Synthesis, in Vitro Antiviral Evaluation, and Stability Studies of Novel r-Borano-Nucleotide Analogues of 9-[2-(Phosphonomethoxy)ethyl]adenine and (R)-9-[2-(Phosphonomethoxy)propyl]adenine Karine Barral, ² Ste ´phane Priet, ‡ Jose ´phine Sire, ‡ Johan Neyts, § Jan Balzarini, § Bruno Canard, ² and Karine Alvarez* ,² Laboratoire d’Architecture et Fonction des Macromole ´ cules Biologiques, UMR CNRS 6098, Equipe “Re ´ plicases Virales: Structure, Me ´ canisme, et Drug-design”, UniVersite ´ s Aix-Marseille I et II, Parc scientifique de Luminy, 163 aV. de Luminy, 13288 Marseille Cedex 9, France, Unite ´ des Virus E Ä mergents, Faculte ´ de Me ´ decine, Marseille, France, and Rega Institute for Medical Research, Katholieke UniVersiteit, LeuVen, Belgium ReceiVed January 11, 2006 We describe here the synthesis of 9-[2-(Boranophosphonomethoxy)ethyl]adenine (6a) and (R)-9-[2- (Boranophosphonomethoxy)propyl]adenine (6b), the first R-boranophosphonate nucleosides in which a borane (BH 3 ) group substitutes one nonbridging oxygen atom of the R-phosphonate moiety. H-phosphinates 5a and 5b and R-boranophosphonates 6a and 6b were evaluated for their in vitro activity against human immunodeficiency virus (HIV) infected cells and against a panel of DNA or RNA viruses. Compounds 5a, 5b, 6a, and 6b exhibited no significant antiviral activity in vitro and cytotoxicity. To measure the chemical and enzymatic stabilities of the target compounds 6a and 6b, kinetic data of decomposition for derivatives 5a, 5b, 6a, 6b, and standard compounds were studied at 37 °C in several media. The R-Boranophosphonates 6a and 6b were metabolized in culture medium into H-phosphinates 5a and 5b, with half-live values of 5.3 h for 6a and 1.3 h for 6b. Introduction Many antiviral drugs directed against HIV-1, the etiologic agent of acquired immunodeficiency syndrome (AIDS), focus on the inhibition of HIV-1 reverse transcriptase (RT), one of the key enzymes in the replicative cycle of the virus. Most of the available drugs targeting the HIV-1 RT are nucleoside and nucleotide analogues. Intracellular phosphorylation is required to convert a nucleoside into its active triphosphate form in order to be selectively incorporated into the viral DNA, leading to the antiviral effect by chain termination. Dideoxynucleosides such as zidovudine (AZT), stavudine (d4T), or lamivudine (3TC) have been extensively used as antiviral nucleosides targeting HIV RT. 1 However, under therapeutic pressure, the viral RT gene mutates and creates RT variants bearing amino acid substitutions responsible for the loss of nucleotide analogue efficacy. The majority of resistance-associated mutations are located near the RT active site, and these resistance mechanisms are now well-characterized. 2-4 Nucleoside 5′-(R-P-borano)triphosphates are nucleotide ana- logues in which a borane (BH 3 ) group substitutes one of the nonbridging R-phosphate oxygens in nucleoside 5′-triphosphate (Chart 1). Potential use of these derivatives was originally described in boron neutron capture therapy for cancer treatment and antisense technologies. 5-7 Later, some interesting results were obtained using borane-nucleotide analogues as inhibitors of HIV-1 RT, 8 which were capable to overcome resistance recovering sensitiv- ity of the mutant RTs to these inhibitors. 9-12 Indeed, the presence of an R-boranophosphate group in the triphosphate form of clinically relevant compounds, such as AZT, d4T, or dideoxyadenosine (ddA), does not influence the binding of the analogue to the RT active site but specifically provides (or restores) a high incorporation rate of the analogue by wild-type and mutant HIV-1 RTs. 11,12 The concept of the acyclic nucleoside phosphonate (ANPs) has been used to design chain terminators 13,14 for antiviral therapy and proved to be valid. 15-17 Currently, tenofovir disoproxil fumarate [a bis-POC prodrug of (R)-9-(2-phospho- nomethoxypropyl)adenine, (R)-PMPA] 1 and adefovir dipivoxil [a bis-POM prodrug of 9-(2-phosphonomethoxyethyl)adenine, PMEA], 18 two ANPs, are used clinically as anti-HIV and anti- HBV (hepatitis B virus) drugs, respectively. There is consider- able interest in phosphonates as biologically active mimics of natural phosphates. Phosphonate replacement is attractive, since the carbon-phosphorus bond is not susceptible to enzymatic cleavage by phosphatases, thus enhancing physiological stability. Moreover, the presence of a R-phosphonate allows circumvent- ing the often rate-limiting first phosphorylation step. According to the powerful antiviral activity of the acyclic nucleoside phosphonate analogues and the advantages granted to triphosphate nucleosides by the presence of an R-P-borane group, we have developed chimeric R-boranophosphonate nucleosides. We have explored the synthesis of 9-[2-(borano- phosphonomethoxy)ethyl]adenine (6a) and (R)-9-[2-(borano- phosphonomethoxy)propyl]adenine (6b), where one of the nonbridging oxygen atoms of the R-phosphonate group is replaced by a BH 3 group (Chart 2). The aim of this concept is to obtain synergy between R-P-borano derivative properties and * To whom correspondence should be addressed. Tel: +33-4-91-82- 55-71. Fax: +33-4-91-26-67-20. E-mail: karine.alvarez@afmb.univ-mrs.fr. ² Universite ´s Aix-Marseille I et II. ‡ Unite ´ des Virus E Ä mergents. § Katholieke Universiteit. Chart 1. Example of 5′-(R-P-Borano)triphosphate Nucleoside 7799 J. Med. Chem. 2006, 49, 7799-7806 10.1021/jm060030y CCC: $33.50 © 2006 American Chemical Society Published on Web 11/29/2006