infantum, phosphonium salt, in vitro activity, in vivo activity, pharmacokinetics, mechanism of action. Abstract We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were

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... ished. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of 4 daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant ROS production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum indicating its potential as antileishmanial lead. form (BSF) trypanosomes which lack the canonical oxidative phosphorylation processes and rely exclusively on TAO for respiration. 15 However, based on the literature reports and our own experience, we envisaged that our mitochondrion-targeted (TAO inhibitor) compounds may have chemotherapeutic potential against Leishmania parasites. Accordingly, we carried out a focused screening of an in-house series of TAO inhibitors against L. donovani parasites which resulted in the discovery of potent leishmanicides (Table S1 ). As TAO is not a validated target in Leishmania, we set about to discover the mechanism of action (MoA) of these mitochondrion-directed compounds. As the most active and selective compounds presented potential metabolic instability, new derivatives were synthesized and tested against promastigote and amastigote forms of L. donovani (Chart 1). We report here the discovery of new phosphonium salts active against L. donovani in vitro, pilot preclinical pharmacological studies and strong in vivo activity in a mouse model of visceral leishmaniasis with an exploration of uptake and mechanism of action of lead compound 1. RESULTS Preliminary screening and SAR studies. Thirty TAO inhibitors 11, 12, 14 were screened in vitro against promastigotes and the human-relevant intracellular amastigote forms of L. donovani HU3, and their cytotoxicity against MRC-5 or THP-1 cells was determined (Table S1 ). Eleven cationic compounds showed IC50 < 1 µM against intracellular amastigotes, among which eight were phosphonium salts (15-18, 23-24, 26, 31) with selectivity indices (SI) versus mammalian cells ranging from 2 to 27. In general, the quinolinium salt derivatives were more cytotoxic and only three compounds (10, 11, 32) presented a fair activity/selectivity profile (IC50 ≈ 0.8 µM, SI = 3.7-5). Remarkably, compounds with a C14 methylene chain were slightly more selective towards Leishmania than shorter analogues with C10 or C12 (compare 10/11, 15/16, 29/30). Three phosphonium hit compounds displaying EC50 < 0.2 µM against promastigotes and intracellular amastigotes of L. donovani and satisfactory selectivities (SI > 10) were selected as possible leads for in vivo studies (Table S1 and Chart 1: 16, 17, and 18) . Since the selected compounds contain an ester bond that can be hydrolyzed by serum hydrolases (e.g. the half-life of compound 16 is 1 h in mouse serum 12 ), which may potentially limit