Original article Ester prodrugs of acyclic nucleoside thiophosphonates compared to phosphonates: Synthesis, antiviral activity and decomposition study Loïc Roux a , Stéphane Priet a , Nadine Payrot a , Clément Weck a , Maëlenn Fournier b , Fabien Zoulim b , Jan Balzarini c , Bruno Canard a , Karine Alvarez a, * a Laboratoire d’Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 7257, Equipe “Chimie Médicinale et Virologie Structurale”, Université Aix-Marseille, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France b INSERM, U871, Université de Lyon, Hospices Civils de Lyon, 69003 Lyon, France c Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven, Belgium article info Article history: Received 6 December 2012 Received in revised form 30 January 2013 Accepted 26 February 2013 Available online 14 March 2013 Dedicated to the memory of Dr Antonin Holý. Keywords: Nucleotide analogue Prodrug Thiophosphonate Antiviral activity Metabolism abstract 9-[2-(Thiophosphonomethoxy)ethyl]adenine [S-PMEA, 8] and (R)-9-[2-(Thiophosphonomethoxy)propyl] adenine [S-PMPA, 9] are acyclic nucleoside thiophosphonates we described recently that display the same antiviral spectrum (DNA viruses) as approved and potent phosphonates PMEA and (R)-PMPA. Here, we describe the synthesis, antiviral activities in infected cell cultures and decomposition study of bis(pivaloyloxymethoxy)-S-PMEA [Bis-POM-S-PMEA, 13] and bis(isopropyloxymethylcarbonyl)-S-PMPA [Bis-POC-S-PMPA, 14] as orally bioavailable prodrugs of the S-PMEA 8 and S-PMPA 9, in comparison to the equivalent “non-thio” derivatives [Bis-POM-PMEA, 11] and [Bis-POC-PMPA, 12]. Compounds 11 , 12, 13 and 14 were evaluated for their in vitro antiviral activity against HIV-1-, HIV-2-, HBV- and a broad panel of DNA viruses, and found to exhibit moderate to potent antiviral activity. In order to determine the decomposition pathway of the prodrugs 11 , 12, 13 and 14 into parent compounds PMEA, PMPA,8 and 9, kinetic data and decomposition pathways in several media are presented. As expected, bis-POM-S-PMEA 13 and bis-POC-S-PMPA 14 behaved as prodrugs of S-PMEA 8 and S-PMPA 9. However, thiophosphonates 8 and 9 were released very smoothly in cell extracts, in contrast to the release of PMEA and PMPA from “non-thio” prodrugs 11 and 12. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Acyclic nucleoside phosphonates (ANPs) [1e3] are outstanding molecules in the fight against DNA virus and retrovirus infections. Three of those compounds have been formally licenced for the treatment of HCMV infections [4] in AIDS patients (HPMPC, cido- fovir, VistideÒ), chronic HBV infections [1,5,6] (PMEA, Adefovir dipivoxil, HepseraÒ, (R)-PMPA, Tenofovir disoproxil fumarate, VireadÒ) and HIV infections (Tenofovir disoproxil fumarate, VireadÒ). Tenofovir is also available in a combination formulation with emtricitabine (TruvadaÒ), or emtricitabine and efavirenz (AtriplaÒ) or emtricibatine and rilpivirine (CompleraÒ/EvipleraÒ) for the treatment of AIDS. In addition to these indications, there are various other clinical conditions in which ANPs have proven to be active [1,7]. ANPs are nucleotide analogues, and thus a phosphonate group is attached to the nucleoside moiety. Hence ANPs: 1) should resist any attack by esterases, phosphatases or any catabolic enzymes, thus enhancing physiological stability and half-life in biological fluids and cells, and 2) require only two phosphorylation steps to be converted into their diphosphate active form [8], thus circum- venting the often rate-limiting first phosphorylation step of nucleoside analogues. With respect to their mode of action, these nucleoside phos- phonate analogues are phosphorylated by cellular kinases into nucleoside phosphonate diphosphates after endocytosis-mediated penetration into the infected cells. While they are generally poor substrates for cellular polymerases, these nucleotide analogues are, Abbreviations: HIV, human immunodeficiency virus; HBV, hepatitis B virus; RT, reverse transcriptase; dNTP, deoxynucleotide; ANP, acyclic nucleoside phospho- nate; PMEA, 9-[2-phosphonomethoxyethyl]adenine; S-PMEA, 9-[2-thiophosphono methoxyethyl]adenine; (R)-PMPA, (R)-9-[2-phosphonomethoxypropyl]adenine; (R)-S-PMPA, (R)-9-[2-thiophosphonomethoxypropyl]adenine; bis-POC-PMPA, (R)- 9-{2-[O,O 0 -bis(isopropoxycarbonyloxymethyl)phosphonomethoxy]propyl}adenine; bis-POC-S-PMPA, (R)-9-{2-[O,O 0 -bis(isopropoxycarbonyloxymethyl)thiophosphono methoxy]propyl}adenine; bis-POM-PMEA, 9-{2-[O,O 0 -bis(pivaloyloxymethyl)phos- phonomethoxy]ethyl}adenine; bis-POM-S-PMEA, 9-{2-[O,O 0 -bis(pivaloyloxymeth- yl)thiophosphonomethoxy]ethyl}adenine. * Corresponding author. Tel.: þ33 491 825 570; fax: þ33 491 266 720. E-mail address: Karine.Alvarez@afmb.univ-mrs.fr (K. Alvarez). Contents lists available at SciVerse ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.02.039 European Journal of Medicinal Chemistry 63 (2013) 869e881