pubs.acs.org/jmc Published on Web 06/08/2010 r 2010 American Chemical Society J. Med. Chem. 2010, 53, 4949–4957 4949 DOI: 10.1021/jm1003792 Phosphoramidate ProTides of 2 0 -C-Methylguanosine as Highly Potent Inhibitors of Hepatitis C Virus. Study of Their in Vitro and in Vivo Properties Christopher McGuigan,* ,† Arnaud Gilles, † Karolina Madela, † Mohamed Aljarah, † Sabrina Holl, † Sarah Jones, † John Vernachio, ‡ Jeff Hutchins, ‡ Brenda Ames, ‡ K. Dawn Bryant, ‡ Elena Gorovits, ‡ Babita Ganguly, ‡ Damound Hunley, ‡ Andrea Hall, ‡ Alexander Kolykhalov, ‡ Yule Liu, ‡ Jerry Muhammad, ‡ Nicholas Raja, ‡ Robin Walters, ‡ Jin Wang, ‡ Stanley Chamberlain, ‡ and Geoffrey Henson ‡ † Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, U.K., and ‡ Inhibitex Inc., 9005 Westside Parkway, Alpharetta, Georgia 30004 Received March 26, 2010 Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2 0 -C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. L-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally L-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentra- tions of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date. Introduction An estimated 200 million people, or ca. 3.5% of the world’s population, are chronically infected with the hepatitis C virus (HCV a ) and at risk of developing life threatening liver disease such as cirrhosis or liver carcinoma. HCV infection is the major cause of liver transplantation in industrialized coun- tries. The current therapy for HCV consists of pegylated interferon and ribavirin, neither of which are specific inhibi- tors of HCV, and a regimen which has side effects and limited efficacy in at least half of the patient population. 1 The HCV genome offers several clear targets for specific anti-HCV therapy; these include an RNA polymerase and a serine protease, both of which have attracted considerable academic and industrial attention. 2 The RNA dependent HCV polymerase is considered to be an attractive target for therapy on account of a high degree of conservation across the six HCV genotypes, and agents tar- geted at the active site, such as nucleoside analogues, may be particularly advantageous with respect to the barrier to resistance. 3 Several families of nucleoside analogues have emerged with apparent selectivity for HCV, these include 4 0 -modified agents such as 4 0 -azidocytidine (R1479, 1a, Figure 1) developed as an oral pro-drug by Roche 4 and 2 0 -C-methyl adenosine (1b, Figure 1) and related deaza compounds developed by Merck. 5 Interestingly, the Merck team observed that the guanine analogue 1c was more potent (>10-fold) as an inhibitor of the HCV RNA polymerase, as its bioactive 5 0 -triphosphate form, than was the triphosphate of the adenosine lead 1b. However, poor cell entry by 1c coupled with apparently poor phospho- rylation lead to it being >10-fold less active than 1b in HCV replicon (1b EC 50 0.26 μM, 1c 3.5 μM). 5 Unfortunately, despite the reasonable in vitro potency and selectivity of 1b, the compound could not be progressed due to it being a good substrate for metabolic enzymes (adenosine deaminase and purine nucleoside phosphorylase), leading to low oral bioavailability. 5 To overcome these PK liabilities of 1b, the Merck team progressed with several unnatural base modifications, such as the 7-deaza family, leading to their late preclinical candidate MK-0608. As recently highlighted, a lack of data on this family for over three years now may place a query over their progres- sion. 3 We were concerned that such base modifications could carry with them the possibility of toxicity, and we sought to retain a natural base and to seek to overcome the limitations of the guanosine compound 1c in particular. Because the first phosphorylation of 1c was considered to be rate-limiting, we *To whom correspondence should be addressed. Phone/Fax: þ44 29 20874537. E-mail: mcguigan@cardiff.ac.uk. a Abbreviations: HCV, hepatitis C virus; TLC, thin layer chromato- graphy; HPLC, high performance, liquid chromatography; ClogP, calculated logarithm of octanol-water partition coefficient; PK, phar- macokinetics; SI, selectivity index; DNAUC, dose normalized area under the curve; NTP, nucleoside triphosphate; THF, tetrahydrofuran.