pubs.acs.org/jmc Published on Web 09/01/2010 r 2010 American Chemical Society J. Med. Chem. 2010, 53, 6825–6837 6825 DOI: 10.1021/jm901828c Synthesis of Ester Prodrugs of 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) as Anti-Poxvirus Agents Marcela Kre cmerov a,* ,† Antonı´n Hol y, † Graciela Andrei, ‡ Karel Pomeisl, † Tom a  s Tich y, † Petra B rehov a, † Milena Masojı´dkov a, † Martin Dra cı´ nsk y, † Radek Pohl, † Genevieve Laflamme, § Lieve Naesens, ‡ Hon Hui, § Tomas Cihlar, § Johan Neyts, ‡ Erik De Clercq, ‡ Jan Balzarini, ‡ and Robert Snoeck ‡ † Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Gilead Sciences and IOCB Research Centre, Flemingovo n  am. 2, CZ-166 10, Prague 6, Czech Republic, ‡ Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and § Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404 Received December 11, 2009 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form were selected for further evaluation as potential drug candidates against poxvirus infections. To increase bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out: alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM), 2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP, with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or ∼10-fold lower than those observed for the parent compounds. Introduction The family of Poxviridae represents a group of enveloped, double-stranded DNA viruses with unique morphology and cytoplasmic site of replication. 1 They are very large (about 300  200 nm) and brick-shaped. Poxvirus-associated diseases are a serious problem for human health as well as veteri- nary medicine. The most dangerous human pathogen of this group is a member of the Orthopoxvirus genus: variola virus (VARV), a the causative agent of smallpox. Smallpox was one of the main causes of morbidity and mortality until recent times. In the 20th century alone, smallpox deaths worldwide numbered in the millions. In 1980, after an intensive program of immunization with a related but relatively nonpathogenic virus, i.e., vaccinia virus (VACV), the WHO declared the disease eradicated. The last naturally occurring outbreak of smallpox was in Somalia in 1977. After the disease was eliminated from the world, routine vaccination against small- pox among the general public was stopped because it was no longer necessary for prevention. By 1983, all known stocks of VARV were kept in two WHO collaborating centers: the U.S. Center for Disease Control and Prevention (CDC) in Atlanta and the Russian State Research Center of Virology and Biotechnology in Novosibirsk. However, in the aftermath of the events of September 2001, there is increasing concern that undeclared stocks of VARV might exist and that they might be used as a bioterrorist weapon. The discontinuation of routine vaccination against smallpox has rendered the human population more susceptible to this disease. Moreover, VARV is highly stable and retains its infectivity for relatively long periods of time outside the host. For all of these reasons, special attention has been paid to precautions for dealing with a smallpox outbreak. On the other hand, VACV vaccination is supposed to cause several complications, especially among immunocompromised patients. Therefore, there are increased demands for anti-poxvirus effective drugs to treat acute infections. Despite of progressive investigation in this area, and the number of compounds in preclinical and/or early clinical stage of development, no FDA approved drugs are currently available on the market. 2,3 Only three drugs have been used in the clinic for the treatment of complications associated with vaccination against smallpox. 4 These include the acyclic nucleoside analogue cidofovir and its oral prodrug, hexadecyloxypropyl ester (HDP-CDV), which inhibit viral DNA replication and compound ST-246, an orally bioavail- able compound that targets orthopoxvirus morphogenesis. 5 *Corresponding author: phone, þ420 220183475; fax, þ420 220183560; e-mail, marcela@uochb.cas.cz. a Abbreviations: ANP, acyclic nucleoside phosphonate; bis(POC)- PMPA, bis(isopropyloxycarbonyloxymethyl) ester of 9-(R)-[2-(phos- phonomethoxy)propyl]adenine; bis(POM)-PMEA, bis(pivaloyloxymethyl) ester of 9-[2-(phosphonomethoxy)ethyl]adenine; DCC, N,N 0 -dicyclohexyl- carbodiimide; HATU, O-7-(azabenzotriazol-1-yl)-N,N,N 0 ,N 0 -tetramethy- luronium hexafluorophosphate; HCMV, human cytomegalovirus; HDP- CDV, hexadecyloxypropyl ester of cidofovir; HOBt, 1-hydroxybenzotria- zole; HPMPA, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine; HPMPC, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir); HPMPDAP, 9-(S)-[3-hydroxy-2-(phosphonomethoxy)- propyl]-2,6-diaminopurine; HSV-1, herpes simplex virus type 1; HSV-2, herpes simplex virus type 2; iPr, 2-propyl; POM, pivaloyloxy- methyl; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; PyBroP, bromotripyrrolidinophosphonium hexafluorophosphate; TEA, triethylammonium; TEAB, triethylam- monium hydrogen carbonate; VACV, vaccinia virus; VARV, variola virus.