26 new 2,4-diaminopteridine derivatives and the reference compounds 1b and 1c were (re)synthesized as reported in Schemes 1-8. We applied our methodology for improved reaction yield of the chemical pteroid step to provide a key intermediate for most of the designed compounds. The PABA amine functionalization was achieved by selective alkylation of primary amines to secondary amines using nitriles as an alkylating reagent with Pd/C for intermediates 32-33. Conventional alkylation of the latter with propargyl bromide or (bromomethyl)benzene resulted in derivatives 34 and 35, respectively (Scheme 2). The reductive alkylation of amines using nitriles was also used to obtain 51 and 74 in Schemes 3 and 7. The isonipecotic acid derivatization was achieved via Fischer esterification using the reagent-solvents propanol (37) and EtOH (38), respectively;