Fused pyrimidines, especially pyrazolo [3,4-d]pyrimidines, are among the most preferred building blocks for pharmacology studies, as they exhibit a broad spectrum of biological activity. In this study, new derivatives of pyrazolo [3,4-d]pyrimidine were synthesized by alkylation of the N1 nitrogen atom. We synthesized 3-iodo-1H-pyrazolo [3,4-d] pyrimidin-4-amine 2 from commercially available aminopyrazolopyrimidine 1 using N-iodosuccinimide as an iodinating agent. The synthesis of compound 2 started with nucleophilic substitution of 3-iodo-1H-pyrazolo [3,4-d]pyrimidin-4-amine with R-X (X:-OMs, -Br, -Cl), affording N-alkylated pyrazolo [3,4-d]pyrimidine. We performed this synthesis using a weak inorganic base and the mild temperature was also used for a two-step procedure to generate N-alkylated pyrazolo [3,4-d]pyrimidine derivatives. Also, all compounds were tested for their ability to inhibit acetylcholinesterase (AChE) and the human carbonic anhydrase (hCA) isoforms I and II, with K i values in the range of