β-d-2′-C-Methyl-2,6-diaminopurine Ribonucleoside Phosphoramidates are Potent and Selective Inhibitors of Hepatitis C Virus (HCV) and Are Bioconverted Intracellularly to Bioactive 2,6-Diaminopurine and Guanosine 5′-Triphosphate Forms

Abstract

The conversion of selected β-d-2,6-diaminopurine nucleosides (DAPNs) to their phosphoramidate prodrug (PD) substantially blocks the conversion to the G-analog allowing for the generation of two bioactive nucleoside triphosphates (NTPs) in human hepatocytes. A variety of 2′-C-methyl DAPN-PDs were prepared and evaluated for inhibition of HCV viral replication in Huh-7 cells, cytotoxicity in various cell lines, and cellular pharmacology in both Huh-7 and primary human liver cells. The DAPN-PDs were pangenotypic, effective against various HCV resistant mutants, and resistant variants could not be selected. 2′-C-Me-DAPN-TP and 2′-C-Me-GTP were chain terminators for genotype 1b HCV-pol, and single nucleotide incorporation assays revealed that 2′-C-Me-DAPN-TP was incorporated opposite U. No cytotoxicity was observed with our DAPN-PD when tested up to 50 μM. A novel, DAPN-PD, 15c, has been selected for further evaluation because of its good virologic and toxicologic profile and its ability to deliver two active metabolites, potentially simplifying HCV treatment.

Graphical Abstract

INTRODUCTION

Over the past 5 decades nucleoside analogs have played an ever-increasing role in the treatment of viral infections. To date there are more than a dozen nucleoside analogs approved by the FDA for the treatment of viral infections. Approximately 170 million people worldwide are infected with the seven major genotypes of hepatitis C virus (HCV), and 3–4 million individuals become newly infected each year.¹ HCV infection often leads to reduced liver function, cirrhosis, and cancer and is the leading cause of liver transplantation.² The first FDA-approved treatment for chronic HCV was limited to pegylated interferon-α alone or in combination with ribavirin,³ which was effective in 40–60% of patients and is often associated with significant side effects, such as fatigue, nausea, depression, and thinning hair.⁴ In May 2011 the introduction of protease inhibitors (PI) boceprevir and telaprevir to the above standard of care marked the introduction of the first direct acting antiviral agents (DAAs) for HCV.^5,6 Unfortunately, these treatments have limited efficacy for the genotype 1 virus, the most prevalent in the U.S. and China. They are also associated with various side effects, especially when combined with pegylated interferon-α and ribavirin.⁷

Three major drug targets have reached evaluation in humans for chronic HCV infection:^8,9 the NS3/4A serine protease, the large phosphoprotein NS5A, and NS5B RNA-dependent RNA polymerase (RdRp) NS5B (nucleoside¹⁰ and non-nucleoside inhibitors¹¹). The HCV NS5B RNA-dependent RNA polymerase (RdRp) is essential for HCV replication, and both nucleoside and non-nucleoside antiviral agents targeting this enzyme are in preclinical and clinical development. For a nucleoside analog to be incorporated into a growing RNA chain by a polymerase, it must first enter the cell by either passive or active transport and undergo three separate phosphorylation steps to ultimately provide the nucleoside analog in its triphosphate form (NTP). It has been reported that many nucleoside analogs are poor substrates for the phosphorylation kinases, which convert the nucleosides to the corresponding nucleoside monophosphate.¹² The initial phosphorylation step is often rate limiting en route to the formation of the active NTP and in many cases does not occur with sufficient efficiency to provide the active NTP form at therapeutically effective concentrations. In order to bypass this rate-limiting step, many nucleoside prodrug analogs have been developed as more lipophilic nucleoside monophosphate prodrugs (NMP-PDs).^10,13,14 NMP-PDs antiviral drugs have been reported to increase the oral bioavailability, liver exposure, and brain penetration of parent drugs. The most advanced nucleoside NMP-PDs are the aryloxy phosphoramidates (“ProTide” or “McGuigan PD”),¹⁵ 1-aryl-1,3-propanyl esters (“HepDirect”),¹⁶ and lipid conjugates¹⁷ which have all have been evaluated in humans. Pronucleotide (ProTide), first introduced in 1992,¹⁵ is the most investigated NMP-PD and demonstrated improved pharmacological activity of many parent nucleosides both in vitro and in vivo.¹⁸ Currently the most advanced NMP-PDs have the following limitations: (1) intracellular delivery of potentially toxic agents; (2) a racemic phosphorus center; (3) the nucleoside-MP phosphorus–oxygen bond being weak and therefore prone to cleavage; (4) low to very low yields for the preparation of NMP-PDs.¹⁴

Nucleoside analog polymerase inhibitors are anticipated to become the backbone of HCV therapy, and to date, at least 14 have entered clinical trials but only one has been approved (Figure 1).¹⁰ Sofosbuvir, which received FDA approval in December 2013, has highly potent clinical antiviral activity across all genotypes in combination with pegylated interferon and/or ribavirin or an NS5A inhibitor (e.g., Harvoni). Sofosbuvir containing regimens with other DAAs can be shorter in duration and achieve very high-sustained virologic response (SVR). However, the continued high cost of DAAs will preclude their prompt worldwide use especially when more than one DAA is needed to cure an HCV-infected person. In addition, the non-nucleoside inhibitors, unlike the nucleoside analogs, are prone to have a low genetic barrier to resistance. Therefore, there is an urgent need for more cost-effective treatments that are pan-genotypic that can be globally utilized.¹⁹

Figure 1.

Chemical structures (where available) of nucleoside analog inhibitors that have entered human clinical trials for HCV therapy.

Early clinical trials of direct acting antiviral agents for HCV treatment²⁰ made clear the need for combination DAA therapy in order to achieve high SVR and as the ultimate solution for pan genotypic treatment of HCV. The ability to deliver more than one active metabolite from a single drug could help increase potency for inhibition of HCV replication and potentially inhibit the formation of viral mutants.²¹ Like Orthrus, the fearsome two-headed dog of Greek mythology and guardian of Geryon’s herd of red cattle, an antiviral drug that delivers two potent metabolites should increase potency and potentially suppress the formation of viral mutants. The typical utilization of the aryloxy phosphoramidate prodrug approach, such as in the case of sofosbuvir, involves the intracellular delivery of phenol. While phenol is used in some over-the-counter products as an oral anesthetic/analgesic and is commonly used to temporarily treat pharyngitis (inflammation of the throat), it has an LD₅₀ of 270 mg/kg in mice and phenol may cause harmful effects to the central nervous system and heart, resulting in dysrhythmia, seizures, and coma. Chronic exposure to phenol in humans has been linked to harmful effects to the liver and kidneys.²² We sought to overcome these potential liabilities by the introduction of a propyl ester side chain at the ortho position of the phenol portion of our prodrugs. Cellular pharmacology in human peripheral blood mononuclear (PBM) cells showed that unmasking to the monophosphate from this substituted phosphoramidate results in the formation of hydroxyphenylpropanoic acid, which is a known nontoxic metabolite²³ of dihydrocoumarin, a common flavoring agent widely used in food²⁴ and cosmetics.²⁵

Herein we report the synthesis and biological evaluation of β-d-2′-C-methyl-2,6-diaminopurine ribonucleoside phosphoramidate prodrugs that intracellularly deliver two active nucleoside triphosphates along with nontoxic prodrug metabolites.

CHEMISTRY

The 2,6-diaminopurine tribenzoylated nucleoside 4 was synthesized from known sugar 2²⁶ and 2,6-diaminopurine 3 with TMSOTf in the presence of DBU. The unprotected 2,6-diaminopurine nucleoside 7 was prepared by reaction of 4 with ammonia in methanol. Boc protected nucleoside 6 was necessary to significantly improve the yield of the phosphoramidate forming reaction and prepared by reaction of 4 with (Boc)₂O at rt, followed by careful debenzylation with sodium methoxide at rt (Scheme 1). The crude debenzylation reaction was cautiously neutralized with Dowex resin (H⁺ form) during workup to avoid unwanted deprotection of the Boc groups.

Scheme 1^a.

^aReagents and conditions: (a) 2,6-diaminopurine (3), DBU, TMSOTf, CH₃CN, −40 to 65 °C, 5 h, 92%; (b) (Boc)₂O, DMAP, THF, rt, 90%; (c) NaOMe/MeOH, rt, 30 min, 92%; (d) NH₃/MeOH, rt, 93%.

Hydrolysis of dihydrocoumarin, 8, in anhydrous ethanol or isopropanol gave esters 9a and 9b, respectively. The phenyl dichlorophosphates 10a, 10b, and 10c were prepared from the appropriate phenol and phosphorus oxychloride. Aryloxy phosphorochloridates 12 were freshly prepared by treatment of l-alanine esters 11 with readily prepared aryloxy phosphorochloridates 10 (Scheme 2). Diaryloxy phosphorochloridate 13 was freshly synthesized with the same reaction conditions (Scheme 2). Since the phosphoryl chlorides 12 were produced as an approximate 1 to 1 mixture of the two possible diastereomers at the phosphorus center, the subsequent phosphoramidates 15 (Scheme 3) were also obtained as a diastereomeric mixture. This is due to the often clean inversion of configuration at the phosphorus atom during the S_NP reaction (similar to the inversion at electrophilic carbon centers in the more well-known S_N2 reaction). The phosphorochloridates 12 and 13 could be stored for extended periods when diluted in dry THF to 1 M and stored in a well-sealed container at −80 °C under nitrogen or argon.

Scheme 2^a.

^aReagents and conditions: (a) ROH, cat. H₂SO₄, reflux, overnight, for 9a, 94%; for 9b, 89%; (b) POCl₃, Et₃N, diethyl ether, −70 °C to rt, overnight; (c) amino acid esters hydrochloride (11), Et₃N, DCM, −70 °C to rt, two steps for 12a, 72%; for 12b, 75%; for 12c, 70%; for 12d, 69%; for 12e, 70%; (d) Et₃N, diethyl ether, −70 °C to rt, 35%.

Scheme 3^a.

^aReagents and conditions: (a) 12a–e, N-methylimidazole, THF–CH₃CN, rt; (b) 80% TFA, 0 °C to rt, two steps for 15a, 38%; for 15b, 56%; for 15c, 40%; for 15d, 41%; for 15e, 43%; (c) N,N′-carbonyl diimidazole, CH₃CN, rt, 71%; (d) 13, N-methylimidazole, THF–CH₃CN, rt; (e) 80% TFA, 0 °C to rt, two steps for 22, 55%.

Phosphoramidate prodrugs derived from the tetra-Boc nucleoside 6 were prepared initially following literature nucleoside phosphoryl chloride displacement methods, which conveniently afforded the desired products as a mixture of two diastereomers at the phosphorus center (14a–e).²⁷ For example, compound 15b was prepared by reacting 6 in the presence of N-methylimidazole with the phosphoryl chloride 12b in THF/ACN to produce protected prodrug 14b. Subsequent deprotection with 80% aqueous TFA gave phosphoramidate 15b in high overall yield from this two-step process (Scheme 3). Acetonitrile was used as a cosolvent to improve the solubility of the reactants, which resulted in a more complete reaction and much better isolated yields. Similar reaction conditions were employed for the synthesis of prodrug 17, which contains an achiral phosphorus center. For the more lipophilic and potentially better absorbed 2′,3′-carbonate prodrug, 16,²⁸ compound 13c was simply treated with carbonyldiimidazole.

The diastereomeric mixtures 15b–e were separated by careful column chromatography on silica gel to obtain the two single diastereomers. However, preparation of 15b(R_p) in larger quantities required a more practical method. A single diastereomeric phosphoramidating reagent containing a p-nitrophenyl ester was used as an alternative to the chloridate 12b for the stereospecific synthesis of 15b(R_p).^28,29 Accordingly, the diastereomeric mixture 19(R_p) and 20(S_p) was prepared and subjected to fractional crystallization to obtain the single isomers 19(R_p) and 20(S_p) (see Experimental Section) (Scheme 4). Their absolute stereochemistry was determined by single crystal X-ray analysis (Figure 2). Considering the change in stereochemical nomenclature priorities, it is the reagent having the S_p configuration that would lead, after inversion through a nucleophilic displacement reaction with 6, to the desired product 15b(R_p). Thus, nucleoside 6 was treated with tert-butylmagnesium chloride in THF, and subsequent treatment of the resulting suspension with 20(S_p) at ambient for 3 days afforded the single diastereomer R_p, which was deprotected with aqueous TFA to afford 15b(R_p) (Scheme 4). The R_p reagent 19 gave the phosphoramidate product 15b(S_p) in similar yield (Scheme 4). The absolute configurations of the phosphorus diastereomers of 15c–e were assigned by comparison to 15b(S_p) and 15b(R_p).

Scheme 4^a.

^aReagents and conditions: (a) p-nitrophenol, Et₃N, diethyl ether, rt, 85% (19 + 20, 1:1 ratio); (b) 6, t-BuMgCl, THF, −78 °C, then rt 3 days; (c) 80% TFA, 0 °C, then rt 4 h. 15b(R_p): 41% two steps. 15b(S_p): 39% two steps.

Figure 2.

X-ray crystal structures of 19(R_p) and 20(S_P).

RESULTS AND DISCUSSION

Antiviral Activity and Cytotoxicity.

2′-C-Methyl-2,6-diaminopurine phosphoramidate prodrugs (DAPN-PD) were evaluated for inhibition of HCV RNA replication in Huh-7 cells using a subgenomic HCV replicon system.³⁰ Cytotoxicity in Huh-7 cells were determined simultaneously by extraction and amplification of both HCV RNA and cellular ribosomal RNA (rRNA) (Table 1).³¹ DAPN-PD compounds showed greater potency against HCV replication in Huh-7 cells when compared to the parent DAPN nucleoside 7 (up to 14-fold; e.g., EC₅₀ = 0.2 for 15b(R_p) vs 5.4 μM for parent compound 7. The difference in activity between the various pairs of phosphorus diastereomers could be due to different rates of deesterification that lead to different levels of unmasked monophosphate. In general one of the diastereomers was producing the majority of the antiviral activity when compared with the diastereomeric mixture. This was not the case for 15c, as the two phosphorus diastereomers had very similar anti-HCV potency. The 2′,3′-carbonate phosphoramidate 16 retained reasonable activity (EC₅₀ = 2.8 μM), but later it was found that the carbonate group falls off quickly in simulated intestinal fluid (T^1/2 < 5 h). The symmetrical phenolic ester 18 was inactive and presumed to be too stable in cell culture to unmask to the monophosphate. 1 (BMS-986094) was included as a positive control and, consistent with the literature,³² demonstrated nanomolar potency. Conversely, this compound was found to be cytotoxic in Huh-7 replicon cells as indicated by significant reductions in cellular rRNA levels (CC₅₀ = 0.8 μM). No cytotoxic effect was observed with up to 10 μM with the DAPN compounds in the same cell line.

Table 1.

HCV Replicon Activity and Cytotoxicity of Phosphoramidate Prodrugs

compd	replicon Huh-7 (μM)a			cytotoxicity, CC50 (μM)b
	EC50	EC90	CC50	PBM	CEM	Vero
7	5.4	>10	>33	>100	>100	>100
15a	0.9 ± 0.3	4.0 ± 2.8	>10	>100	>100	>100
15b	2.3	7.3	>10	>100	>100	>100
15b(Rp)	0.2 ± 0.1	0.7 ± 0.3	>10	>100	>100	>100
15b(Sp)	2.7	8.7	>10	>100	>100	>100
15c	0.7 ± 0.2	2.5 ± 0.2	>10	>100	>100	>100
15c(Rp)	1.0	2.7	>10	>100	>100	>100
15c(Sp)	0.9	2.8	>10	>100	40	>100
15d	2.2	9.0	>10	>100	>100	>100
15d(Rp)	2.4	8.2	>10	>100	>100	>100
15d(Sp)	4.7	9.7	>10	>100	>100	>100
15e	1.7	8.0	>10	>100	>100	>100
15e(Rp)	1.4	6.9	>10	>100	>100	>100
15e(Sp)	9.1	26	>10	>100	>100	>100
16	2.8	8.5	>10	>100	>100	>100
18	>10	>10	>10	64	25 ± 9.9	90
1	0.02 ± 0.2	0.04 ± 0.2	0.8	4.7 ± 3.3	8.7 ± 9.1	14 ± 5.1

^aEC₅₀, EC₉₀, and CC₅₀ values obtained from Huh-7 replicon were generated in triplicate from one or two separate experiments (±SD where applicable).

^bCC₅₀ values of compounds reported for PBM, CEM, and Vero cells were generated from one to four separate experiments (±SD where applicable).

DAPN compounds were further evaluated for cytotoxicity at higher concentrations in human PBM cells, human lymphoblastoid CEM, and African Green monkey Vero cells (Table 1).³³ In contrast to 1 (BMS-986094) that showed cytotoxicity in all cell types tested, we did not observe a cytotoxic effect with the majority of DAPN compounds. Prototype DAPN-PD with the highest antiviral activity, 15b(R_p), was also further evaluated for toxicity in PC3 cells (a human prostate cancer cell line) as well as its effect on cellular GAPDH levels and thymidine uptake. At 100 μM 15b(R_p) we did not observe any toxicity, effect on GAPDH levels, or effect on thymidine uptake (data not shown).

It has previously been reported that some nucleotide analog compounds may also inhibit Huh-7 cell growth³⁴ and exhibit mitochondrial toxicity.³⁵ In order to address this issue, compounds 7, 15a, 15b(R_p), and 15c were evaluated for their effects on mitochondrial DNA levels. HepG2 cells were propagated in the presence of nucleotide analogs (up to 50 μM) for 14 days prior to quantification of mitochondrial COXII DNA (mtDNA) and β-actin DNA using real-time PCR. Lamivudine (3TC) and β-d-2′,3′-dideoxycytidine (ddC) (at 10 μM) were included as negative and positive controls, respectively (Table 2). Neither the parent nor the DAPN prodrug compounds tested showed measurable mitochondrial toxicity up to 50 μM in HepG2 cell line. On the other hand, 1 (BMS-986094) showed mitochondrial toxicity with a CC₅₀ value of 4.7 ± 1.8 μM. Lactic acid levels were also measured in the culture supernatant after 14 days of incubation with each prodrug. The total amount of lactic acid produced was determined for each sample. Increased production of lactic acid (generally above 100% when normalized to β-actin control) is associated with mitochondrial toxicity.³⁶ We did not observe increased lactic acid production with up to 50 μM parent DAPN or DAPN-PD compounds. As expected, treatment with ddC resulted in increased lactic acid production (Table 2). We were unable to accurately measure lactic acid levels with 1 (BMS-986094) because treatment with 10 μM of this compound led to excessive cell death.

Table 2.

Mitochondrial Toxicity and Lactic Acid Production with DAPN Prodrugs

compd	CC50 (μM)a		lactic acid levels (% of β-actin control)b
	mtDNA	β-actin DNA	1 μM	10 μM	50 μM
7	>50	>50	110 ± 0.9	170 ± 3.7	110 ± 6.1
15a	>50	>50	100 ± 13	140 ± 2.0	90 ± 4.5
15b (Rp)	>50	>50	150 ± 1.7	120 ± 5.6	110 ± 1.9
15c	>50	>50	90 ± 5.1	81 ± 0.1	80 ± 3.0
1	3.4, 5.9, <1c	<1, 2.3, 0.86c	190 ± 20	UMd	UMd
3TC	>10	>10	NDe	91 ± 2.5	NDe
ddC	<10	<10	NDe	220 ± 11	NDe

^aCC₅₀ values obtained from HepG2 cells were generated from two separate experiments (±SD where applicable). mtDNA was measured and compared to nuclear β-actin DNA.

^bLactic acid measurements are the mean of triplicates from a single 96-well plate (±SD).

^cDetermined in three separate experiments.

^dUM: unmeasurable at 100 and 10 μM, as the percent of cells at these concentrations was <1.

^eND: not determined

Finally, activity of candidate DAPN-PD compound 15b(R_p) was also tested against a panel of genetically diverse HCV replicons. We found that 15b(R_p) was equally active against all HCV genotype replicons tested (Table 3). Furthermore, this compound remained effective against a panel of HCV genotype 1b replicons harboring mutations associated with drug resistance (Table 4).

Table 3.

Pan-Genotypic Activity of DAPN pProdrug 15b(R_p) Tested in Cell-Based Assays

Huh-7 replicon	EC50 (μM)	EC90 (μM)
GT 1a	0.35	0.74
GT 1b	0.26	0.39
GT 2a	0.26	0.70
GT 2b (chimera)a	0.29	0.42
GT 3a (chimera)a	0.23	0.39
GT 4a (chimera)a	0.39	0.89
GT 5a (chimera)a	0.32	0.64

^aChimeric replicons harbored NS5B gene from indicated genotypes in backbone of HCV GT 1b con1 strain.

Table 4.

Cell-Based Antiviral Activity of 15b(R_p) against Mutant HCV GT 1b Strains Commonly Associated with Drug Resistance

Huh-7 replicona	EC50 (μM)	EC90 (μM)
NS5A Y93H	0.30	3.0
NS3 R155K	0.32	1.3
NS5B S282T	0.10	0.23
NS5B C316Y	0.27	0.77
NS5B C445F	0.12	0.25
NS5B M414I	0.28	1.1
NS5B P495A	0.08	0.14

^aValues averaged from two to three experiments except GT 1a Y93H (n = 1).

Cellular Pharmacology and Biochemical Evaluation.

It has been well established that 6-modified nucleosides can be substrates for human deaminases.³⁷ We therefore set out to evaluate the intracellular metabolism of DAPN-PD in both Huh-7 cells and primary human hepatocytes.³⁸ Parent DAPN compound 7 and DAPN-PD 15b(R_p) were incubated in Huh-7 cells at 50 μM for 4 h at 37 °C. Intracellular metabolites were extracted with 70% ice-cold methanol in water and subsequently identified by LC–MS/MS. Incubation of the parent DAPN in Huh-7 revealed the generation of two triphosphate metabolites, namely, 2′-C-Me-DAPN-TP and 2′-C-Me-GTP at a 1:3 ratio (Figure 3A). Conversely, incubation of DAPN-PD 15b(R_p) increased the intracellular levels of 2′-C-Me-DAPN-TP in Huh-7 cells over 13-fold compared to the levels achieved with parent nucleoside DAPN (0.74 ± 0.3 pmol/10⁶ cells and 9.9 ± 5.5 pmol/10⁶ cells, respectively). The same trend was observed in primary human hepatocytes where 7-fold higher triphosphate metabolites were observed when compared to Huh-7 cells (Figure 3B). Very similar results were obtained in Huh-7 cells and primary human hepatocytes for 15c. Together, these data indicate that DAPN-PD was capable of delivering two nucleoside triphosphate metabolites intracellularly.

Figure 3.

Intracellular metabolism of parent DAPN and DAPN-PD in one donor hepatocytes. (A) An amount of 50 μM of either parent DAPN (gray bars) or DAPN-PD (15b(R_p)) (white bars) was incubated with Huh-7 cells for 4 h at 37 °C. Intracellular generation of parent nucleotide, NI-MP, NI-DP, and NI-TP was analyzed using LC–MS/MS. (B) An amount of 50 μM DAPN-PD (15b(R_p)) was incubated with primary human hepatocytes (PHH, gray bars) cells for 4 h at 37 °C. Generation of intracellular metabolites was determined as described above. Error bars represent standard deviation from three separate replicates from a single 12-well plate.

In Vitro Inhibition of Viral RNA Synthesis by NTP Metabolites Generated by DAPN-PD.

2′-C-Me-GTP is a known inhibitor of HCV NS5B polymerase.³⁹ On the other hand, 2′-C-Me-DAPN-TP has not been previously examined for its effect on RNA polymerization. In order to determine whether 2′-C-Me-DAPN-TP alone can inhibit NS5B-mediated RNA polymerization, we separately synthesized each of the 5′-triphosphate analogs and evaluated the1m in cell-free assays using purified recombinant NS5B enzyme (Figure 4). The IC₅₀ values for 2′-C-Me-GTP and 2′-C-Me-DAPN-TP were determined to be 5.6 ± 1.6 and 3.4 ± 1.1, respectively. Under the conditions tested, we found that both NTP metabolites inhibited polymerization as indicated by the reduction in full-length RNA product formation with increased TP inhibitor concentrations. As expected, distinct pausing was observed at sites of GMP incorporation for 2′-C-Me-GTP. Conversely, incubation with 2′-C-Me-DAPN-TP resulted in the appearance of distinct pausing bands at AMP incorporation sites. These data suggest that 2′-C-Me-DAPN-TP acts as an A-analog. This finding was consistent with previous observations that nucleotides with the 2,6-diaminopurine modification are incorporated opposite a template T or U.⁴⁰ Further biochemical characterization is underway to evaluate the kinetic properties of 2′-C-Me-DAPN-TP in vitro.

Figure 4.

Incorporation of NI-TPs by NS5B RNA-dependent RNA polymerase. Increasing amounts of either 2′-C-Me-DAPN-TP or 2′-C-Me-GTP were incubated with NS5B polymerase, RNA template, radiolabeled GpG primer, and rNTP nucleotide mix. Amount of RNA product formed was measured after a 2 h incubation and visualized on a 20% denaturing polyacrylamide gel. The RNA template sequence is indicated, and sites of either A analog or G analog incorporation are highlighted with an asterisk. Arrow represents full length RNA product formed in the absence of inhibitor.

CONCLUSIONS

The chemical synthesis, antiviral activity, cytotoxicity, and cellular metabolism of DAPN prodrug compounds were described. The intracellular metabolism of DAPN-PD resulted in the generation two NI-TP metabolites, namely, 2′-C-Me-DAPN-TP and 2′-C-Me-GTP, both of which inhibited HCV NS5B polymerase-mediated RNA synthesis in vitro. Furthermore, DAPN-PDs did not exhibit any of the in vitro toxicities observed with 1 (BMS-986094) and appear to be differentiated in their toxicity profile additionally with the release of nontoxic prodrug metabolites. Further studies are underway to evaluate to enzymatic properties of each of the nucleoside 5′-triphosphate metabolites, as well as the intracellular metabolism of DAPN-PD in various cell lines such as cardiomyocytes. On the basis of these results, compound 15c has been selected for further in depth preclinical studies.

EXPERIMENTAL SECTION

Anhydrous solvents were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Reagents were purchased from commercial sources. Unless noted otherwise, the materials used in the examples were obtained from readily available commercial suppliers or synthesized by standard methods known to one skilled in the art of chemical synthesis. ¹H and ¹³C NMR spectra were taken on a Varian Unity Plus 400 spectrometer at rt and reported in ppm downfield from internal tetramethylsilane. Deuterium exchange, decoupling experiments were done to confirm proton assignments. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). All J-values are in Hz. Purity of final compounds was determined to be >95%, using analytical HPLC analyses performed on a Hewlett-Packard 1100 HPLC instrument with a Phenomenex Gemini-NX column (2 mm × 50 mm, 3 μm, C18, 110 Å) and further supported by clean NMR spectra. Mobile phase flow was 0.5 mL/min with a 3.5 min initial hold, a 6.5 min gradient from 96% aqueous media (0.05% formic acid) to 96% CH₃CN (0.05% formic acid), and a 15 min total acquisition time. Photodiode array detection was from 190 to 360 nm. Mass spectra were determined on a Micromass Platform LC spectrometer using electrospray ionization. Analytic TLC was performed on Whatman LK6F silica gel plates, and preparative TLC was performed on Whatman PK5F silica gel plates. Column chromatography was carried out on silica gel or via reverse-phase high performance liquid chromatography.

(2R,3R,4R,5R)-5-((Benzoyloxy)methyl)-2-(2,6-diamino-9H-purin-9-yl)-3-methyltetrahydrofuran-3,4-diyl Dibenzoate, 4.

To a stirred suspension of (3R,4S,5R)-5-((benzoyloxy)methyl)-3-methyltetrahydrofuran-2,3,4-triyl tribenzoate 2 (2.9 g, 5 mmol) and 2,6-diaminopurine 3 (830 mg, 5.5 mmol) in anhydrous acetonitrile at −78 °C was added DBU (2.3 mL, 15 mmol), followed by a slow addition of TMSOTf (3.8 mL, 20 mmol). The reaction mixture was stirred at −78 °C for 20 min and then raised to 0 °C. After it was stirred for 30 min at 0 °C, the reaction mixture was heated gradually to 65 °C and stirred overnight. The reaction mixture was cooled, diluted with CH₂Cl₂ (200 mL), and washed with saturated NaHCO₃. The layers were separated, and the resulting aqueous layer was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic layers were dried over Na₂SO₄. After removal of the solvent, the residue was purified by silica gel column chromatography (0–10% MeOH in EtOAc). 2.8 g of compound 4 was obtained (92% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.15–8.17 (m, 2H), 7.97–8.02 (m, 4H), 7.77 (s, 1H), 7.45–7.61 (m, 5H), 7.32–7.37 (m, 4H), 6.62 (s, 1H), 6.53 (d, 1H, J = 6.6 Hz), 5.91 (s, 2H), 5.04–5.10 (m, 3H), 4.82–4.86 (m, 1H), 4.70–4.74 (m, 1H), 1.62 (s, 3H). ¹³C NMR (CDCl₃): δ 17.5, 64.0, 76.0, 79.4, 85.8, 88.5, 114.9, 128.3, 128.4, 128.5, 128.9, 129.5, 129.7, 129.8, 129.9, 133.1, 133.5, 133.6, 137.4, 151.3, 155.9, 159.7, 165.3, 165.4, 166.3. LC/MS calcd for C₃₂H₂₉N₆O₇ (M + 1)⁺, 609.2; observed, 609.2.

(2R,3R,4R,5R)-5-((Benzoyloxy)methyl)-2-(2,6-bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-3-methyltetrahydrofuran-3,4-diyl Dibenzoate, 5.

A solution of 4 (1.4 g, 2.3 mmol), Boc anhydride (3.0 g, 14 mmol), and DMAP (56 mg, 0.46 mmol) in THF (12 mL) was stirred at rt for 30 h. After the reaction was complete, the solvent was removed under reduced pressure and the residue was purified by flash column chromatography (0–40% EtOAc in hexane). 2.1 g of white solid 5 was obtained (90% yield). ¹H NMR (400 MHz, CD₃OD) δ 8.39 (s, 1H, H₈), 8.07–8.20 (m, 4H, Ar–H), 7.87–7.89 (m, 2H, Ar–H), 7.42–7.61 (m, 7H, Ar–H), 7.25–7.30 (m, 2H, Ar–H), 6.74 (s, 1H, H₁′), 6.00 (d, 1H, J = 4.8 Hz), 4.95–4.96 (m, 2H), 4.72–4.76 (m, 1H), 1.58 (s, 3H), 1.44 (s, 18H), 1.38 (s, 18H). ¹³CNMR (100 MHz, CD₃Cl) δ 17.8, 27.7, 27.7, 63.6, 76.0, 81.2, 83.3, 83.9, 84.2, 89.2, 127.7, 128.4, 128.5, 128.6, 128.7, 129.3, 129.4, 129.7, 129.75, 130.0, 133.4, 133.6, 133.7, 143.7, 150.0, 150.6, 151.4, 152.4, 153.3, 165.1, 165.2, 166.3.

Di-tert-butyl (9-((2R,3R,4R,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-9H-purine-2,6-diyl)bis-(tert-butoxycarbonylcarbamate), 6.

To a solution of 5 (1.7 g, 1.7 mmol) in anhydrous methanol (50 mL) was added a solution of sodium methoxide (4.4 M, 0.3 mL, 1.3 mmol) at rt for 30 min (monitored by TLC and LC–MS). After the reaction was complete, Dowex resin (H⁺ form) was added portionwise to adjust the pH to 7.0. The resin was filtered and washed with methanol, the filtrate was concentrated, and the residue was purified by flash column chromatography (0–10% MeOH in CH₂Cl₂) to afford 1.1 g of white solid 6 (92% yield). ¹H NMR (400 MHz, CD₃OD): 9.09 (s, 1H), 6.19 (s, 1H), 4.22 (d, 1H, J = 8.8 Hz), 4.03–4.11 (m, 2H), 3.89 (dd, 1H, J = 2.8 Hz, J = 12.4 Hz), 1.41 (s, 18H), 1.40 (s, 18H), 0.92 (s, 3H). ¹³C NMR (CD₃OD): 20.2, 27.9, 28.1, 60.9, 73.1, 80.2, 84.6, 84.9, 85.4, 93.3, 128.8, 147.0, 151.2, 151.9, 152.0, 152.9, 155.0. LC/MS calcd for C₃₁H₄₉N₆O₁₂ (M + 1)⁺, 697.3; observed, 697.4.

(2R,3R,4R,5R)-2-(2,6-Diamino-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol, 7.

Nucleoside 4 (1.1 g, 1.7 mmol) was treated with saturated ammonia/methanol (100 mL). The reaction mixture was stirred at rt for 36 h to completion. After removal of the solvent under reduced pressure, 150 mL of dichloromethane was added to the residue. The white solid 7 was collected and dried (500 mg, 93%). ¹H NMR (400 MHz, DMSO-d₆): 8.01 (s, 1H), 6.73 (s, 2H), 5.81 (s, 2H), 5.76 (d, J = 2.8 Hz, 1H), 5.18–5.22 (t, J = 6.8 Hz), 5.05 (s, 1H), 0.78 (s, 3H), 3.98–4.02 (m, 1H), 3.78–3.84 (m, 2H), 3.63–3.67 (m, 1H). LC/MS calcd for C₁₁H₁₇N₆O₂ (M + 1)⁺, 297.1; observed, 297.2.

Ethyl 3-(2-Hydroxyphenyl)propanoate, 9a.

Dihydrocoumarin (52 g, 350 mmol) was added to 300 mL of anhydrous ethanol. H₂SO₄ (0.5 mL) was added, and the resulting solution was heated overnight at reflux. To the solution was added Ambersep 900 OH resin with stirring until approximately pH 7.0 was reached, and then the solution was filtered. The filtrate was evaporated under reduced pressure, and the residue was dissolved in ether acetate (300 mL), washed with water (3 × 50 mL), brine (50 mL), and dried over MgSO₄. The solution was filtered, and the filtrate was evaporated under reduced pressure. The residue was dried to give 64 g of solid product 9a (94% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.40 (s, 1H), 7.05–7.15 (m, 2H), 6.84–6.90 (m, 2H), 4.14 (q, J = 6.8 Hz, 2H), 2.90 (m, 2H), 2.72 (m, 2H), 1.23 (t, J = 6.8 Hz, 3H). LC/MS calcd for C₁₁H₁₅O₃ (M + 1)⁺, 195.1; observed, 195.2.

Isopropyl 3-(2-Hydroxyphenyl)propanoate, 9b.

Same procedure was employed for the preparation of compound 9b in 89% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.47 (brs, 1H), 7.09–7.27 (m, 2H, Ar–H), 6.87–6.91 (m, 2H, Ar–H), 5.00–5.06 (m, 1H), 2.90–2.93 (m, 2H), 2.68–2.72 (m, 2H), 1.22 (d, 6H, J = 8.0 Hz). ¹³C NMR (100 MHz, CD₃Cl) δ 21.7, 24.7, 35.5, 68.9, 117.1, 120.6, 127.4, 127.9, 130.5, 154.4, 175.2. LCMS calcd for C₁₂H₁₇O₃ (M + 1)⁺, 209.1; observed, 209.2.

Ethyl 3-(2-((Chloro(((S)-1-ethoxy-1-oxopropan-2-yl)amino)-phosphoryl)oxy)phenyl)propanoate, 12b.

To a solution of phosphorus oxychloride (2.4 mL, 26 mmol) in 70 mL of anhydrous diethyl ether was added dropwise a solution of 9a (5.0 g, 26 mmol) and triethylamine (3.6 mL, 26 mmol) in 80 mL of anhydrous diethyl ether at −78 °C under an Ar atmosphere over 2 h. After stirring for 1 h at −78 °C under Ar atmosphere, the solution was additionally stirred for 15 h toward rt, and then the solids were removed by filtration under a N₂ atmosphere. The solid salt was washed with anhydrous diethyl ether. The filtrate was concentrated under reduced pressure and dried under high vacuum overnight at rt. To a mixture of the oil 10b and dried L-alanine ethyl ester hydrogen chloride 11 (3.9 g, 26 mmol) in 20 mL of anhydrous CH₂Cl₂ was added a solution of Et₃N (7 mL, 51 mmol) in 20 mL of anhydrous CH₂Cl₂ over 2 h at −78 °C under Ar atmosphere. The solution was stirred for 16 h at rt, and the solid was filtered. The filtrate was concentrated under reduced pressure and purified on a silica gel column (EtOAc/hexane; gradient 0–50%, v/v) to give 9.5 g of compound 12b in 75% yield in two steps. ¹H NMR (400 MHz, CDCl₃) (1:1 mixture of P diastereomers): δ 7.14–7.49 (m, 4H), 4.70–4.80 (m, 1H), 4.09–4.27 (m, 5H), 2.92–3.08 (m, 2H), 2.61–2.65 (m, 2H), 1.50–1.55 (m, 3H), 1.21–1.32 (m, 6H). ³¹P NMR (162 MHz, CDCl₃) δ 8.88, 8.72.

The same procedure was employed for the preparation of compounds 12a, 12c, 12d, and 12e in 72%, 70%, 69% and 70% yields, respectively.

12a.

¹H NMR (400 MHz, CDCl₃): δ 7.17–7.40 (m, 1H, 5H), 4.10–4.50 (m, 4h), 1.50–1.53 (m, 3H), 1.28–1.33 (m, 3H). ³¹PNMR (162 MHz, CDCl₃): 8.13, 9.15.

12c:

¹H NMR (400 MHz, CDCl₃): δ 7.43–7.48 (m, 1H), 7.13–7.26 (m, 3H), 4.97–5.12 (m, 2H), 4.50–4.53 (m, 1H), 4.08–4.23 (m, 1H), 2.93–3.02 (m, 2H), 2.58–2.62 (m, 2H), 1.18–1.29 (m, 12H). ³¹PNMR (162 MHz, CDCl₃): 8.64, 8.87.

12d.

¹H NMR (400 MHz, CDCl₃): δ 7.44–7.48 (m, 1H), 7.14–7.26 (m, 3H), 5.04–5.12 (m, 1H), 4.54–4.57 (m, 1H), 4.09–4.56 (m, 3H), 2.95–3.04 (m, 2H), 2.61–2.64 (m, 2H), 1.48–1.52 (m, 3H), 1.20–1.29 (m, 9H). ³¹PNMR (162 MHz, CDCl₃): 7.35, 7.57.

12e.

¹H NMR (400 MHz, CDCl₃): δ 7.44–7.48 (m, 1H), 7.16–7.26 (m, 3H), 4.97–5.03 (m, 1H), 4.47–4.53 (m, 1H), 4.16–4.28 (m, 3H), 2.94–3.04 (m, 2H), 2.58–2.62 (m, 2H), 1.50–1.55 (m, 3H), 1.29–1.33 (m, 3H), 1.17–1.22 (m, 6H). ³¹PNMR (162 MHz, CDCl₃): 7.84, 8.08.

Diethyl 3,3′-(((Chlorophosphoryl)bis(oxy))bis(2,1-phenylene))dipropanoate, 13.

To a solution of phosphorus oxychloride (0.47 mL, 5.0 mmol) in 30 mL of anhydrous diethyl ether were added dropwise a solution of 9a (1.9 g, 10 mmol) and triethylamine (1.4 mL, 10 mmol) in 50 mL of anhydrous diethyl ether at −78 °C under an Ar atmosphere in 1.5 h. After stirring for 1 h at −78 °C under Ar atmosphere, the solution was additionally stirred for 15 h toward rt and then the solid was removed by filtration under a N₂ atmosphere. The solid salt was washed anhydrous diethyl ether. The filtrate was concentrated under reduced pressure and purified on a silica gel column (EtOAc/hexane; gradient 0–25%, v/v) to give 815 mg of compound 13 as a colorless oil in 35% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.44–7.47 (m, 2H), 7.18–7.31 (m, 6H), 4.09–4.14 (q, J = 12.0 Hz, J = 8.0 Hz, 4H), 3.00 (t, J = 6.0 Hz, 4H), 2.60 (t, J = 8.0 Hz, 4H), 1.22 (t, J = 8.0 Hz, 6H). ³¹P NMR (162 MHz, CDCl₃) δ −5.85.

(2S)-Ethyl 2-((((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphorylamino)propanoate, 15a.

To a solution of 6 (780 mg, 1.1 mmol) and N-methylimidazole (0.45 mL, 5.8 mmol) in THF (5 mL) at 0 °C was added dropwise a 1 M solution of (2R)-ethyl 2-(chloro(phenoxy)phosphorylamino)propanoate¹ (5.8 mL, 5.8 mmol). The resulting mixture was stirred overnight at rt. After solvent removal under reduced pressure, the residue was purified by flash column chromatography with a gradient of MeOH (0–10% MeOH in CH₂Cl₂) to afford 580 mg of white solid 14a (54% yield). A precold solution of TFA (80%, 23 mL) was added to a precold 14a (550 mg, 0.58 mmol) in an ice bath. The solution was stirred from 0 °C to rt and stirred at rt for 4 h (monitored by TLC and LC/MS). After the reaction was completed, the solvent was removed under reduced pressure and the residue was coevaporated with methanol (4 × 15 mL). The residue was dissolved in methanol (20 mL) and neutralized by saturated NaHCO₃. After solvent removal, the residue was purified by flash column chromatography with a gradient of MeOH (0–15% MeOH in CH₂Cl₂) to afford 225 mg of white solid 15a (71%) (38% yield over two steps). ¹H NMR (400 MHz, CD₃OD) (1:1 mixture): 7.86 (s, 2H), 7.14–7.34 (m, 10H), 5.96 (s, 1H), 5.93 (s, 1H), 3.90–4.58 (m, 14H), 1.16–1.31 (m, 6H), 1.13–1.19 (m, 6H), 0.97 (s, 3H), 0.94 (s, 3H). ³¹P NMR (162 MHz, CD₃OD): 4.77, 4.89. LC/MS calcd for C₂₂H₃₁N₇O₈P (M + 1)⁺, 552.2; observed, 552.3.

Ethyl 3-(2-(((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)-propanoate, 15b(S_p), and Ethyl 3-(2-(((R)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl)methoxy)(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)propanoate 15b(R_p).

A procedure similar to that used for 15a was employed for the synthesis of prodrug 15b (56% over two steps). Two diasteroisomers were separated and identified.

15b(S_p).

Optical rotation [α]²⁴_D −7.08 (c 0.24, MeOH). ¹H NMR (400 MHz, CD₃OD) δ 7.86 (s, 1H, H8), 7.10–7.39 (m, 4H, Ar–H), 5.93 (s, 1H, H1′), 3.96–4.51 (m, 9H), 2.99 (t, 2H, J = 8.0 Hz, CH₂), 2.62 (t, 2H, J = 8.0 Hz, CH₂), 1.34 (d, 3H, J = 7.2 Hz, CH₃), 1.15–1.17 (m, 6H, 2 × CH₃), 0.97 (s, 3H, CH₃). ³¹PNMR (162 MHz, CD₃OD) δ 5.03; LRMS calcd for C₂₇H₃₉N₇O₁₀ P (M + 1)⁺ 652.25, found 652.32.

15b(R_p).

Optical rotation [α]²⁴_d +12.12 (c 0.13, MeOH). ¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 8.0 Hz, J = 1.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 5.91 (s, 1H), 4.51–4.48 (m, 2H), 4.25–3.95 (m, 7H), 2.99 (t, J = 8.0 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.34 (d, J = 6.8 Hz, 3H), 1.18 (t, J = 7.2 Hz, 3H), 1.16 (t, J = 7.2 Hz, 3H), 0.97 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 4.98. LCMS calcd for C₂₇H₃₉N₇O₁₀P (M + 1)⁺ 652.3, found 652.3.

Isopropyl 3-(2-(((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)-phenyl)propanoate 15c(S_p) and Isopropyl 3-(2-(((R)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)propanoate 15c(R_p).

A procedure similar to that used for 15a was employed for the synthesis of prodrug 15c (40% over two steps). Two diastereoisomers were separated and identified.

15c(S_p).

¹H NMR (400 MHz, CD₃OD) δ 7.86 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.17 (dt, J = 8.0 Hz, J = 1.6 Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 5.05–4.84 (m, 2H), 4.62–4.46 (m, 2H), 4.22 (s, 2H), 3.95–3.91 (m, 1H), 2.99 (t, J = 8.0 Hz, 2H), 2.60 (t, J = 8.0 Hz, 2H), 1.33 (d, J = 7.2 Hz, 3H), 1.20 (d, J = 6.4 Hz, 3H), 1.19 (d, J = 6.0 Hz, 3H), 1.15 (d, J = 6.0 Hz, 3H), 1.14 (t, J = 6.4 Hz, 3H), 0.96 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 5.08. LRMS calcd for C₂₉H₄₃N₇O₁₀P (M + 1)⁺ 680.28, found 680.12.

15c(R_p).

¹H NMR (400 MHz, CD₃OD) δ 8.03 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 7.6 Hz, J = 1.6 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 5.91 (s, 1H), 4.95–4.79 (m, 2H), 4.53–4.46 (m, 2H), 4.18 (s, 2H), 3.95–3.91 (m, 1H), 2.99 (t, J = 8.0 Hz, 2H), 2.61 (t, J = 8.0 Hz, 2H), 1.35 (d, J = 7.2 Hz, 3H), 1.19–1.16 (m, 12H), 0.99 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 5.02. LRMS calcd for C₂₉H₄₃N₇O₁₀P (M + 1)⁺ 680.28, found 680.14.

Ethyl 3-(2-(((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)-phenyl)propanoate, 15d(S_p), and Ethyl 3-(2-(((R)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)propanoate, 15d(R_p).

A procedure similar to that used for 15a was employed for the synthesis of prodrug 15d (41% over two steps). Two diasteroisomers were separated and identified.

15d(S_p).

¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.16 (dt, J = 8.0 Hz, J = 1.6 Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 5.02–4.84 (m, 1H), 4.62–4.49 (m, 2H), 4.21 (s, 2H), 4.07–3.91 (m, 3H), 2.99 (t, J = 8.0 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.34–1.14 (m, 12H), 0.96 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 5.04. LRMS calcd for C₂₈H₄₁N₇O₁₀P (M + 1)⁺ 666.27, found 666.10.

15d(R_p).

¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 5.91 (s, 1H), 4.62–4.48 (m, 4H), 4.25–3.91 (m, 4H), 2.99 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.33 (d, J = 7.2 Hz, 3H), 1.18 (t, J = 7.2 Hz, 3H), 1.17 (d, J = 6.0 Hz, 3H), 1.14 (d, J = 6.4 Hz, 3H), 0.97 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 4.97. LRMS calcd for C₂₈H₄₁N₇O₁₀P (M + 1)⁺ 666.27, found 666.08.

Ethyl ((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(2-(3-isopropoxy-3-oxopropyl)phenoxy)phosphoryl)-l-alaninate, 15e(S_p), and Ethyl ((R)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)-methoxy)(2-(3-isopropoxy-3-oxopropyl)phenoxy)-phosphoryl)-l-alaninate, 15e(R_p).

A procedure similar to that used for 15a was employed for the synthesis of prodrug 15e (41% over two steps). Two diasteroisomers were separated and identified.

15e(S_p).

¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.17 (dt, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 4.93–4.84 (m, 1H), 4.61–4.49 (m, 2H), 4.22 (s, 2H), 4.18–3.91 (m, 3H), 2.99 (t, J = 8.0 Hz, 2H), 2.60 (t, J = 8.0 Hz, 2H), 1.34 (d, J = 6.4 Hz, 3H), 1.20 (t, J = 7.2 Hz, 3H), 1.15 (d, J = 6.0 Hz, 3H), 1.14 (d, J = 6.0 Hz, 3H), 0.97 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 5.02; LRMS calcd for C₂₈H₄₁N₇O₁₀P (M + 1)⁺ 666.27, found 666.19.

15e(R_p).

¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 5.91 (s, 1H), 4.93–4.84 (m, 1H), 4.51–4.48 (m, 2H), 4.25–3.95 (m, 5H), 2.98 (t, J = 8.0 Hz, 2H), 2.60 (t, J = 8.0 Hz, 2H), 1.34 (d, J = 7.2 Hz, 3H), 1.18–1.49 (m, 9H), 0.97 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 4.97. LRMS calcd for C₂₈H₄₁N₇O₁₀P (M + 1)⁺ 666.27, found 666.09.

Ethyl 3-(2-(((((3aS,4R,6R,6aR)-6-(2,6-Diamino-9H-purin-9-yl)-6a-methyl-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-methoxy)(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)-oxy)phenyl)propanoate, 16.

To a solution of compound 15b (110 mg, 0.17 mmol) in acetonitrile (10 mL) was added carbodiimidazole (60 mg, 0.37 mmol), and the reaction mixture was stirred at rt overnight. Water (0.2 mL) was added, and the reaction mixture was stirred for 0.5 h. The mixture was concentrated and portioned between ethyl acetate (100 mL) and water (15 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated, and the residue was purified by silica gel column chromatography in a gradient of MeOH (0–10% MeOH in CH₂Cl₂) to afford white solid 16 (84 mg, 71%). ¹H NMR (400 MHz, CD₃OD) δ 7.86 (s, 0.5H), 7.81 (s, 0.5H), 7.34–7.39 (m, 1H), 7.06–7.27 (m, 3H), 6.32 (s, 0.5H), 6.30 (s, 0.5H), 5.32–5.36 (m, 1H), 4.71–4.77 (m, 1H), 4.38–4.57 (2H), 3.97–4.16 (m, 5H), 2.93–3.00 (m, 2H), 2.57–2.65 (m, 2H), 1.32–1.39 (m, 6H), 1.13–1.23 (m, 6H). ³¹P NMR (162 MHz, CD₃OD) δ 4.10, 3.89. LCMS calcd for C₂₈H₃₇N₇O₁₁P (M + 1)⁺ 678.2, found 678.3.

Ethyl 3-(2-(((((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)oxy)phenyl)propanoate, 17.

To a solution of 6 (630 mg, 0.91 mmol) and N-methylimidazole (0.35 mL, 4.5 mmol) in THF (3 mL) at 0 °C was added dropwise a 1 M solution of diethyl 3,3′-(((chlorophosphoryl)bis(oxy))bis(2,1-phenylene))dipropanoate, 13 in THF (9 mL, 4.5 mmol). The resulting mixture was stirred overnight at rt. After solvent removal under reduced pressure, the residue was purified by flash column chromatography in a gradient of MeOH (0–10% MeOH in CH₂Cl₂) to afford 540 mg of white solid 17 (53% yield). A precold solution of TFA (80%, 26 mL) was added to a precold 17 (540 mg, 0.58 mmol) in an ice bath. The solution was stirred from 0 °C to rt and stirred at rt for 4 h (monitored by TLC and LC/MS). After the reaction was complete, the solvent was removed under reduced pressure and the residue was coevaporated with methanol (4 × 15 mL). The residue was dissolved in methanol (20 mL) and neutralized by saturated NaHCO₃. After solvent removal, the residue was purified by flash column chromatography in a gradient of MeOH (0–15% MeOH in CH₂Cl₂) to afford 270 mg of white solid 18 (77%). ¹H NMR (400 MHz, CD₃OD) δ 7.80 (s, 1H), 7.09–7.32 (m, 8H), 5.90 (s, 1H), 4.72–4.93 (m, 1H), 4.26–4.38 (m, 2H), 4.02–4.08 (m, 4H), 2.86–2.92 (m, 4H), 2.46–2.54 (m, 4H), 1.16 (t, 6H), 1.00 (s, 3H). ³¹P NMR (162 MHz, CD₃OD) δ 12.21. LCMS calcd for C₂₂H₃₁N₇O₈P (M + 1)⁺ 729.2; observed, 729.3.

Ethyl 3-(2-(((R)-(((S)-1-Ethoxy-1-oxopropan-2-yl)amino)(4-nitrophenoxy)phosphoryl)oxy)phenyl)propanoate, 19(R_p), and Ethyl 3-(2-(((S)-(((S)-1-Ethoxy-1-oxopropan-2-yl)amino)(4-nitrophenoxy)phosphoryl)oxy)phenyl)propanoate, 20(S_p).

A solution of Et₃N in anhydrous diethyl ether (100 mL) was added dropwise to a solution of 12b (10 g, 26 mmol) and p-nitrophenol (3.8 g, 27 mmol) in diethyl ether (200 mL) at 0 °C in 30 min. The reaction mixture was stirred at 0 °C for 1 h and at rt for 15 h. The solids were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified on a silica gel column (EtOAc/CH₂Cl₂, EtOAc gradient 0–10%, v/v) to give 10.8 g of a mixture of 19 and 20 in 85% yield in 1:1 ratio. The mixture was recrystallized in 2% CH₃CN in diisopropyl ether. Pure 19 was obtained by seeding the recrystallization solution with pure 19 crystal seeds which were obtained by column chromatography. The resulting solution was stored for 18 h at 0 °C. Filtration of the resulting solid from the mother liquor gave 2.2 g of isomer 19. ¹H NMR (400 MHz, CDCl₃) δ 8.22–8.24 (dd, J = 10 Hz, J = 2.0 Hz, 2H), 7.11–7.44 (m, 6H), 4.06–4.20 (m, 6H), 2.88–3.00 (m, 2H), 2.54–2.59 (m, 2H), 1.40 (d, J = 6.8 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H). ³¹P NMR (162 MHz, CDCl₃) δ −2.01. LC–MS, m/z 495 (M + 1)⁺. A single crystal of 19 was obtained by crystallization in 2% CH₃CN in diisopropyl ether and an X-ray structure of 19 was obtained to unambiguously confirm the configuration of the phosphorus center as R_p (Figure 2).

The filtrate was concentrated under reduced pressure to dryness, and the mixture was stirred in diisopropyl ether (200 mL) with pure 20 seeds. An amount of 510 mg of pure 20 was collected. ¹H NMR (400 MHz, CDCl₃) δ 8.22–8.24 (dd, J = 10 Hz, 2H), 7.11–7.43 (m, 6H), 4.00–4.18 (m, 6H), 2.93–2.98 (m, 2H), 2.55–2.60 (m, 2H), 1.43 (d, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H). ³¹P NMR (162 MHz, CDCl₃) δ −2.07. LC–MS, m/z 495 (M + 1)⁺. A single crystal of 20 was obtained by crystallization in 2% CH₃CN in diisopropyl ether and an X-ray structure of 20 was obtained to unambiguously confirm the configuration of the phosphorus center as S_p (Figure 2).

Ethyl 3-(2-(((R)-(((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)-propanoate, 15b(R_P).

To a solution of 6 (100 mg, 0.14 mmol) in THF (0.5 mL) was added 0.5 mL of t-BuMgCl solution (1 M, 0.5 mmol) at −78 °C under Ar atmosphere. The reaction mixture was stirred for 30 min at this temperature and then warmed to rt. A solution of 20 (210 mg, 0.42 mmol) in 1 mL of anhydrous THF was added. The reaction mixture was stirred at rt for 3 days under Ar atmosphere to completion. The solvent was evaporated under reduced pressure, and to the residue was added a precooled 80% TFA solution (10 mL) at 0 °C. The reaction mixture was stirred for an additional 4 h toward rt to completion. After evaporation of the solvents under reduced pressure, to the residue was added a small amount of saturated solution of aqueous NaHCO₃ to adjust to pH 7.0. The mixture was concentrated under reduced pressure and then purified by a silica gel column (MeOH/DCM, MeOH gradient 0–10%, v/v) to afford 37.5 mg of 15b(R_P) in 41% yield over two steps. Optical rotation [α]²⁴_d −7.08 (c 0.24, MeOH). ¹H NMR (400 MHz, CD₃OD) δ 0.97 (s, 3H, CH₃), 1.15–1.20 (m, 6H, 2 × CH₃), 1.34 (d, 3H, J = 7.2 Hz, CH₃), 2.62 (t, 2H, J = 8.0 Hz, 2H, CH₂), 2.99 (t, 2H, J = 8.0 Hz, 2H, CH₂), 3.95–4.58 (m, 9H), 5.94 (s, 1H, H₁′), 7.07–7.38 (m, 4H, Ar–H), 7.86 (s, 1H, H₈). ¹³C NMR (100 MHz, CD₃OD) δ 14.5, 14.6, 20.4, 20.6, 26.8, 35.4, 51.7, 61.7, 62.5, 67.0, 74.4, 80.1, 81.9, 92.8, 114.4, 121.0, 126.2, 128.8, 131.8, 133.2, 137.5, 150.6, 152.7, 157.7, 162.0, 174.8, 175.1. ³¹P NMR (162 MHz, CD₃OD): 5.03. LC/MS calcd for C₂₇H₃₉N₇O₁₀P (M + 1)⁺, 652.2; observed, 552.2.

Ethyl 3-(2-(((S)-(((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)-propanoate 15b(S_P).

A procedure similar to that used for 15a was employed for the preparation of 15b(S_P) in 39% yield. Optical rotation [α]²⁴_D +12.12 (c 0.13, MeOH). ¹H NMR (400 MHz, CD₃OD) δ 0.97 (s, 3H, CH₃), 1.15–1.17 (m, 6H, 2 × CH₃), 1.34 (d, 3H, J = 7.2 Hz, CH₃), 2.62 (t, 2H, J = 8.0 Hz, 2H, CH₂), 2.99 (t, 2H, J = 8.0 Hz, 2H, CH₂), 3.96–4.51 (m, 9H), 5.93 (s, 1H, H₁′), 7.10–7.39 (m, 4H, Ar–H), 7.86 (s, 1H, H₈). ¹³C NMR (100 MHz, CD₃OD) δ 14.5, 14.6, 20.4, 20.8, 26.8, 35.4, 51.6, 61.6, 62.4, 67.7, 74.7, 80.0, 82.1, 93.0, 114.4, 121.1, 126.2, 128.8, 131.7, 133.1, 137.7, 150.5, 152.6, 157.6, 161.9, 174.7, 174.8. ³¹P NMR (162 MHz, CD₃OD): 4.98. LC/MS calcd for C₂₇H₃₉N₇O₁₀P (M + 1)⁺, 652.2; observed, 552.3.

((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl Tetrahydrogen Triphosphate Tetratriethylammonium Salt (DAPN-TP) and ((2R,3R,4R,5R)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl Tetrahydrogen Triphosphate Tetratriethylammonium Salt (2′-Me-GTP).

A mixture of 2,6-diaminopurine ribonucleoside 7 (14 mg, 1 equiv), trimethyl phosphate (0.5 mL), and molecular sieves were stirred at rt overnight. 2,4,6-Collidine (9 μL, 1.5 equiv) was added and the mixture stirred for 10 min before phosphorus oxychloride (9 μL, 2 equiv) was added. The reaction solution was stirred for 1 h at rt. A solution of tributylammonium pyrophosphate (0.26 mL of 1 M solution in DMF, 5.5 equiv) and tributylamine (45 μL, 4 equiv) were added simultaneously. The mixture was stirred for 30 min at rt. The reaction was then quenched by addition of 2 mL of triethylammonium bicarbonate buffer (0.25M) and stirred for 45 min. After extraction with dichloromethane (2 × 3 mL), the aqueous phase was concentrated under reduced pressure. The crude was subjected to HPLC purification using ion-exchange DNAPac PA200 (9 mm × 250 mm) column (Dionex), with water and triethylammnoium bicarbonate (0.5M) as eluent. LC/MS calcd for C₁₁H₁₈N₆O₁₃P₃ (M − 1)⁺, 535.0; observed, 535.0.

The same procedure was applied for 2′-C-Me-GTP, using 2′-C-Me guanosine (20 mg, 1 equiv), trimethylphopsphate (0.65 mL), 2,4,6-collidine (14 μL, 1.5 equiv), phosphorus oxychloride (14 μL, 2.1 equiv), tributylamine (63 μL, 4 equiv), tributylammonium pyrophosphate (0.37 mL of 1 M solution in DMF, 5.5 equiv), triethylammonium bicarbonate buffer (2.5 mL, 0.25 M). LC/MS calcd for C₁₁H₁₇N₅O₁₄P₃ (M − 1)⁺, 536.0; observed, 535.9.

X-ray Crystallography of Diastereomers.

Results from X-ray structure determination of compound 19 are the following. Crystal data for C₂₂H₂₇N₂O₉P (M = 494.43): 0.29 × 0.11 × 0.09, triclinic, space group P1, a = 5.0028(3) Å, b = 8.9645(7) Å, c = 14.5700(14) Å, β = 90.753(7)°, V = 603.56(8) ų, Z = 1, μ(Cu Kα) = 1.484 mm⁻¹, D_calc = 1.360 g/mm³, temperature 173 K. Intensity data were collected on a Bruker APEX II CCD diffractometer with monochromated Cu Kα radiation (λ = 1.541 78 Å) at 173 K in the 2θ range 3.18–64.34°. The user interface Olex2 was used for the crystallographic calculations and crystal structure visualization.⁴¹ The structure was solved with Superflip by charge flipping and refined by least-squares minimization using SHELXL.^42,43 A total of 3668 reflections were measured (3.18 ≤ 2θ ≤ 64.34), while 2214 unique data (R_int = 0.0273) were used in the refinements. The final R₁ was 0.0475 (I > 2σ(I)) and the weighted R value wR2 was 0.1255 (all data). All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were included but not refined. The maximum and the minimum peak on the final difference Fourier map corresponded to 0.46 and −0.27 e/ų, respectively.

Results from the X-ray structure determination of compound 20 are the following. Crystal data for C₂₂H₂₇N₂O₉P (M = 494.43): 0.28 × 0.26 × 0.11, monoclinic, space group P2(1), a = 4.87400(10) Å, b = 28.8315(6) Å, c = 8.9118(2) Å, β = 104.3380(10)°, V = 1213.32(4) ų, Z = 2, μ(Cu Kα) = 1.477 mm⁻¹, D_calc = 1.353 g/mm³, temperature 173 K. Intensity data were collected on a Bruker APEX II CCD diffractometer with monochromated Cu Kα radiation (λ = 1.541 78 Å) at 173 K in the 2θ range 5.12–69.48°. The user interface Olex2 was used for the crystallographic calculations and crystal structure visualization.⁴¹ The structure was solved with Superflip by charge flipping and refined by least-squares minimization using SHELXL.^42,43 A total of 8394 reflections were measured (5.12 ≤ 2θ ≤ 69.48), while 3469 unique data (R_int = 0.0159) were used in the refinements. The final R₁ was 0.0267 (I > 2σ(I)) and the weighted R value wR2 was 0.0728 (all data). All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were included but not refined. The maximum and the minimum peak on the final difference Fourier map corresponded to 0.239 and −0.255 e/ų, respectively.

NS5B-Mediated RNA Polymerization Assay.

C-terminal Histagged NS5BΔ21 enzyme was purified as previously described (Powdrill et al., 2010). 1 μM NS5BΔ21 was incubated at 30 °C with synthetic 1 μM 20-mer RNA templates (IDT) and 1 μM ³²P-radiolabeled GpG primer (Trilink) in a buffer containing 40 mM Tris, pH 7.5, 6 mM NaCl, and 2 mM MgCl₂. Reactions were initiated with the addition of 10 μM NTP mix, 1 μM of competing NTP, and varying concentrations of inhibitor ranging from 0 to 100 μM. Reactions were allowed to proceed for 120 min and subsequently stopped with the addition of 10 mM EDTA and formamide. Samples were visualized on 20% denaturing polyacrylamide gel and quantified using QuantityOne software. IC₅₀ values were calculated using KaleidaGraph software.

Intracellular Metabolism.

Huh-7 cells and fresh plated human primary hepatocytes (BioreclamationIVT, Baltimore, MD) were seeded at 1 × 10⁶ per well in 12-well plates. After attachment (Huh-7 cells) or acclimatation overnight (hepatocytes), cells were exposed to 50 μM compounds, respectively. At 4 h, medium was removed from the cell layers and cells were washed twice with ice-cold phosphate buffered saline (PBS) to remove any residual medium. Cells were resuspended in 70% methanol containing 20 nM ddATP overnight at −20 °C. The supernatants were dried under a flow of air and dried samples stored at −20 °C until LC–MS/MS analysis.

Mitochondrial Toxicity Assa.

Human hepatocarcinoma cells (HepG2) were propagated at 37 °C and 5% CO₂ atmosphere in Dulbecco’s modified Eagle medium. Media contained 4.5 g/L d-glucose and were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. All cell reagents were from Invitrogen Corporation (Carisbad, CA, USA), Cellgro/Mediatech, Inc. (Manassas, VA, USA), and Hyclone (Logan, UT, USA). Low-passage-number HepG2 cells were seeded at 5000 cells/well onto collagen-coated 24-well tissue culture plates. After 1 day of cell seeding, test compounds were added to the medium to obtain final concentrations varying from 1 to 50 μM. Media and compounds were replenished every 3–4 days until the end of 14 days. All samples were performed in replicates of two in two independent experiments. On culture day 14, cells and cell culture supernatants were harvested. Total nucleic acid was isolated from cells by using commercially available kits (MagNA Pure LC DNA Isolation Kit, Roche). The mitochondrial cytochrome c oxidase subunit II (COXII) gene and the rRNA gene were amplified in parallel by a real-time PCR protocol that uses suitable primers and probes for both target and reference amplifications (Stuyver et al., 2002).⁴⁴ Real-time PCR was performed by using a TaqMan 9700 HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The primers and probes for the rRNA gene were purchased from Applied Biosystems. The amount of target (mtDNA) was normalized to the amount of an endogenous control (nuclear DNA) and was relative to the untreated control. The 50% inhibitory concentration (IC₅₀) of test compounds was determined by using CalcuSyn program (Biosoft, Cambridge, U.K.).

Lactate Production Assay.

Following a 14-day incubation with compounds at various concentrations, lactic acid quantification from cultured supernatant was performed in a 96-well plate using the d-lactic acid/L-lactic acid test kit (Boehringer Mannheim, Indianapolis, IN; USA/R-Biopharam, South Marshall, MI, USA/Roche). Extracellular levels of lactic acid were measured using a Multiscan Spectrum (Thermo Electron Corporation). The total amount of lactic acid produced and the fold change in lactic acid production (% of lactic acid/% of nuclear DNA) were determined for each sample.

HCV Replicon Assay.

Huh-7 clone B cells containing HCV replicon RNA were seeded in a 96-well plate at 3000 cells/well, and the compounds were added in dose response in triplicate immediately after seeding.⁴⁵ Following 5 days of incubation (37 °C, 5% CO₂), total cellular RNA was isolated by using the RNeasy 96-well extraction kit from Qiagen. Replicon RNA and an internal control (TaqMan rRNA control reagents, Applied Biosystems) were amplified in a single step multiplex real time RT-PCR assay. The antiviral effectiveness of the compounds was calculated by subtracting the threshold RT-PCR cycle of the test compound from the threshold RT-PCR cycle of the no-drug control (ΔCt HCV). A ΔCt of 3.3 equals a 1 log reduction (equal to 90% less starting material) in replicon RNA levels. The cytotoxicity of the compounds was also calculated by using the ΔCt rRNA values. 2′-C-Me cytidine was used as the positive control. To determine EC₉₀ and CC₅₀ values, ΔCt values were first converted into fraction of starting material and then were used to calculate the % inhibition.

Toxicity Assays.

For detailed descriptions of cytotoxicity assays, see ref 33.

ABBREVIATIONS USED

DAPN

β-d-2,6-diaminopurine nucleoside

PD

prodrug

NTP

nucleoside triphosphate

PI

protease inhibitor

DAA

direct acting agent

RdRp

RNA-dependent RNA polymerase

NMPD

nucleoside monophosphate prodrug

SVR

sustained virologic response

PBM

peripheral blood mononuclear

SD

standard deviation

mtDNA

mitochondrial DNA

ND

not determined

NA

not applicable

GT

genotype

NI

nucleoside inhibitor

DBU

1,8-diazabicycloundec-7-ene

TMSOTf

trimethylsilyl trifluoromethanesulfonate

(Boc)₂O

di-tert-butyl dicarbonate

DMAP

4-dimethylaminopyridine

DCM

dichloromethane

THF

tetrahydrofuran