Novel O-methylpyrimidine prodrugs of phenolic compounds bioactivated by aldehyde oxidase: Enhancing metabolic stability against first-pass conjugative metabolism in the intestine

Phenol-containing drugs may exhibit limited oral bioavailability due to first-pass conjugation in the intestine and liver, and potentially unfavorable biopharmaceutical properties imparted by the hydrogen-bond donor. We present a novel prodrug strategy in which O-methylpyrimidine modification masks the phenolic moiety and employs aldehyde oxidase (AO) to release the parent drug. Prototypical prodrugs of 4-hydroxy-tamoxifen (4OH-TAM), raloxifene (RAL), rotigotine, 5-hydroxy-tolterodine, and phentolamine were all substrates for AO-mediated parent drug release in liver cytosol from humans and every preclinical species evaluated. Reaction phenotyping confirmed the role of AO; hydralazine inhibited production of 4OH-TAM and RAL from their respective prodrugs in the human liver cytosol, and recombinant human AO activated those same prodrugs. Based on the identified byproduct, 5-(hydroxymethyl)uracil, and characterized 4OH-TAM prodrug metabolite intermediates, a mechanism is proposed, involving oxidation of the pyrimidine 4-position, followed by rate-limiting oxidation at the 2-position and subsequent C-O bond cleavage via an imine-methide intermediate. To determine a preclinical animal for proof-of-concept prodrug activation in vivo, we measured both absolute AO protein concentration and parent release for 2 prodrugs in the liver cytosol of multiple species and found that hamster was a promising candidate to model humans. After confirming a similar balance of AO-mediated prodrug conversion versus nonproductive/subsequent biotransformation in human and hamster hepatocytes, the 4OH-TAM prodrug and RAL prodrug 1 were progressed to a pharmacokinetic study in hamsters. A 30 mg/kg oral dose of RAL prodrug 1 demonstrated a 2-fold increase in RAL exposure compared with dosing parent RAL, indicating that this novel prodrug strategy has the potential to improve bioavailability in humans. SIGNIFICANCE STATEMENT: An aldehyde oxidase-mediated biotransformation that cleaves O-linked methylpyrimidine-masked phenolic moieties was identified, and this system employed for a novel prodrug bioactivation strategy. The research herein expands existing knowledge surrounding the metabolism capabilities of this enzyme and provides medicinal chemists with a tool to enhance the oral bioavailability of phenolic compounds that otherwise would be limited due to extensive phase II metabolism and possibly low permeability.