Synthesis and Antifungal Activity of ( z )-3-( bromomethylene ) thiochroman-4-ones

Three (z)-3-(bromomethylene)thiochroman-4-ones were designed and synthesized. Their structures were confirmed byMS and 1H NMR. In vitro antifungal activities of these synthesized compounds were evaluated against ten species offungi, and the results showed that the target compounds exhibited activity against fungi tested to some extent. Themaximum inhibitory activity was found for 3a against C.neoformans, 3a and 3c exhibited more potent antifungalactivities against C.Krusei than Fluconazole.


Chemistry material
Substituted benzenethiols (chemically pure) were from SHOUERFU LLC (ZHEJIANG, China), and the other reagents were almost from TIANJIN Chemical LLC (TIANJIN, China). 1 H-NMR spectra were recorded in CDCl 3 on Bruker Avance DMX 400 using TMS as an internal standard (Bruker, Billerica, MA, USA).Mass spectral data were obtained by LC-MSD Trap XCT G2446A (Agilent Technologies, USA).Melting points were determined SGW X-4 microscopic melting point (Shanghai Precision & Scientific Instrument Co., Ltd, China).The IR spectra were recorded in potassium bromide on IRPrestige-21/FTIR-8400S (SHIMADZU CORPORATION, Kyoto, Japan).

Synthesis of (z)-3-(bromomethylene)thiochroman-4-one (3a-3c)
In a sealed tube, compound 2 (100 mmol) and dichloromethane (10 mL) were placed, acetyl bromide (150 mmol) was added within 2 min, sealed the tube.After warming it in an oil bath at 50℃ for 2h, the organic phase was extracted twice with 0.5 mol/L sodium carbonate solution (15 mL per time), the organic extracts was removed under vacuum to give the crude product and then purified by silica-gel column chromatography, eluting with dichloromethane: petroleum ether= 1:10 (v/v).

Antifungal Activity in Vitro
In vitro antifungal activities were measured by means of the minimal inhibitory concentrations (MIC) by consecutive double dilution method.The MIC means the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in broth dilution susceptibility test (Marcelo C. Murguı´a, 2008).The MIC was determined according to the national committee for clinical laboratory standards (NCCLS) recommendations.Ten human opportunistic pathogenic fungi were tested, Fluconazole was taken as the reference drug for positive control.The compounds were dissolved in dimethyl sulfoxide (DMSO) (1 mL), further progressive dilutions by RPMI 1640 gave the required concentrations ( 64,32,16,8,4, 2, 1, 0.5, 0.25, 0.125μg/mL); the fungi were prepared and adjusted to a final concentration of 0.5×10 4 -2.5×10 4 CFU /mL.MIC values were determined by visual observation after 2-7d of incubation.

Reaction mechanism
Compounds 3 were synthesized by bromination reaction of Compounds 2. In addition, this kind of bromination reaction had no been reported, a plausible mechanism showing the formation of this reaction was supposed in Scheme 2. The proposed pathway involves initial tautomerization of the intermediate enol compounds 4, it was possible that the nucleophilic attack of the enolate oxygen on the acetyl bromide to generate the intermediate compounds 5, the latter step presumably involves compounds 5 was attacked by bromine ionic to give compounds 3.

Antimicrobial Activity in vitro
The results of antifungal activities in vitro were shown in Table 1.The results showed that the target compounds were effective against all of the tested fungi.The maximum inhibitory activity was found for 3a against C.neoformans; 3a and 3c had higher inhibitory effects on the growth of C.Krusei; 3a, 3b, 3c, showed a similar level of activity with fluconazole when against M.gypseum and showed moderate activity against C.parapsilosis.In particular, 3a, 3b, 3c were active for E.floccosum and S.schenekn while the activity of Fluconazole was lower.However, all of the tested compounds and Fluconazole were low active against A.niger.
In conclusion, the target compounds had an antifungal effect on most tested fungi in vitro.Further biological evaluation of the three compounds is in progress.Moreover, the results should encourage us to design and synthesize more potent antifungal agents.