheme 1).

Antifungal Activity
Antifungal activity of compounds 5a-5k against Pythium ultimum Trow, Phytophthora infestans (Mont.)de Bary, Corynespora cassiicola, Botrytis cinerea and Rhizoctonia solani were evaluated according to reference [3].A potted plant test method was adopted.Germination was conducted by soaking cucumber seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °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 cotyledons, and tomato plants were five euphyllas.Tested compounds and commercial fungicides were sprayed with a hand sprayer on the surface of the leaves and on a fine morning, at the standard concentration of 100 μg/mL, and each plant was sprayed compounds and commercial fungicides 200 μL.Dimethomorph, Fludioxonil, Chlorothalonil, Validamycin, Zhongshengmycin were used as a control.After 2 h, inoculations of Phytophthora infestans, Corynespora cassiicola and Botrytis cinerea were carried out by spraying fungal spore suspension with 1 × 10 4 spore/mL, inoculation of Rhizoctonia solani and Pythium ultimum were carried out by spraying mycelial suspension of 2 × 10 4 CFU/mL, which was smashed with IKA T10 basic ULTRA-TURRAX ® (Guangzhou, China).Each kind of inoculum was sprayed 300 μL/plant.Each treatment was replicated 4 times.After inoculation, the plants were maintained at 18-30 °C (mean temperature of 24 °C and above 80% relative humidity (RH)).The antifungal activity were evaluated when the nontreated plant (blank) fully developed symptoms.The area of inoculated treated leaves covered by disease symptoms was assessed and compared to that of nontreated ones to determine the average disease index.The relative control efficacy of compounds compared to the blank assay was calculated via the following equation: relative control efficacy (%) = (CK − PT)/CK × 100% where CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing.All experiments were replicated three times.

Figure 1 .
Figure 1.Some representative commercial drugs or pesticides.

Scheme 1 .
Scheme 1.The synthetic route of title compounds.

Table 2 .
The antifungal activity of title compounds in vivo at 100 ppm (%).