Discovery of an Orally Efficacious Pyrazolo[3,4-<i>d</i>]pyrimidine Benzoxaborole as a Potent Inhibitor of <i>Cryptosporidium</i>

Cryptosporidiosis is a diarrheal disease caused by the parasite <i>Cryptosporidium</i> resulting in over 100,000 deaths annually. Here, we present a structure-activity relationship study of the benzoic acid position (R⁶) of pyrazolo[3,4-<i>d</i>]pyrimidine lead SLU-2815 (<b>1</b>), an inhibitor of parasite phosphodiesterase <i>Cp</i>PDE1, resulting in the discovery of benzoxaborole SLU-10906 (<b>63</b>) as a benzoic acid bioisostere. Benzoxaborole <b>63</b> is 10-fold more potent than <b>1</b> against the parasite in a cell-based infection model (EC₅₀ = 0.19 μM) and non-cytotoxic. Furthermore, <b>63</b> has a fast rate of parasite-killing and is orally efficacious in a <i>Cryptosporidium</i> mouse infection model (50 mg/kg BID), although relapse was observed 7 days post-drug treatment. The partial selectivity profile versus human phosphodiesterases is preserved with the benzoxaborole motif and represents an important feature to improve in future optimization. Benzoxaborole <b>63</b> represents an important advance toward the optimization of the pyrazolo[3,4-<i>d</i>]pyrimidine series and the identification of a drug to treat cryptosporidiosis.