Synthesis of a New Purine Analogue Class with Antifungal Activity and Improved Potency against Fungal IP_3–4K

New antifungals are urgently needed to treat deadly fungal infections. Targeting the fungal inositol polyphosphate kinases IP_3-4K (Arg1) and IP₆K (Kcs1) is a promising strategy as it has been validated genetically to be crucial for fungal virulence but never pharmacologically. We now report the synthesis of <b>DT-23</b>, an analogue of <i>N</i>2-(<i>m</i>-trifluorobenzylamino)-<i>N</i>6-(<i>p</i>-nitrobenzylamino)purine (<b>TNP</b>), and demonstrate that it more potently inhibits recombinant Arg1 from the priority pathogen <i>Cryptococcus neoformans</i> (<i>Cn</i>) (IC₅₀ = 0.6 μM) than previous analogues (IC₅₀ = 10-30 μM). <b>DT-23</b> also inhibits recombinant Kcs1 with similar potency (IC₅₀ = 0.68 μM) and Arg1 and Kcs1 activity <i>in vivo</i>. Unlike previous analogues, <b>DT-23</b> inhibits fungal growth (MIC₅₀ = 15 μg/mL) and only 1.5 μg/mL synergizes with Amphotericin B to kill <i>Cn in vitro</i>. <b>DT-23</b>/Amphotericin B is also more protective against <i>Cn</i> infection in an insect model compared to each drug alone. Transcription profiling shows that <b>DT-23</b> impacts early stages in IP synthesis and cellular functions impacted by IPK gene deletion, consistent with its targeted effect. This study establishes the first pharmacological link between inhibiting IPK activity and antifungal activity, providing tools for studying IPK function and a foundation to potentially develop a new class of antifungal drug.