The conversion of carbon dioxide (CO2) and bicarbonate (HCO3-) to each other is very important for living metabolism. Carbonic anhydrase (CA, E.C.188.8.131.52), a metalloenzyme familly, catalyzes the interconversion of these ions (CO2 and HCO3-) and are very common in living organisms. In this study, a series of novel 2-amino-3-cyanopyridines supported with some functional groups was synthesized and tested as potential inhibition effects against both cytosolic human CA I and II isoenzymes (hCA I and II) using by Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography. The structural elucidations of novel 2-amino-3-cyanopyridines were achieved by NMR, IR, and elemental analyses. K-i values of the novel synthesized compounds were found in range of 2.84-112.44M against hCA I and 2.56-31.17M against hCA II isoenzyme. While compound 7d showed the best inhibition activity against hCA I (K-i: 2.84M), the compound 7b demonstrated the best inhibition profile against hCA II isoenzyme (K-i: 2.56M).