A Cyclic Phosphoramidate Prodrug of 2'-Deoxy-2'-Fluoro-2'- C-Methylguanosine for the Treatment of Dengue Virus Infection

Abstract

Monophosphate prodrug analogs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue virus activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are among the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate, prodrug 17, demonstrating well-balanced anti-dengue virus cellular activity and in vitro stability profiles. We further determined the PBMC concentration of active triphosphate needed to inhibit virus replication by 50% (TP₅₀). Compound 17 was assessed in an AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reductions at 100 and 300 mg/kg twice a day (BID), respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs exceeded the TP₅₀ value, demonstrating TP₅₀ as the target exposure for efficacy. In dogs, oral administration of compound 17 resulted in high PBMC triphosphate levels, exceeding the TP₅₀ at 10 mg/kg. Unfortunately, 2-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that "no observed adverse effect level" (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of compound 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue virus infection.

Keywords: cyclic phosphoramidate; dengue; monophosphate prodrug; nucleoside triphosphate; nucleotide analog; polymerase inhibitor.

FIG 1

Structures and in vitro biological activities of selected dengue antiviral compounds. (A) Structures and in vitro biological profile of NITD-008 (1), R-1479 (2), and balapiravir (3). (B) Structures and in vitro biological profile of PSI-352938 (4), PSI-353661 (5), and their corresponding triphosphate 6 and nucleoside 7 metabolites. (C) Synthesis of cyclic phosphoramidate prodrugs of 6-O-alkyl-2′-deoxy-2′-fluoro-2′-C-methylguanosine. Reactions conditions are as follows: (i) t-BuMgCl in tetrahydrofuran; (ii) t-BuOK in DMSO; (iii) separation by preparative reverse-phase HPLC.

FIG 2

Single X-ray crystal structure of compound 17, represented as ball and stick model. The oxygen, nitrogen, fluorine, and phosphorous atoms are colored in red, blue, green, and orange, respectively.

FIG 3

Correlation between triphosphate levels versus potencies in human PBMCs. For the PBMC triphosphate level, different prodrugs (compounds 12, 5, 17, 14, and 18) were incubated in human PBMCs at 10 μM for 24 h, and then the intracellular triphosphate concentration was measured by LC-MS/MS. Data were obtained from at least two biological replicates. The DENV-2 EC₅₀ was obtained from the human PBMCs plaque assay. See Materials and Methods for further details.

FIG 4

Pharmacokinetic profiles of triphosphate 6 in PBMCs. Selected prodrugs (compounds 12, 14, 17, and 18) were dosed orally (3 mg/kg) to Beagle dogs, and the PBMC triphosphate concentration from each prodrug was measured by LC-MS/MS. Error bars represent standard deviations (n = 3 dogs).

FIG 5

TP₅₀ determination. Compound 17 (3, 10, 30, and 100 μM) was incubated in human PBMCs for 24 h, and the intracellular triphosphate concentration was measured. A Michaelis-Menten equation was used for curve fitting. The TP₅₀ was calculated from extrapolating the DENV-2 EC₅₀ of compound 17 to the triphosphate concentration. See Materials and Methods for more details. Experiments were conducted in three biological replicates. The graph represents one of the three biological experiments (error bars represent standard deviations from three technical replicates in each experiment). The average TP₅₀ is 0.22 ± 0.12 pmol/1e6 cells (0.78 ± 0.42 μM) (means ± standard deviations from 3 biological replicates).

FIG 6

Efficacy study and PBMC triphosphate analysis in the dengue viremia mouse model. (A) Dosing scheme of efficacy study in AG129 mice. Compound 17 was dosed p.o. immediately after infection at 10, 30, 100, and 300 mg/kg BID for 3 days (total doses of 20, 60, 200, and 600 mg/kg/day). Each dose group contained 6 mice. Plasma was sampled at 1, 3, 6, 24, 48, 50, 52, 55, and 72 h after the first dose for intact prodrug and nucleoside 7 analysis. A plasma sample was also taken at 72 h after the first dose (terminal sampling) for viremia readout. PBMCs were collected during the terminal time points for triphosphate analysis. (B) Compound 17 shows efficacy in AG129 mice. Viremia readout from each mouse was done on day 3 (at terminal time points) by plaque assay. Error bars represent standard deviations (n = 6 mice). Compound 17 reduced viremia by 3-, 4-, 28- and 54-fold at doses of 10, 30, 100, and 300 mg/kg/day BID, respectively. The viremia reductions at doses of 100 and 300 mg/kg BID are significant (P < 0.0001). The difference in the viremia reduction levels between the 30- and 100- or 300-mg/kg BID groups are also significant (P < 0.01 or P < 0.0001, respectively). *, P < 0.01; ***, P < 0.0001. (C) PBMC triphosphate concentration from 30- and 100-mg/kg BID groups. Blood from 6 mice was pooled at the terminal time point (72 h after the first dose) to collect PBMCs. The terminal triphosphate concentrations were 0.39 and 1.43 μM for the 30- and 100-mg/kg BID groups, respectively. The terminal triphosphate level from the 100-mg/kg BID group exceeded TP₅₀.

FIG 7

Pharmacokinetic profiles of the intact prodrug (A), major metabolite nucleoside 7 in plasma (B), and triphosphate metabolite 6 in PBMCs (C) upon single rising doses of compound 17 in Beagle dogs. Error bars represent standard deviations (n = 3 dogs). The triphosphate concentration in PBMCs was determined only from the 10- and 30-mg/kg groups. At 10 mg/kg, the PBMC triphosphate level exceeded TP₅₀.