Synthesis, Anti-Human Immunodeficiency Virus Activity and Esterase Lability of Some Novel Carboxylic Ester-Modified Phosphoramidate Derivatives of Stavudine (d4T)

McGuigan, Christopher; Sutton, PW; Cahard, Dominique; Turner, Kevin B.; O’Leary, Gerard; Wang, Y; Gumbleton, Mark; Clercq, Erik De; Balzarini, Jan

doi:10.1177/095632029800900603

Cited by 44 publications

(50 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The first step in the metabolism of phosphoramidate prodrugs of nucleoside monophosphate analogs involves the hydrolysis of the carboxyl ester moiety (23,24). Examination of 23 different enzymes, including serine proteases, cysteine proteases, aspartic protease, and serine esterases, resulted in the identification of two serine proteases, cathepsin A and neutrophil elastase, and a serine esterase, carboxylesterase 1 that were capable of hydrolyzing the carboxyl ester of PSI-7851.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Murakami¹,

Tolstykh²,

Bao³

et al. 2010

Journal of Biological Chemistry

268

308

View full text Add to dashboard Cite

A phosphoramidate prodrug of 2-deoxy-2-␣-fluoro-␤-Cmethyluridine-5-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections (1-5). Recent studies have suggested that nucleoside analogs may be useful for treating hepatitis C virus (HCV) 3 infection (4, 6 -8). The most advanced anti-HCV nucleoside, RG7128, is a diisobutyrate nucleoside prodrug of ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylcytidine (PSI-6130) and is currently in phase IIb clinical studies. PSI-6130 demonstrated potent activity in the subgenomic HCV replicon assay (9); the incubation of radiolabeled PSI-6130 with either replicon cells or primary human hepatocytes resulted in the formation of the 5Ј-mono-, di-, and triphosphate metabolites of . The triphosphate metabolite (PSI-6130-TP) was shown to be a potent inhibitor of HCV NS5B RNA-directed RNA polymerase (RdRp) (11). However, incubation of replicon cells with the uridine analog, PSI-6206, resulted in no inhibition of HCV RNA production due to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases to its monophosphate, 12). Biochemical studies showed that PSI-7411 was consecutively phosphorylated to its diphosphate, PSI-7410, by UMP-CMP kinase and its triphosphate, PSI-7409, by nucleoside diphosphate kinase (12). Inhibition studies using the replic...

show abstract

Section: Discussionmentioning

confidence: 99%

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Murakami¹,

Tolstykh²,

Bao³

et al. 2010

Journal of Biological Chemistry

268

308

View full text Add to dashboard Cite

show abstract

“…GS-465124 is a nucleotide phosphoramidate, and the metabolic activation of this type of prodrug when applied to different nucleotide analogs has been characterized (18)(19)(20). Similar to the other phosphoramidate prodrugs targeting HCV, including sofosbuvir (GS-7977) and PSI-353661, GS-465124 was hydrolyzed by CES1 and CatA to form metabolite X, followed by HINT1-mediated removal of L-alanine resulting in the formation of the nucleoside analog monophosphate, GS-558272 (8, 9).…”

Section: Discussionmentioning

confidence: 99%

Metabolism and Pharmacokinetics of the Anti-Hepatitis C Virus Nucleotide Prodrug GS-6620

Murakami

Wang

Babusis

et al. 2014

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with L-alanine-isopropyl ester and phenol moieties attached to the 5=-phosphate that release the nucleoside monophosphate in hepatocytes and a 3=-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3=-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.A pproximately 170 million people worldwide are infected by the hepatitis C virus (HCV), and nearly 2% of the U.S. population is estimated to have HCV (1). HCV is a major health issue since approximately 15 to 35% of those who are chronically infected will develop cirrhosis and 1 to 3% will progress to hepatocellular carcinoma over 30 years (2). Currently, the recently approved protease inhibitors (boceprevir and telaprevir) are used in combination with pegylated interferon alpha plus ribavirin (PEG-RBV) for the treatment of chronic HCV infection. While these treatments show improved response rates and the potential for shorter duration of treatment over PEG-RBV alone, they lack efficacy against genotypes other than genotype 1, require thricedaily dosing, and cause additional side effects from the new directacting antivirals on top of the already-challenging tolerability profile of PEG-RBV. Therefore, there is a need for more potent anti-HCV compounds with improved clinical efficacy and greater tolerability in order to more broadly address the unmet medical needs of those with chronic HCV infection. Combinations of antiviral agents targeting viral proteins essential to HCV replication have the potential to achieve increased clinical efficacy across HCV genotypes, ...

show abstract

“…While GS-7340 and other amidate prodrugs of tenofovir successfully validated the concept of enhanced in vivo intracellular delivery of parent nucleotide, GS-9131 has been selected as a clinical development candidate based on its unique activity against HIV-1 strains resistant to approved antiretroviral nucleosides and its favorable in vivo pharmacological properties, including the ability to effectively deliver the active GS-9148 diphosphate metabolite into PBMCs (9,36). The prodrug moieties of GS-7340 and GS-9131 are structurally similar to a class of nucleoside monophosphate pro-drugs referred to as phosphoramidate pronucleotides (27,28,43,47,51). The initial step in the proposed activation mechanism for the amidate prodrugs is the hydrolysis of the carboxyester bond by an unidentified cellular hydrolase ( Fig.…”

Section: Selectively Hydrolyzed Inside Cells To Antiviral Nucleotidesmentioning

confidence: 99%

Cathepsin A Is the Major Hydrolase Catalyzing the Intracellular Hydrolysis of the Antiretroviral Nucleotide Phosphonoamidate Prodrugs GS-7340 and GS-9131

Birkuš

Wang

Liu

et al. 2007

Antimicrob Agents Chemother

159

162

View full text Add to dashboard Cite

GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-Tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV), an acyclic nucleotide analog of dAMP, is a potent in vitro and in vivo inhibitor of human immunodeficiency virus type 1 (HIV-1) replication (2). TFV is sequentially phosphorylated in the cell by AMP kinase and nucleoside diphosphate kinase to the active species, tenofovir diphosphate (23, 39), which acts as a potent inhibitor of HIV-1 reverse transcriptase (7,49). The presence of a nonhydrolyzable phosphonic acid moiety in tenofovir circumvents an initial phosphorylation step which can be rate limiting for the activation of nucleoside analog inhibitors of HIV reverse transcriptase (3, 4).In order to increase the cellular permeability and oral bioavailability of TFV, which is a dianion at physiological pH, neutral prodrugs of TFV have been synthesized. Tenofovir disoproxil fumarate (TDF) is a bis-isopropoxycarbonyloxymethyl ester prodrug of TFV approved for the treatment of HIV. TDF is well tolerated, with infrequent development of resistance and a favorable long-term toxicity profile (2,8, 16). The oral administration of TDF results in high systemic levels of TFV (5); however, the rapid systemic degradation of TDF to TFV limits its uptake into target cells.Investigations to develop plasma-stable prodrugs which would be selectively hydrolyzed inside cells to antiviral nucleotides led to the design of GS-7340 and GS-9131{9--fluoro-1Ј-furanyladenine, respectively}, alkylalaninyl amidate phenyl ester prodrugs of TFV and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2Ј-fluoro-2Ј,3Ј-dideoxydidehydroadenosine), respectively. Both GS-7340 and GS-9131 exhibit potent in vitro anti-HIV-1 activities, favorable resistance profiles, and low cytotoxicities (9, 25). Compared to TDF, GS-7340 is significantly more stable in plasma and delivers ϳ30-fold-greater levels of active diphosphate metabolites into peripheral blood mononuclear cells (PBMCs) in vitro and in vivo (12). Compared to what was seen for TDF, oral administration of an equal dose of GS-7340 resulted in significantly higher levels of TFV accumulation both in lymphatic tissues and in PBMCs (24). While GS-7340 and other amidate prodrugs of tenofovir successfully validated the concept of enhanced in vivo intracellular delivery of parent nucleotide, GS-9131 has been selected as a clinical development candidate based on its unique activity against HIV-1 strains resistant to approved antiretroviral nucleosides and its favorable in vivo pharmacological properties, including the ability to effectively deliver the active GS-9148 diphosphate metabolite into PBMCs (9, 36). The prodrug moieties of GS-7340 and GS-9131 are structurally similar to a class of nucleoside monophosphate pro-* Corresponding author. Mailing address: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404.

show abstract

Synthesis, Anti-Human Immunodeficiency Virus Activity and Esterase Lability of Some Novel Carboxylic Ester-Modified Phosphoramidate Derivatives of Stavudine (d4T)

Cited by 44 publications

References 5 publications

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Metabolism and Pharmacokinetics of the Anti-Hepatitis C Virus Nucleotide Prodrug GS-6620

Cathepsin A Is the Major Hydrolase Catalyzing the Intracellular Hydrolysis of the Antiretroviral Nucleotide Phosphonoamidate Prodrugs GS-7340 and GS-9131

Contact Info

Product

Resources

About