Acidic Dissociation Constants of Folk Acid, Dihydrofolic Acid, and Methotrexate

The acidic dissociation constants in the range H,, -1.3 to pH 7 of folic acid, dihydrofolic acid, methopterin (N(lO)methylfolic acid), and methotrexate have been measured by potentiometric and spectrophotometric titrations. Assignment of these dissociations was made by comparison to model compounds, by proton magnetic resonance measurements, and by examination of associated ultraviolet absorbance changes. For folk acid, the dissociation constants are as follows: N(l), pK’ 2.35; N(lO), pK’ 0.20; N(5), pK’ <-1.5. For dihydrofolic acid: N(3), pK’ 3.84; N(l), pK’ 1.38; X(10), pK’ 0.28. For methotrexate: N(l), pK’ 5.71; y-carboxyl, pK’ 1.70; Lu-carboxyl, pK’ 3.36; N(lO), pK’ 0.30; N(S), pK’ <1.5. For methopterin: acidic ionization of amide, pK 7.68; y-carboxyl, pK’ 4.62; N(l), pK’ 2.40; N(lO), pK’ 0.36; N(3), pK’ <-1.5. The pK’ values were determined directly for the four compounds at 23” near 0.1 ionic strength, or in 0.1 to 4 M HCI for pK’ less than 1. The carboxyl pK’ values were measured potentiometricallg at 25” in 0.1 M NaCI.