Synthesis of 3-substitutedmethylene-2 H-thiopyrano [ 2 , 3-b ] Pyridine-4 ( 3 H )-ones and Their Antifungal Activity In Vitro

Six (Z)-3-substitutedmethylene-2H-thiopyrano[2,3-b]pyridin-4(3H)-ones were designed and synthesized. Theirstructures were confirmed by MS and 1H-NMR and element analysis. Their antifungal activity was tested by microdilution broth susceptibility for eight kinds of fungi, and the results showed that the target compounds exhibitedactivity against fungi tested to some extent. The compound 5a had the best antifungal effect among of the targetcompounds.

Firstly the intermediate of 2H-thiopyrano[2,3-b]pyridin-4(3H)-one was synthesized from the 2-chloronicotinic acid.Secondly, the target compounds were obtained by the reactions of aldehyde with 2H-thiopyrano[2,3-b]pyridin-4(3H)-one in ethanol.The antifungal activity of the target compounds in vitro was measured by consecutive double dilution.The synthetic route was outlined in Figure 1.

Chemistry material
2-chloronicotinic acid (chemically pure) were from SHANDONG KEHUI Chemical Co., LTD (SHANDONG, 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 600 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).Elemental Analysis (C, H, N, S) was realized on Carlo Erba 1106 EAinstrument.

Preparation of 2-mercaptonicotinic acid
A suspension of 2-chloronicotinic acid 1 (15.7 g, 100 mmoles) and thiocarbamide (13.7 g, 180 mmoles) in 170 mL of water was strong mixing reflux for 4 hours.After cooling, the solid precipitate product was collected and washed with water to give 14.8 g (95% yield) of pure 2.

Preparation of 2-(2-carboxyethylthio)nicotinic acid
3-chloropropionic acid (11.7 g,108 mmoles) and sodium iodide in 50 mL of water and sodium hydrogen carbonate (9 g, 108 mmoles) were added to a solution of 2-mercaptopyridine-3-carboxylic acid (13.9 g, 90 mmoles) in 90 mL of 10% potassium hydroxide aqueous solution.The reaction mixture was stirred at 60℃ for 4 hours, cooled and acidified with concentrated hydrochloric acid to pH 3. The solid precipitate product was collected and washed with water to give 18.7 g (92% yield) of pure 3.

Preparation of 2H-thiopyrano[2,3-b]pyridin-4(3H)-one
A solution of 3 (19.3g, 85 mmoles) and anhydrous sodium acetate (13.9 g, 170 mmoles) in 72mL of acetic anhydride was refluxed at 160℃ for 1.5 hours.After cooling, the reaction mixture was diluted with water, basified with 30% ammonium hydroxide solution to pH 8-9, extracted with ethyl acetate.The combined extracts were washed with water, dried and evaporated to give 9.7 g of crude 4. Purification was made by filtration on a silica gel chromatographic column, using petroleum ether 60-80℃/ethyl acetate 10:1 as the eluting system.The product recovered from the less mobile fraction gave 3.5 g (25％ yield) of pure 4.

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) recommendation.Eight human opportunistic pathogenic fungi (C.parapsilosis, C.glabrata, C.albicas, C.tropicalis, C.neoformans, C.Krusei, A.niger, M.gypseum) were tested, All experiments were performed in comparison with Fluconazole, a known antifungal agent (Odds, F. C, 1986, Hoban, D. J, 1999).The six new compounds were dissolved in dimethyl sulfoxide (DMSO) (1 mL), further progressive dilutions by RPMI 1640 gave there quired concentrations (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg/mL); the fungi were prepared and adjusted to a final concentration of 0.5×104-2.5×104CFU/mL.MIC values were determined by visual observation after 2-7 d of incubation.

Results and discussions
In order to make the reaction proceed easy, we put sodium iodide into the synthetic process of compound 3 on the basis of literature (A.D. Settimo, 2000;P. L. Ferrarini, 2000).The compounds 5a-5f were synthesized by Knoevenagel reaction of compounds 4. With active methylene compound 4 and aldehyde condensation in the presence of alkaline catalysts are α,β-unsaturated ketones.
The results of antifungal activities in vitro were shown in Table 1.The results showed that the target compounds exhibited activity against fungi tested to some extent.And all the target compounds had no activity against C.neoformans.The compound 5a showed a similar level of activity with Fluconazole when against M.gypseum and C.Krusei, and showed moderate activity against C.glabrata.
In conclusion, The target compounds had an antifungal effect on most tested fungi in vitro.Compound 5a had the best antifungal effect among of the target compounds.Further biological evaluation of the compounds is in progress.