Synthesis and stability studies of phosphonoformate–amino acid conjugates: a new class of slowly releasing foscarnet prodrugs Mong S. Marma, Boris A. Kashemirov and Charles E. McKenna* Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA Received 20 November 2003; accepted 10 January 2004 Abstract—Prodrugs of phosphonoformic acid (PFA), an anti-viral agent used clinically as the trisodium salt (foscarnet), are of interest due to the low bioavailability of the parent drug, which severely limits its utility. Neutral PFA triesters are known to be susceptible to P–C bond cleavage under hydrolytic de-esterification conditions, and it was previously found that P,C-dimethyl PFA P–N conjugates with amino acid ethyl esters did not release PFA at pH 7, and could not be fully deprotected under either acid or basic conditions, which led, respectively, to premature cleavage of the P–N linkage (with incomplete deprotection of the PFA ester moiety), or to P–C cleavage. Here we report that novel, fully deprotected PFA-amino acid P–N conjugates 4 can be prepared via coupling of C-methyl PFA dianion 2 with C-ethyl-protected amino acids using aqueous EDC, which gives a stable monoanionic intermediate 3 that resists P–C cleavage during subsequent alkaline deprotection of the two carboxylate ester groups. At 37  C, the resulting new PFA-amino acid (Val, Leu, Phe) conjugates (4a–c) undergo P–N cleavage near neutral pH, cleanly releasing PFA. A kinetic investigation of 4a hydrolysis at pH values 6.7, 7.2, and 8.5 showed that PFA release was first-order in [4a] with respective t 1/2 values of 1.4, 3.8, and 10.6 h. # 2004 Elsevier Ltd. All rights reserved. Foscarnet, the trisodium salt of phosphonoformic acid (PFA), has been used as an antiviral agent 1 4 to treat AIDS-related cytomegalovirus infection, and also exerts an inhibitory action against HIV. 5 The antiviral effect of PFA is attributed to inhibition of viral nucleic acid polymerases. 6,7 Because of its multiple negative charges at physiological pH, PFA has low oral bioavailability; this poor ability to penetrate cell membranes has long been a major impediment to its overall effectiveness as an anti-viral drug. 8,9 Past efforts to address this short- coming by prodrug strategies have included partial esterification of PFA, 8 13 including its incorporation into cyclic esters, 11 and the replacement of an oxygen atom in PFA by a sulfur atom. 14 16 It has been shown that all three acidic groups of PFA must be available for maximal nucleic acid polymerase inhibition activity, and thus the ultimate antiviral activity of PFA prodrugs wherein one or more of these groups has been deriva- tized by esterification (or amidation) is likely to depend on a hydrolytic activation process in vivo to release the active drug. 8 Thus, PFA–amino acid conjugates might be useful as prodrugs of foscarnet, provided that such compounds are synthetically accessible and facile clea- vage of the PFA–amino acid linkage occurs in vivo. It was recently reported that a fully alkyl-protected, neu- tral P–N linked PFA–amino acid phosphoramide produced by coupling (MeO)(Cl)P(O)CO 2 Me with an l-amino acid Et ester, decomposed with P–C cleavage under basic deprotection conditions. 17 19 The same workers found that acidic conditions applied to the same protected conjugate hydrolyzed its P–N linkage instead of dealkylating the PFA diester. 17 This perplex- ing result is not unanticipated, given that the P–C bond in PFA esters is known to be highly activated due to juxtaposition of the carbonyl and phosphoryl groups, and is highly susceptible to cleavage under hydrolytic conditions to produce a phosphate or phosphite, depending on whether the water nucleophile attacks at carbon or phosphorus. 20 24 We reasoned that an inter- mediate product from condensation between C-methyl PFA and a C-alkyl-protected amino acid should be sta- bilized to unwanted P–C bond cleavage at high pH by virtue of its retention of one negative charge on the phosphonate group, thereby permitting alkaline depro- tection of both carboxylate esters. We report here successful application of this simple strategy to synthesis of the first examples of PFA– amino acid conjugates (4), and describe the surprisingly 0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2004.01.016 Bioorganic & Medicinal Chemistry Letters 14 (2004) 1787–1790 *Corresponding author. Tel.: +1-213-740-7007; fax: +1-213-740- 0930; e-mail: mckenna@usc.edu