Discovery of Novel Nucleotide Prodrugs with Improved Potency

Article pubs.acs.org/jmc

Discovery of Novel Nucleotide Prodrugs with Improved Potency Against HCV Variants Carrying NS5B S282T Mutation Le Zhen,§,†,‡ Liang Dai,§,† Xiaoan Wen,§,† Lan Yao,‡ Xiaoliang Jin,‡ Xiao-Wen Yang,† Wenfeng Zhao,† Sheng-Qi Yu,† Haoliang Yuan,† Guangji Wang,*,‡ and Hongbin Sun*,† †

Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China ‡ Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing, 210009 Jiangsu, China S Supporting Information *

ABSTRACT: Resistant HCV variants carrying NS5B S282T mutation confer reduced sensitivity to sofosbuvir, the sole marketed NS5B polymerase inhibitor. On the basis of the finding that 2′-α-F-2′-β-C-methylcytidine 5′-triphosphate (8) was more potent than sofosbuvir’s active metabolite on inhibition of both wild-type and S282T mutant polymerase, a dual-prodrug approach has been established. Twenty-nine phosphoramidates with N4-modified cytosine were designed, synthesized, and evaluated for anti-HCV activity. The results showed that compounds 4c−4e and 4m (EC50 = 0.19−0.25 μM) exhibited comparable potency to that of sofosbuvir (EC50 = 0.15 μM) on inhibition of wild-type replicons. Notably, 4c (EC50 = 0.366 μM) was 1.5-fold more potent than sofosbuvir (EC50 = 0.589 μM) on inhibition of S282T mutant replicons. In vitro metabolic studies disclosed the possible metabolic pathways of 4c. The toxicity study results indicated a good safety profile of 4c. Together, 4c−4e and 4m hold promise for drug development for the treatment of HCV infection, especially the resistant variants with NS5B S282T mutation.

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INTRODUCTION More than 185 million people worldwide become infected with the hepatitis C virus (HCV).1 One fourth of those who have chronic infection predictably progress to liver cirrhosis or hepatocellular carcinoma.2 Traditional interferon-based treatments cause unbearable side effects while only curing half of the treated patients.3 Nowadays, more interferon-free regimens with effective direct-acting antivirals have resulted in steadily higher cure rates (90−100%) in clinical trials.4,5 Among the antiviral targets, HCV NS5B RNA-dependent RNA polymerase efficiently accomplishes RNA replication but with no errorcorrecting processes.6,7 This deficiency in proofreading is responsible for the high spontaneous mutation rate, aggravating resistant mutations selected under antiviral drug pressure.8,9 These particular natures dramatically affect the outcomes during or after antiviral therapy.10 Therefore, further drug discovery against resistance mutations is desirable to maintain optimal response to therapy. In the ongoing battle between antivirals and drug resistance, nucleoside polymerase inhibitors remain the mainstay of antiviral therapies. HCV polymerase inhibitor sofosbuvir (1, Scheme 1) shows a high resistance barrier and pan-genotypic efficacy because its active nucleoside triphosphate metabolite binds to the highly conserved catalytic site of NS5B, leading to efficient chain termination during HCV genome replication.11 Nevertheless, the inhibitor inevitably induces an S282T mutation in NS5B.12 The mutation drastically reduces the binding affinity of the active triphosphate metabolite for NS5B © 2017 American Chemical Society

Scheme 1. Dual Prodrug Design Based on Active Metabolite 8 from Mericitabine 2

and accordingly diminishes replicon activity of 1.13−15 Moreover, the S282T mutation was also detected in clinical trials.16 Received: February 22, 2017 Published: June 26, 2017 6077

DOI: 10.1021/acs.jmedchem.7b00262 J. Med. Chem. 2017, 60, 6077−6088

Journal of Medicinal Chemistry

Article

to the literature reports. The synthesis of phosphoramidate dual prodrugs 4 is depicted in Scheme 2. Selective acylation of N4amino group of fluorine-containing cytidine 530,31 with the corresponding anhydrides in ethanol was carried out following the literature procedure33 to afford N-acylated cytidine derivatives 6a−6e. Alternatively, the corresponding carboxylic acid could also be used as acylating agents for the preparation of 6f−6s. Transient hydroxyl protection of 5 with trimethylsilyl chloride in pyridine followed by reaction with excess alkyl chloroformate gave N4-alkyloxycabonyl derivatives 6aa−6aj in moderate yields.34 Treatment of 6a−6s or 6aa−6aj with tertbutylmagnesium chloride in THF afforded the corresponding magnesium salts by deprotonation at the 5′-hydroxyl group. Reaction of the resulting magnesium salt with the chiral phosphoramidating reagents 732 furnished nucleotide phosphoramidates 4a−4s and 4aa−4aj in high diastereomeric purities, respectively. Inhibition of Wild-Type HCV Replicon and SAR Analysis. Thirty phosphoramidate derivatives were evaluated for the inhibition of wild-type HCV replicon, and 1 and 2 were chosen as the reference compounds. The assay results are summarized in Table 2. Compound 3 (EC50 = 0.66 μM) exhibited a 2-fold potency increase over 2 (EC50 = 1.10 μM), indicating that the ProTide strategy could improve the potency probably by bypassing the rate-limiting phosphorylation step to release active metabolite 8. Notably, N4-acylated derivatives 4a−4s are generally more potent than N4-alkyloxycabonyl derivatives 4aa−4aj (Table 2), implying that the former could be better substrates than the latter for hydrolase-mediated amide hydrolysis to generate active metabolites. The key factors affecting the amide hydrolysis by the catalytic triad of a hydrolase include steric and electronic effects.35 It seems that the proper length of the alkanoyl chain is critical for the potency of 4a−4s. For example, compounds 4c−4e (EC50 = 0.19−0.25 μM) with 4−6 carbon chain lengths showed comparable potencies with that of 1 (EC50 = 0.15 μM), whereas compounds 4a and 4b (EC50 = 1.04−1.48 μM) with 2−3 carbon chain lengths were less potent. With increasing bulkiness of the alkanoyl chain, the compounds (4f−4l, 4n) tended to exhibit decreased potency. Interestingly, compound 4m (EC50 = 0.19 μM) with a cyclobutanecarboxamide group was very potent. The N4-benzoyl derivatives (4o and 4p) exhibited moderate activity, and compound 4q with the p-OMe group on the benzene ring almost lost potency, indicating that an electron-donating group could weaken the carbonyl polarization and thus negatively affect amide hydrolysis to release the active metabolite. Considering that the carbamate prodrug approach has been successfully applied to the anticancer drug capecitabine,36 a series of carbamate-containing prodrugs 4aa−4aj were synthesized and tested for their anti-HCV activity. To our surprise, only a couple carbamates (4aa, 4ab, 4ac, and 4ad) showed weak anti-HCV activity, and most of the carbamate prodrugs lost potency. This indicates that the carbamates may not be suitable substrates of the hydrolase in contrast to the N4acylated derivatives. Notably, in comparison with 3 (EC50 = 0.66 μM) with a free N4-amino group, N4-amidation prodrugs 4c−4e and 4m are significantly more potent, further demonstrating the feasibility of the dual prodrug strategy. Together, on the basis of a dual prodrug strategy, some novel anti-HCV agents (4c−4e, 4m) have been discovered, whose potency on inhibition of the wild-type HCV replicon is

Although HCV drug cocktails have retarded the development of resistance, therapeutic implications of antiviral resistance remain to be seen in parallel with long-term and expanded access to 1.17 Therefore, more attention should be paid to the acquired drug resistance imparted by the S282T mutation. Encouraged by the commentary from Michael Sofia that “to identify a novel nucleos(t)ide to be an alternative nucleos(t)ide therapy in the event that sofosbuvir resistance becomes a problem”,18 we became interested in drug discovery aiming at S282T mutation-induced resistance. To the best of our knowledge, very few compounds have been specifically designed to overcome S282T mutation. Although the resistance mechanism associated with NS5B S282T variants remains elusive,19 it was strikingly found that not all nucleos(t)ides with the same figured sugar moiety shared the resistance profile of 1.18,20,21 In particular, anti-HCV drug candidate mericitabine (2, Scheme 1) captured our attention because its active metabolite 2′-α-F-2′-β-C-methylcytidine 5′-triphosphate (8) was more efficient than its uridine metabolite (9) on inhibiting both of wild-type and S282T mutant NS5B polymerase (Table 1).11,13,20 Notably, uridine metabolite 9 is the active metabolite Table 1. Inhibitory Activity of 8 and 9 against Wild-Type (WT) and S282T Mutant NS5B Polymerase 8 9

WT NS5B Ki (μM)

S282T NS5B Ki (μM)

fold shift

0.06 0.42

0.31 22

5 52

of 1.14,22 Moreover, the presence of the S282T mutation, alone or in combination with other mutations, led to more dramatic reduction in drug susceptibility to 1 relative to 2.23 Therefore, we reasoned that triphosphate metabolite 8 might be a promising lead compound for novel nucleotide inhibitors with improved potency against both wild-type and S282T mutant HCV. Herein, we describe a dual prodrug strategy based on 8. Thus, a series of dual prodrugs 4 (Scheme 1) were designed, synthesized, and biologically evaluated as anti-HCV agents. SAR analysis around the N4-position of cytosine is also presented.

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RESULTS AND DISCUSSION Molecular Design. Given the fact that 8 was much more potent than its deaminated counterpart 9 on inhibiting both the wild-type and S282T mutant NS5B polymerase, we wanted to establish a dual prodrug approach based on 8 to overcome drug resistance caused by HCV NS5B S282T mutation. First, the phosphoramidate pronucleotide (ProTide) approach that has been successfully translated into clinical solutions24−26 was employed to carry out nucleoside monophosphate delivery of 8. Second, 8 was notably less stable (t1/2 = 4.7 h) than 9 (t1/2 = 38 h), leading to insufficient accumulation and function at viral reservoirs.13,27 This instability might be due to the deamination of 828 or other xenobiotic metabolism.13 In this regard, various acyl and alkoxycarbonyl groups were introduced at the N4position of the cytosine29 to block possible deamination of cytosine and to orchestrate the hydrolytic exposure of the free N4-amino group at appropriate rates. In this regard, a series of dual prodrugs 4 (Scheme 1) containing a phosphoramidate moiety and an N-protected fluorine-substituted cytidine derived from 1 were designed. Chemistry. The control compounds 1,15 2,30,31 and 3 (a cytidine counterpart of 1, Table 2)32 were prepared according 6078



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Table 2. Inhibition of Wild-Type HCV Replicon by Phosphoramidate Dual Prodrugs 4a

The tested compounds showed no sign of cytotoxicity up to 100 μM. bEC50 values (mean ± SD) are the average of 2−10 independent determinations. cE:Z isomer = 3:2. a

Scheme 2. Synthesis of Phosphoramidate Dual Prodrugs 4a

a Reagents and conditions: (a) (R1CO)2O, EtOH, reflux or R1CO2H, EDCI, HOBt, NMM, DMF, 25−65 °C or TMSCl, pyridine, 0 °C and then R1COCl, DCM, 25 °C; (b) 7, tBuMgCl, dry THF, 0 °C to rt.

comparable to that of 1 and is much better than clinical drug candidate 2. Inhibition of S282T Mutant HCV Replicon. To examine the resistance profile of the dual prodrugs 1, compounds 3 and 4c were tested in wild-type and S282T mutant HCV replicon assays (Table 3). As expected, the inhibitory activity of the compounds against wild-type HCV replicon followed the same trend as shown in Table 2. To our delight, compound 4c (EC50 = 0.366 μM) was 1.5-fold more potent than 1 (EC50 = 0.589 μM) against S282T mutant HCV replicon. In terms of the resistance fold shift caused by S282T mutation, 1 was subjected to at least an 8-fold decrease in potency (EC50 = 0.073−0.589 μM), whereas 3 and 4c displayed 4-fold (EC50 = 0.208−0.857 μM) and 3-fold (EC50 = 0.113−0.366 μM) reduction. This demonstrates that the cytosine-based anti-HCV agents could be

Table 3. Inhibition of Wild-Type (WT) and S282T Mutant HCV Replicon by 1, 3, and 4c cpda

WT EC50 (μM)b

S282T EC50 (μM)c

fold shiftd

1 3 4c

0.073 ± 0.05 0.208 ± 0.09 0.113 ± 0.06

0.589 ± 0.14 0.857 ± 0.35 0.366 ± 0.12

8.1 4.1 3.2

a

EC50 values are the average of duplicate determinations. bWild-type replicon activity for compounds. cS282T mutant replicon activity for compounds. dRatio of S282T EC50 to WT EC50 was calculated.

more efficacious than the uracil-based ones against drug resistance caused by S282T mutation. Together, besides its strong inhibition against wild-type HCV replicon, compound 6079



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4c exhibited more potent activity than 1 against S282T mutant HCV replicon. In Vitro Metabolic Study. For the metabolic stability of the dual prodrugs to be examined, evaluation of their stability in human plasma and liver S9 fractions was performed (Table 4). Table 4. Stability of 1, 3, and 4c in Human Plasma and Human Liver S9 Fractions stability t1/2 (min) compd

human plasma

human liver S9

1 3 4c

>231 >231 >231

48.1 106.6 9.6

Figure 1. Time course of metabolite formation of 4c in Huh-7 cells. Huh-7 cells were incubated with compound 4c for 0−48 h (as indicated in the figure). The Δ mean peak area ratio was employed to measure the relative content of metabolites in the same system (see Supporting Info).

It was found that compounds 1, 3, and 4c all possessed high metabolic stability in human plasma (t1/2 > 231 min). On the other hand, different extents of metabolic degradation of the test compounds in human hepatic S9 fractions were observed. Notably, compound 4c was decomposed most rapidly (t1/2 = 9.6 min), whereas compound 3 was 10-fold more stable (t1/2 = 106.6 min). Compound 1 had a moderate metabolic stability in hepatic S9 fractions (t1/2 = 48.1 min). On the basis of the metabolic study results of 122 and 2,13 the metabolic pathways of 4c are proposed as shown in Scheme 3 (see Supporting Info S2 for an integrated pathway). The isopropyl ester of 4c could be hydrolyzed by cathepsin A and carboxylesterase 1 in human hepatocytes, which triggers a nonenzymatic self-cyclization/hydrolysis reaction to afford 10. Carboxylesterase-mediated amide hydrolysis29,35 of 10 gives 11. Phosphoramidate cleavage of 10 catalyzed by Hint122 gives 12, which is converted to 13 via amide hydrolysis. Alternatively, phosphoramidate cleavage of 11 gives 13. Triphosphate formation from 13 is catalyzed by UMP-CMP kinase and nucleoside diphosphate kinase13,22 to afford active metabolite 8. Alternatively, carboxylesterase-catalyzed amide hydrolysis of 4c gives 3, which is further converted to 11 via isopropyl ester hydrolysis and a nonenzymatic self-cyclization/hydrolysis reaction. For these pathways to be preliminarily validated, the metabolic profile of 4c in Huh-7 cells37 was investigated. Each proposed metabolite was examined and semiquantified by LC/ MS/MS. During the 48 h assay (Figure 1), the major metabolite detected was 11, whereas 10 was found to be a minor metabolite. Notably, the time for both 10 and 11 to reach peak concentrations was approximately 24 h. Moreover, a small amount of 3 was also detected. The subsequent metabolites 13 and 8 were only detected once in two assays

(Supporting Info S7 and S8) probably due to technical limitations. The active metabolite 9 of 1 was not detected in this assay (Supporting Info S9). Parent compound 4c and potential metabolite 12 were not detectable either. Together, in Huh-7 cells, compound 4c seemed to be quickly subjected to ester hydrolysis to afford 10, which was further converted to 11 via amide hydrolysis. Because 12 was not detectable, 13 should be generated from phosphoramidate cleavage of 11. Active metabolite 8 could be produced via triphosphate formation from 13. Given the fact that a tiny amount of compound 3 was detected as a metabolite of 4c, metabolic generation of 8 from 3 should also be considered. In Vivo Pharmacokinetic Study. We determined the PK parameters of 4c using Beagle dogs. Six dogs (three male and three female) were orally dosed with 4c (150 mg/kg), and the plasma samples were collected over a 24 h period. No dog showed any clinical symptoms or adverse events during the study. The pharmacokinetic parameters are summarized in Table 5. Plasma concentrations of 4c reached Cmax (51.3 ng/ Table 5. Pharmacokinetic Parameters of 4c Following Single Oral Dose (150 mg/kg) to Dogs PK parameters t1/2 (h) Tmax (h) Cmax (ng/mL) AUClast (h ng/mL) AUC0−∞ (h ng/mL)

mean ± SD 14.2 0.9 51.3 248.1 411.3

± ± ± ± ±

2.8 0.6 16.9 103.8 143.7

Scheme 3. Proposed Metabolic Pathways of 4c in Huh-7 Cells

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mL) within a Tmax of 0.9 h after oral administration on average. The mean half-life (t1/2) from plasma was 14.2 h, suggesting that 4c may possess modest blood stability and slow elimination. The AUClast and AUC0−∞ were 248.1 and 411.3 h ng/mL, respectively. Further pharmacokinetic studies on 4c, especially the challenging work on the detection of the active metabolites, is ongoing in our laboratory. Toxicity Studies. For the safety profile of 4c to be assessed, acute and subacute toxicity studies were conducted in healthy ICR mice (Supporting Info S10). In acute toxicity studies, no mortality or body weight loss were observed throughout a period of 14 days at each dose of 150, 500, 1200, and 1800 mg/ kg, indicated that the established no observed adverse effect level (NOAEL) was more than 1800 mg/kg. The 14 day subacute toxicity study revealed that 4c did not produce mortality, clinical signs of toxicity, body weight loss, organ weight changes (liver, kidney), or macroscopic pathologic findings at any dose level (100, 500, and 1000 mg/kg) or vehicle control. Furthermore, mice treated with 4c did not display any significant alteration in the basic levels of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) compared to those of the control group. A histological examination of the liver and kidney was conducted at the end of the subacute study period. The micrographs (H&E stain) demonstrated normal hepatic architecture with a normal appearance of hepatocytes and perilobular vein. An H&E stained section of the kidney also showed a normal renal architecture, glomerulus, and tubules. The primarily toxicological studies presented above indicated a good safety profile of 4c.

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EXPERIMENTAL SECTION

Materials and Methods. The purities of all target compounds tested in vitro were determined by HPLC analysis as being ≥95%. See the Supporting Information for full details of HPLC methods and data. All in vitro biological assays were conducted at WuXi AppTec Co., Ltd. (Shanghai). 1 H and 13C NMR spectra were recorded on an ACF* 300Q Bruker or ACF* 500Q Bruker spectrometer. Low- and high-resolution mass spectra (LRMS and HRMS) were recorded in ESI mode. The mass analyzer type used for the HRMS measurements was TOF. Reactions were monitored by TLC on silica gel 60 F254 plates. Column chromatography was carried out on silica gel (200−300 mesh). Data for 1H NMR are recorded as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet or unresolved, br = broad singlet, coupling constant (s) in Hz, integration). Data for 13C NMR are reported in terms of chemical shift (δ, ppm). General Synthetic Procedure for Preparation of the N4-Acyl-2′deoxy-2′-fluoro-2′-C-methylcytidine Derivatives (6a−6e): Procedure A. To a stirred suspension of 5 (1.0 equiv) in EtOH (1 mL) was added butyric anhydride (1.0 equiv). The reaction mixture was heated under reflux for 3 h. Additional butyric anhydride (1.0 equiv each time) was added after 1, 2, and 3 h. The resulting solution was refluxed for an additional hour after all anhydride was added. After the solvent was concentrated in vacuo, the residue was purified by flash column chromatography (CH2Cl2:MeOH = 100:1−20:1) to afford 6a−6e. General Synthetic Procedure for Preparation of the N4-Acyl-2′deoxy-2′-fluoro-2′-C-methylcytidine Derivatives (6f−6s): Procedure B. To a stirred solution of N-methylmorpholine (1.1 equiv) in DMF/ DMSO (3:1, 3 mL) were sequentially added 5, HOBt (1.1 equiv), appropriate carboxylic acid (1.1 equiv), and EDCI (1.3 equiv) at ambient temperature. The reaction mixture was heated to 60 °C (external temperature) for 16 h under N2. Upon completion, the reaction solution was poured into ice water, and the resulting mixture was extracted with EtOAc. The combined organic phase was washed with 20% LiCl solution, saturated NaHCO3 solution, and brine sequentially, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude product was subjected to column chromatography (CH2Cl2:MeOH = 100:1−20:1) to afford 6f−6s. General Synthetic Procedure for Preparation of the N4Alkoxycarbonyl-2′-deoxy-2′-fluoro- 2′-C-methylcytidine Derivatives (6aa−6aj): Procedure C. Trimethyl chlorosilane (5.0 equiv) was slowly added to a suspension of 5 (1.0 equiv) in dry pyridine (1 mL) under nitrogen with stirring and cooling in an ice bath. Then, the ice bath was removed, and the mixture was stirred for 1 h. The suspension was cooled again with an ice bath, and a solution of chloroformate (5.0 equiv) in CH2Cl2 (2 mL) was added dropwise. The resulting mixture was stirred at room temperature overnight. The reaction was quenched carefully with a small amount of aqueous ammonia. The mixture was dried by azeotropic evaporation with toluene and pumped to dryness, and the residue was purified by flash column chromatography (CH2Cl2:MeOH = 100:1−20:1) to afford 6aa−6aj. General Synthetic Procedure for Preparation of Prodrugs 4: Procedure D. To a suspension of 6a−6aj (1.0 equiv) in dry THF was added a 1.0 M solution of tert-butylmagnesium chloride (2.5 equiv) dropwise at 0 °C under nitrogen. The resulting slurry was stirred for 1 h at room temperature, and then a solution of 7 (1.5 equiv) in dry THF was added gradually with stirring. After an almost clear solution was formed, the reaction was stirred for an additional 10 h and quenched with saturated aqueous NH4Cl. The resulting mixture was dried by azeotropic evaporation with toluene and pumped to dryness. The residue was subjected to column chromatography (CH2Cl2:MeOH = 100:1−20:1) to yield compounds 4a−4aj. (S)-Isopropyl 2-(((R)-(((2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (1, Sofosbuvir). 1 H NMR (500 MHz, CDCl3) δ 7.48 (d, J = 8.1 Hz, 1H), 7.33 (t, J = 7.9 Hz, 2H), 7.24−7.14 (m, 3H), 6.17 (d, J = 18.1 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 5.11−4.92 (m, 1H), 4.59−4.41 (m, 2H), 4.29 (dd, J =

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CONCLUSIONS Although NS5B polymerase inhibitor 1 has become a revolutionary therapy against HCV infection, resistant HCV variants have been identified clinically.13,19 For potential drug resistance caused by HCV variants carrying NS5B S282T mutation to be overcome, a dual prodrug approach based on 2′α-F-2′-β-C-methylcytidine 5′-triphosphate (8) has been established. Notably, 8 was much more potent than sofosbuvir’s active metabolite 9 on inhibition of both wild-type and S282T mutant HCV NS5B polymerase. In this study, twenty-nine phosphoramidate derivatives with N4-modified cytosine were designed, synthesized, and evaluated for their inhibitory activity against wild-type and S282T mutant HCV replicon. The results of HCV replicon assays showed that compounds 4c−4e and 4m (EC50 = 0.19−0.25 μM) exhibited comparable potency to that of 1 (EC50 0.15 μM) on inhibition of wild-type HCV replicon. Intriguingly, compound 4c (EC50 = 0.366 μM) exhibited more potent activity than 1 (EC50 0.589 μM) against S282T mutant HCV replicon. Moreover, these compounds showed no detectable cytotoxicity up to 100 μM. Preliminary in vitro metabolic studies disclosed the possible metabolic pathways of 4c. In addition, an in vivo pharmacokinetic study of 4c was conducted in beagle dogs. Primarily toxicological studies clearly confirmed the safety of 4c without the toxic nature associated with normal body function, liver, or kidney. Comprehensive pharmacokinetic studies on 4c and related compounds are warranted for further drug development for the treatment of HCV infection, especially the resistant variants with NS5B S282T mutation. 6081



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methoxy)(phenoxy)phosphoryl)amino)propanoate (4c). According to general procedures A and D described above, 4c (372 mg, two steps, 62%) was obtained as a white powder. 1H NMR (500 MHz, MeOD) δ 8.04 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.9 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.22 (t, J = 7.4 Hz, 1H), 6.29 (d, J = 18.1 Hz, 1H), 5.06−4.94 (m, 1H), 4.58 (dd, J = 11.1, 5.2 Hz, 1H), 4.49−4.39 (m, 1H), 4.19 (d, J = 8.1 Hz, 1H), 4.02−3.88 (m, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.79−1.65 (m, 2H), 1.39 (d, J = 7.1 Hz, 3H), 1.33 (d, J = 22.3 Hz, 3H), 1.24 (dd, J = 6.2, 2.6 Hz, 6H), 1.01 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, DMSO) δ 173.81, 172.53 (d, J = 5.2 Hz), 162.55, 154.47, 150.65 (d, J = 6.4 Hz), 144.16 (br s), 129.59, 124.54, 120.01 (d, J = 4.8 Hz), 100.43 (d, J = 182.1 Hz), 96.14, 89.23 (br s), 79.51 (br s), 71.27 (d, J = 16.3 Hz), 67.93, 64.50 (br s), 49.77, 38.22, 21.32 (d, J = 3.0 Hz), 19.74 (d, J = 6.4 Hz), 17.82, 16.41 (d, J = 25.1 Hz), 13.36. 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C26H37N4O9FP [M + H]+ m/z 599.2282; found 599.2288. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Pentanamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4d). According to general procedures A and D described above, 4d (24.5 mg, two steps, 40%) was obtained as a white powder. 1H NMR (300 MHz, MeOD) δ 8.01 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.41− 7.34 (m, 2H), 7.29−7.24 (m, 2H), 7.23−7.14 (m, 1H), 6.25 (d, J = 18.7 Hz, 1H), 5.11−4.88 (m, 1H), 4.64−4.48 (m, 1H), 4.47−4.36 (m, 1H), 4.20−4.10 (m, 1H), 4.05−3.80 (m, 2H), 2.44 (t, J = 7.4 Hz, 2H), 1.81−1.55 (m, 2H), 1.49−1.38 (m, 2H), 1.36 (dd, J = 7.2, 0.9 Hz, 3H), 1.30 (d, J = 22.4 Hz, 3H), 1.21 (dd, J = 6.2, 1.6 Hz, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 174.43, 172.90, 163.04, 156.40, 143.94 (br s), 129.53, 124.89, 119.87 (d, J = 4.9 Hz), 100.10 (d, J = 182.8 Hz), 97.45, 79.66 (d, J = 7.1 Hz), 71.48 (d, J = 15.8 Hz), 68.86, 64.21 (br s), 50.30, 36.59, 26.72, 21.86, 20.70 (d, J = 6.8 Hz), 19.41 (d, J = 6.2 Hz), 15.65 (d, J = 25.6 Hz), 12.83. 31P NMR (121 MHz, MeOD) δ 3.95. HRMS calcd for C27H38N4O9FPNa [M + Na]+ m/z 635.2258; found 635.2272. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Hexanamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4e). According to general procedures A and D described above, 4e (18.8 mg, two steps, 30%) was obtained as a white powder. 1H NMR (300 MHz, CDCl3 + MeOD) δ 8.01 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.41−7.34 (m, 2H), 7.31−7.25 (m, 2H), 7.23−7.16 (m, 1H), 6.26 (d, J = 18.1 Hz, 1H), 5.05−4.89 (m, 1H), 4.64−4.48 (m, 1H), 4.47−4.35 (m, 1H), 4.16 (dd, J = 9.7, 1.6 Hz, 1H), 4.03−3.81 (m, 2H), 2.43 (t, J = 7.4 Hz, 2H), 1.80−1.56 (m, 2H), 1.41−1.34 (m, 7H), 1.30 (d, J = 22.3 Hz, 3H), 1.21 (dd, J = 6.3, 1.6 Hz, 6H), 0.92 (t, J = 6.9 Hz, 3H). 13 C NMR (75 MHz, MeOD) δ 174.43, 172.93 (d, J = 5.4 Hz), 163.06, 156.41, 150.60, 143.95 (br s), 129.52, 124.88, 119.87 (d, J = 4.8 Hz), 100.11 (d, J = 182.7 Hz), 97.42, 90.33 (br s), 79.67 (d, J = 6.9 Hz), 71.48 (d, J = 17.6 Hz), 68.83, 64.20 (d, J = 4.3 Hz), 50.29, 36.77, 30.96, 24.29, 22.01, 20.64 (d, J = 6.9 Hz), 19.33 (d, J = 6.1 Hz), 15.59 (d, J = 25.7 Hz), 12.93. 31P NMR (121 MHz, CDCl3 + MeOD) δ 3.95. HRMS calcd for C28H40N4O9FPNa [M + Na]+ m/z 649.2415; found 649.2430. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-Methylpentanamido)2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyl tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl) amino)propanoate (4f). According to general procedures B and D described above, 4f (28.8 mg, two steps, 23%) was obtained as a white powder. 1H NMR (300 MHz, MeOD) δ 8.02 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.43−7.34 (m, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 7.3 Hz, 1H), 6.26 (d, J = 19.6 Hz, 1H), 5.04−4.91 (m, 1H), 4.61−4.50 (m, 1H), 4.48−4.37 (m, 1H), 4.16 (d, J = 9.6 Hz, 1H), 4.05−3.82 (m, 2H), 2.60−2.25 (m, 2H), 1.65−1.49 (m, 3H), 1.36 (d, J = 7.4 Hz, 3H), 1.31 (d, J = 22.4 Hz, 3H), 1.21 (d, J = 6.2 Hz, 6H), 0.94 (d, J = 6.1 Hz, 6H). 31P NMR (121 MHz, MeOD) δ 3.99. HRMS calcd for C28H41N4O9FP [M + H]+ m/z 627.2595; found 627.2599. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(2-Phenylacetamido)-2oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyl tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4g). According to general procedures B and D described above, 4g (32.2 mg, two steps, 25%) was obtained as a white foam. 1H NMR (500

11.8, 9.9 Hz, 1H), 4.12 (d, J = 9.3 Hz, 1H), 3.93 (td, J = 16.3, 7.2 Hz, 2H), 1.37 (d, J = 29.4 Hz, 6H), 1.35 (s, 3H), 1.23 (d, J = 6.3 Hz, 6H). 13 C NMR (75 MHz, MeOD) δ 174.34 (d, J = 5.5 Hz), 165.58, 152.13 (d, J = 6.2 Hz), 141.52 (br s), 130.88, 126.24, 121.32 (d, J = 4.8 Hz), 103.36, 101.44 (d, J = 181.5 Hz), 91.42 (br s), 81.11 (d, J = 7.7 Hz), 73.21 (d, J = 18.3 Hz), 70.20, 65.90 (d, J = 3.7 Hz), 51.66, 21.92 (d, J = 6.5 Hz), 20.59 (d, J = 6.3 Hz), 16.86 (d, J = 25.6 Hz). 31P NMR (121 MHz, MeOD) δ 3.86. HRMS calcd for C22H29N3O9FPNa [M + Na]+ m/z 552.1523; found 552.1535. (2R,3R,4R,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-4-fluoro-2((isobutyryloxy)-methyl)-4-methyltetrahydrofuran-3-yl Isobutyrate (2, Mericitabine). 1H NMR (300 MHz, MeOD) δ 7.74 (d, J = 6.9 Hz, 1H), 6.19 (d, J = 17.8 Hz, 1H), 5.96 (d, J = 6.7 Hz, 1H), 5.22 (d, J = 15.1 Hz, 1H), 4.38 (s, 3H), 2.66 (s, 2H), 1.31 (d, J = 22.2 Hz, 3H), 1.20 (s, 12H). 13C NMR (75 MHz, MeOD) δ 177.83, 177.50, 167.66, 157.94, 141.89 (br s), 101.21 (d, J = 185.0 Hz, 1H), 96.65, 92.82 (br s), 78.67, 73.14 (d, J = 18.9 Hz, 4H), 63.07, 35.07 (d, J = 13.8 Hz, 5H), 19.17 (d, J = 12.9 Hz, 6H), 19.09, 17.59 (d, J = 25.6 Hz, 7H). HRMS calcd for C18H26N3O6FNa [M + Na]+ m/z 422.1703; found 422.1714. (S)-Isopropyl 2-(((R)-(((2R,3R,4R,5R)-5-(4-Amino-2-oxopyrimidin1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (3). 1H NMR (300 MHz, MeOD) δ 7.75 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.24−7.17 (m, 1H), 6.24 (d, J = 19.0 Hz, 1H), 5.99 (d, J = 7.6 Hz, 1H), 5.06−4.92 (m, 1H), 4.55 (dd, J = 11.6, 5.9 Hz, 1H), 4.45−4.33 (m, 1H), 4.12 (d, J = 9.6 Hz, 1H), 3.99− 3.84 (m, 2H), 1.35 (d, J = 7.8 Hz, 3H), 1.32 (d, J = 21.0 Hz, 3H), 1.21 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 172.88 (d, J = 5.4 Hz), 164.55, 155.09, 150.65 (d, J = 6.6 Hz), 141.17, 129.48, 124.86, 119.93 (d, J = 4.9 Hz), 100.07 (d, J = 182.1 Hz), 95.84, 90.33, 79.57 (d, J = 7.8 Hz), 71.59 (d, J = 18.0 Hz), 68.77, 64.34, 50.28, 20.50 (d, J = 6.2 Hz), 19.14 (d, J = 6.4 HzH), 15.57 (d, J = 25.6 Hz). 31P NMR (121 MHz, MeOD) δ 3.79. HRMS calcd for C22H31N4O8FP [M + H]+ m/z 529.1864; found 529.1870. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Acetamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4a). According to general procedures A and D described above, 4a (18.2 mg, two steps, 32%) was obtained as a white powder. 1H NMR (300 MHz, MeOD) δ 8.03 (d, J = 7.6 Hz, 1H), 7.51−7.34 (m, 3H), 7.29 (d, J = 7.9 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 6.27 (d, J = 19.2 Hz, 1H), 5.05− 4.90 (m, 1H), 4.62−4.51 (m, 1H), 4.43 (ddd, J = 9.9, 6.0, 3.4 Hz, 1H), 4.18 (d, J = 9.4 Hz, 1H), 4.02−3.86 (m, 2H), 2.19 (s, 3H), 1.37 (d, J = 7.4 Hz, 4H), 1.32 (d, J = 22.4 Hz, 3H), 1.22 (d, J = 6.2 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 175.02, 172.89 (d, J = 5.2 Hz), 163.09, 156.41, 150.69 (d, J = 6.7 Hz), 143.96 (br s), 129.48, 124.83, 119.88 (d, J = 4.8 Hz), 100.12 (d, J = 183.1 Hz), 97.27, 90.43 (br s), 79.72 (d, J = 7.5 Hz), 71.53 (d, J = 17.8 Hz), 68.76, 64.24, 50.32 (d, J = 0.8 Hz), 23.10, 20.48 (d, J = 6.6 Hz), 19.16 (d, J = 6.4 Hz), 15.42 (d, J = 25.5 Hz). 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C24H33N4O9FP [M + H]+ m/z 571.1968; found 571.1974. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Propionamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4b). According to general procedures A and D described above, 4b (17.5 mg, two steps, 30%) was obtained as a white powder. 1H NMR (300 MHz, MeOD) δ 8.03 (d, J = 7.6 Hz, 1H), 7.41 (dd, J = 16.6, 7.6 Hz, 3H), 7.29 (d, J = 8.5 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.27 (d, J = 19.3 Hz, 1H), 5.07−4.90 (m, 1H), 4.62−4.52 (m, 1H), 4.48−4.39 (m, 1H), 4.18 (d, J = 9.5 Hz, 1H), 4.02−3.86 (m, 2H), 2.48 (q, J = 7.5 Hz, 2H), 1.38 (d, J = 7.5 Hz, 3H), 1.32 (d, J = 22.4 Hz, 3H), 1.23 (d, J = 6.2 Hz, 6H), 1.17 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 175.07, 172.89, 163.09, 156.55, 143.94, 129.50, 124.86, 119.90 (d, J = 4.9 Hz), 100.14 (d, J = 182.6 Hz), 97.20, 79.80, 71.56 (d, J = 18.3 Hz), 68.79, 64.26, 50.31, 29.89, 20.50 (d, J = 6.7 Hz, 2H), 19.16 (d, J = 6.3 Hz, 1H), 15.43 (d, J = 25.6 Hz, 1H), 7.72. 31P NMR (121 MHz, MeOD) δ 4.01. HRMS calcd for C25H35N4O9FP [M + H]+ m/z 585.2125; found 585.2136. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Butyramido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)6082



Article

MHz, MeOD) δ 8.00 (d, J = 7.6 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.37−7.23 (m, 9H), 7.16 (t, J = 7.4 Hz, 1H), 6.25 (d, J = 18.6 Hz, 1H), 5.03−4.90 (m, 1H), 4.63−4.49 (m, 1H), 4.47−4.36 (m, 1H), 4.16 (d, J = 8.5 Hz, 1H), 3.99−3.85 (m, 2H), 3.75 (s, 2H), 1.35 (d, J = 7.1 Hz, 3H), 1.30 (d, J = 22.3 Hz, 3H), 1.20−1.17 (m, 6H). 13C NMR (75 MHz, MeOD) δ 174.29, 173.58, 164.58, 157.77, 151.03, 145.60 (br s), 135.54, 130.90, 130.37, 129.66, 128.24, 126.26, 121.29 (d, J = 4.9 Hz), 101.57 (d, J = 182.5 Hz), 98.69, 90.85 (br s), 81.16 (br s), 72.98 (d, J = 18.2 Hz), 70.21, 65.74 (br s), 51.72, 44.80, 21.90 (d, J = 6.4 Hz), 20.57 (d, J = 6.3 Hz), 16.84 (d, J = 25.6 Hz). 31P NMR (121 MHz, MeOD) δ 3.94. HRMS calcd for C30H37N4O9FP [M + H]+ m/z 647.2282; found 647.2287. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(3-Phenylpropanamido)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4h). According to general procedures B and D described above, 4h (29.5 mg, two steps, 22%) was obtained as a white foam. 1H NMR (500 MHz, MeOD) δ 8.02 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.39 (t, J = 7.9 Hz, 2H), 7.32−7.16 (m, 8H), 6.27 (d, J = 18.7 Hz, 1H), 5.04−4.94 (m, 1H), 4.61−4.53 (m, 1H), 4.48−4.39 (m, 1H), 4.18 (d, J = 9.6 Hz, 1H), 3.95 (dq, J = 10.0, 7.1 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H), 1.38 (d, J = 7.1 Hz, 3H), 1.31 (d, J = 22.3 Hz, 3H), 1.25−1.20 (m, 6H). 31P NMR (202 MHz, MeOD) δ 4.00. HRMS calcd for C31H39N4O9FP [M + H]+ m/z 661.2439; found 661.2445. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Isobutyramido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4i). According to general procedures B and D described above, 4i (22.0 mg, two steps, 18%) was obtained as an off-white semisolid. 1H NMR (500 MHz, MeOD) δ 8.04 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.9 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H), 7.23 (t, J = 7.2 Hz, 1H), 6.29 (d, J = 18.3 Hz, 1H), 5.04−4.90 (m, 1H), 4.57 (dd, J = 11.2, 5.1 Hz, 1H), 4.45 (ddd, J = 11.9, 6.1, 3.7 Hz, 1H), 4.19 (d, J = 8.1 Hz, 1H), 3.96 (ddd, J = 14.2, 10.1, 7.1 Hz, 2H), 2.80−2.67 (m, 1H), 1.39 (d, J = 7.1 Hz, 3H), 1.33 (d, J = 22.3 Hz, 3H), 1.24 (dd, J = 6.2, 2.6 Hz, 6H), 1.20 (d, J = 6.8 Hz, 6H). 31P NMR (202 MHz, MeOD) δ 4.02. HRMS calcd for C26H37N4O9FP [M + H]+ m/z 599.2282; found 599.2286. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(2-Ethylbutanamido)-2oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyl tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4j). According to general procedures B and D described above, 4j (36.1 mg, two steps, 29%) was obtained as a white foam. 1H NMR (500 MHz, MeOD) δ 7.93 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.27 (t, J = 7.9 Hz, 2H), 7.18 (d, J = 7.9 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H), 6.17 (d, J = 18.2 Hz, 1H), 4.92−4.81 (m, 1H), 4.46 (dd, J = 11.2, 5.2 Hz, 1H), 4.33 (ddd, J = 11.9, 6.1, 3.5 Hz, 1H), 4.07 (d, J = 9.5 Hz, 1H), 3.88−3.79 (m, 2H), 2.30−2.20 (m, 1H), 1.63−1.52 (m, 2H), 1.49− 1.39 (m, 2H), 1.27 (d, J = 7.1 Hz, 3H), 1.21 (d, J = 22.4 Hz, 3H), 1.11 (dd, J = 6.2, 3.3 Hz, 6H), 0.82 (t, J = 7.4 Hz, 6H). 31P NMR (202 MHz, MeOD) δ 4.00. HRMS calcd for C28H41N4O9FP [M + H]+ m/z 627.2595; found 627.2603. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(2-Propylpentanamido)2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyl tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4k). According to general procedures B and D described above, 4k (52.7 mg, two steps, 20%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.03 (d, J = 7.6 Hz, 1H), 7.54−7.42 (m, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 6.27 (d, J = 18.9 Hz, 1H), 5.10−4.89 (m, 1H), 4.63−4.50 (m, 1H), 4.50− 4.34 (m, 1H), 4.17 (d, J = 8.9 Hz, 1H), 4.04−3.85 (m, 2H), 2.67−2.41 (m, 1H), 1.78−1.56 (m, 2H), 1.55−1.40 (m, 2H), 1.39−1.26 (m, 10H), 1.21 (dd, J = 6.2, 1.8 Hz, 6H), 0.92 (t, J = 7.2 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 177.81, 172.93 (d, J = 5.0 Hz), 163.03, 156.46, 150.76, 144.20 (br s), 129.51, 124.84, 119.90 (d, J = 4.8 Hz), 100.17 (d, J = 183.1 Hz), 98.96, 97.44, 90.70 (br s), 79.81, 71.52 (d, J = 17.4 Hz), 68.78, 64.36, 50.30, 34.64 (d, J = 1.4 Hz), 20.52 (d, J = 7.5 Hz), 20.20, 19.17 (d, J = 6.2 Hz), 15.45 (d, J = 25.6 Hz), 12.99. 31P NMR (121 MHz, MeOD) δ 3.96. HRMS calcd for C30H45N4O9FP [M + H]+ m/z 655.2908; found 655.2915.

(S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(Cyclopropanecarboxamido)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4l). According to general procedures B and D described above, 4l (17.7 mg, two steps, 15%) was obtained as a white solid. 1H NMR (300 MHz, MeOD) δ 8.01 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 6.27 (d, J = 19.2 Hz, 1H), 5.06−4.90 (m, 1H), 4.63−4.50 (m, 1H), 4.50−4.37 (m, 1H), 4.17 (br d, J = 8.8 Hz, 1H), 4.02−3.85 (m, 2H), 1.96−1.83 (m, 1H), 1.37 (d, J = 6.9 Hz, 3H), 1.32 (d, J = 22.2 Hz, 3H), 1.25−1.17 (m, 6H), 1.06−0.88 (m, 4H). 13C NMR (75 MHz, MeOD) δ 176.40, 174.34 (d, J = 5.4 Hz), 164.34, 157.93, 152.14 (d, J = 6.5 Hz), 145.41 (br s), 130.93, 126.29, 121.34 (d, J = 4.8 Hz), 101.58 (d, J = 182.7 Hz), 98.80, 91.81 (br s), 81.18 (d, J = 8.2 Hz), 73.01 (d, J = 18.8 Hz), 70.21, 65.72 (br s), 51.74, 21.93 (d, J = 6.6 Hz), 20.59 (d, J = 6.4 Hz), 16.87 (d, J = 25.5 Hz), 16.03, 9.67. 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C26H34N4O9FPNa [M + Na]+ m/z 619.1945; found 619.1960. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(Cyclobutanecarboxamido)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4m). According to general procedures B and D described above, 4m (24.5 mg, two steps, 20%) was obtained as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.03 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.8 Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.27 (d, J = 18.9 Hz, 1H), 5.08−4.91 (m, 1H), 4.65−4.52 (m, 1H), 4.48−4.38 (m, 1H), 4.18 (br d, J = 8.9 Hz, 1H), 4.07−3.83 (m, 2H), 2.45−1.76 (m, 7H), 1.38 (d, J = 7.1 Hz, 3H), 1.31 (d, J = 22.3 Hz, 3H), 1.25−1.18 (m, 6H). 13C NMR (75 MHz, MeOD) δ 177.25, 174.34 (d, J = 5.5 Hz), 164.71, 157.86, 152.19, 145.36 (br s), 130.93, 126.29, 121.34 (d, J = 4.7 Hz), 101.57 (d, J = 182.9 Hz), 98.78, 92.18 (br s), 81.20, 81.13, 73.12, 72.87, 70.21, 65.75 (br s), 51.74, 41.77, 25.75, 21.93 (d, J = 6.7 Hz), 20.60 (d, J = 6.3 Hz), 18.86, 16.85 (d, J = 25.5 Hz). 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C27H36N4O9FPNa [M + Na]+ m/z 633.2102; found 633.2116. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(Cyclohexanecarboxamido)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4n). According to the general procedures B and E described above, 4n (19.7 mg, two steps, 15%) was obtained as a white solid. 1H NMR (500 MHz, MeOD) δ 8.01 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 7.9 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 7.20 (t, J = 7.3 Hz, 1H), 6.26 (d, J = 19.1 Hz, 1H), 5.07−4.88 (m, 1H), 4.62−4.50 (m, 1H), 4.48−4.35 (m, 1H), 4.16 (d, J = 9.6 Hz, 1H), 4.01−3.84 (m, 2H), 2.43 (tt, J = 11.4, 3.3 Hz, 1H), 1.88 (d, J = 12.4 Hz, 2H), 1.82 (dd, J = 9.9, 2.9 Hz, 2H), 1.71 (d, J = 12.6 Hz, 2H), 1.47 (dd, J = 24.0, 11.8 Hz, 2H), 1.36 (d, J = 7.0 Hz, 3H), 1.30 (d, J = 22.3 Hz, 3H), 1.21 (dd, J = 6.2, 3.1 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 178.74, 174.34 (d, J = 5.3 Hz), 164.77, 157.85, 145.46 (br s), 130.92, 126.27, 121.32 (d, J = 4.8 Hz), 101.56 (d, J = 182.8 Hz, 2H), 98.78, 92.21 (br s), 81.18 (d, J = 7.6 Hz), 73.02 (d, J = 18.3 Hz), 70.22, 65.74 (br s), 51.74, 47.04, 30.21, 26.78, 26.52, 21.92 (d, J = 6.9 Hz), 20.58 (d, J = 6.3 Hz), 16.86 (d, J = 25.6 Hz). 31P NMR (121 MHz, MeOD) δ 3.97. HRMS calcd for C29H41N4O9FP [M + H]+ m/z 639.2595; found 639.2598. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4o). According to general procedures B and D described above, 4o (32.9 mg, two steps, 26%) was obtained as a white solid. 1H NMR (300 MHz, MeOD) δ 8.10 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 7.3 Hz, 2H), 7.65 (t, J = 7.3 Hz, 1H), 7.61−7.48 (m, 3H), 7.39 (t, J = 7.8 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 7.21 (t, J = 7.4 Hz, 1H), 6.29 (d, J = 19.0 Hz, 1H), 5.06−4.91 (m, 1H), 4.58 (dd, J = 11.1, 5.0 Hz, 1H), 4.50−4.39 (m, 1H), 4.19 (d, J = 8.2 Hz, 1H), 4.05−3.88 (m, 2H), 1.39 (d, J = 6.4 Hz, 3H), 1.34 (d, J = 21.0 Hz, 3H), 1.22 (d, J = 6.2 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 174.86 (d, J = 5.3 Hz), 169.65, 165.56, 158.28, 152.66 (d, J = 6.6 Hz), 146.22 (br s), 135.15, 134.66, 131.45, 130.34, 129.71, 126.80, 121.84 (d, J = 4.9 Hz), 102.11 (d, J = 182.7 Hz), 99.63, 92.65 (br d, J = 31.2 Hz), 81.75 (d, J = 7.4 Hz), 73.52 (d, J = 18.0 Hz), 70.75, 66.25 (br s), 52.27, 22.44 (d, J = 6.8 Hz), 21.12 (d, J = 6.3 Hz), 6083



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17.40 (d, J = 25.5 Hz). 31P NMR (121 MHz, MeOD) δ 4.01. HRMS calcd for C29H35N4O9FP [M + H]+ m/z 633.2126; found 633.2132. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-Chlorobenzamido)-2oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4p). According to general procedures B and D described above, 4p (28.5 mg, two steps, 21%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.10 (d, J = 7.6 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 3H), 7.46−7.35 (m, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 6.29 (d, J = 19.0 Hz, 1H), 5.05−4.91 (m, 1H), 4.64−4.53 (m, 1H), 4.50−4.39 (m, 1H), 4.19 (d, J = 9.5 Hz, 1H), 4.02−3.88 (m, 2H), 1.38 (d, J = 6.7 Hz, 3H), 1.33 (d, J = 22.2 Hz, 3H), 1.22 (d, J = 6.2 Hz, 6H). 31P NMR (121 MHz, MeOD) δ 4.00. HRMS calcd for C29H34N4O9FPCl [M + H]+ m/z 667.1736; found 667.1742. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-Methoxybenzamido)2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4q). According to general procedures B and D described above, 4q (42.4 mg, two steps, 32%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.07 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 9.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.46−7.34 (m, 2H), 7.29 (d, J = 8.7 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 7.06 (d, J = 9.0 Hz, 2H), 6.29 (d, J = 18.5 Hz, 1H), 5.07−4.91 (m, 1H), 4.62−4.52 (m, 2H), 4.49−4.39 (m, 1H), 4.18 (d, J = 9.8 Hz, 1H), 4.02−3.91 (m, 2H), 3.89 (s, 3H), 1.38 (d, J = 5.3 Hz, 3H), 1.33 (d, J = 20.6 Hz, 3H), 1.21 (d, J = 6.3 Hz, 6H). 31P NMR (121 MHz, MeOD) δ 4.01. HRMS calcd for C30H37N4O10FP [M + H]+ m/z 663.2231; found 663.2237. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-Cinnamamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4r). According to general procedures B and D described above, 4r (24.6 mg, two steps, 19%) was obtained as a white solid. 1H NMR (500 MHz, MeOD) δ 8.05 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.78 (d, J = 15.7 Hz, 1H), 7.67−7.60 (m, 2H), 7.57 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.44−7.15 (m, 8H), 7.01 (d, J = 12.6 Hz, 1H), 6.85 (d, J = 15.7 Hz, 1H), 6.33−6.24 (m, 1H), 6.17 (d, J = 12.6 Hz, 1H), 5.08−4.88 (m, 1H), 4.60−4.53 (m, 1H), 4.48−4.39 (m, 1H), 4.22− 4.11 (m, 1H), 4.02−3.85 (m, 2H), 1.45−1.33 (m, 3H), 1.31−1.26 (m, 3H), 1.24−1.18 (m, 6H). 31P NMR (121 MHz, MeOD) δ 4.00. HRMS calcd for C31H37N4O9FP [M + H]+ m/z 659.2282; found 659.2287. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(2-(4-Chlorophenoxy)-2methylpropanamido)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4s). According to general procedures B and D described above, 4s (62.4 mg, two steps, 43%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.11 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.44−7.34 (m, 2H), 7.29 (d, J = 8.8 Hz, 4H), 7.21 (t, J = 7.3 Hz, 1H), 6.97 (d, J = 8.9 Hz, 1H), 6.26 (d, J = 19.4 Hz, 1H), 5.14−4.90 (m, 1H), 4.71−4.50 (m, 1H), 4.49−4.35 (m, 1H), 4.18 (d, J = 9.9 Hz, 1H), 4.05−3.83 (m, 2H), 1.57 (s, 6H), 1.37 (d, J = 7.5 Hz, 3H), 1.31 (d, J = 22.4 Hz, 3H), 1.24−1.18 (m, 6H). 13C NMR (75 MHz, MeOD) δ 176.42, 174.31, 164.24, 154.29, 146.19, 130.93, 130.42, 126.28, 123.63, 121.33 (d, J = 4.8 Hz), 116.82, 101.58 (d, J = 183.0 Hz), 98.56, 92.38, 81.20, 72.97 (d, J = 18.9 Hz), 70.23, 65.73, 51.75, 24.89 (d, J = 6.2 Hz), 21.94 (d, J = 6.9 Hz), 20.60 (d, J = 6.2 Hz), 16.85 (d, J = 25.6 Hz). 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C32H38N4O10FPCl [M − H]+ m/z 723.1998; found 723.2003. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Ethoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4aa). According to general procedures C and D described above, 4aa (32.4 mg, two steps, 27%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 7.99 (d, J = 7.6 Hz, 1H), 7.45−7.33 (m, 2H), 7.23−7.12 (m, 1H), 6.25 (d, J = 19.0 Hz, 1H), 5.09−4.87 (m, 1H), 4.65−4.48 (m, 1H), 4.48−4.36 (m, 1H), 4.23 (q, J = 7.1 Hz, 2H), 4.16 (br d, J = 8.3 Hz, 1H), 4.03−3.77 (m, 2H), 1.43− 1.25 (m, 9H), 1.20 (d, J = 6.2 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 174.32 (d, J = 5.5 Hz), 165.14, 157.61, 154.53, 152.12 (d, J = 7.0 Hz),

145.08 (br s), 130.90, 126.25, 121.31 (d, J = 4.9 Hz, 5H), 101.55 (d, J = 182.8 Hz), 97.45, 92.11 (br s), 81.14 (d, J = 8.1 Hz), 73.00 (d, J = 17.7 Hz), 70.20, 65.74, 63.17, 51.71, 21.91 (d, J = 6.6 Hz), 20.59 (d, J = 6.3 Hz), 16.86 (d, J = 25.6 Hz), 14.57. 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C25H34N4O10FPNa [M + Na]+ m/z 623.1894; found 623.1911. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Propoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ab). According to general procedures C and D described above, 4ab (22.6 mg, two steps, 18%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 7.99 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.9 Hz, 2H), 7.31−7.25 (m, 3H), 7.20 (t, J = 7.2 Hz, 1H), 6.25 (d, J = 19.5 Hz, 1H), 5.09−4.88 (m, 1H), 4.61−4.49 (m, 1H), 4.48−4.36 (m, 1H), 4.20−4.09 (m, 3H), 4.03−3.82 (m, 2H), 1.79− 1.63 (m, 2H), 1.36 (d, J = 7.7 Hz, 3H), 1.30 (d, J = 22.5 Hz, 3H), 1.21 (d, J = 6.2 Hz, 6H), 0.99 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 174.34 (d, J = 5.5 Hz), 165.21, 157.64, 154.66, 145.18 (br s), 130.91, 126.27, 121.32 (d, J = 4.9 Hz), 101.56 (d, J = 182.4 Hz), 97.48, 91.87 (br s), 81.21, 73.03 (d, J = 17.2 Hz), 70.21, 68.72, 65.76 (br s), 51.75, 23.10, 21.91 (d, J = 6.7 Hz), 20.58 (d, J = 6.3 Hz), 16.85 (d, J = 25.7 Hz), 10.52. 31P NMR (121 MHz, MeOD) δ 3.97. HRMS calcd for C26H36N4O10FPNa [M + Na]+ m/z 637.2051; found 637.2067. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4((Isopropoxycarbonyl)amino))-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3hydroxy-4-methyl tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ac). According to general procedures C and D described above, 4ac (29.0 mg, two steps, 24%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 7.99 (d, J = 7.7 Hz, 1H), 7.43−7.16 (m, 6H), 6.25 (d, J = 19.4 Hz, 1H), 5.09−4.91 (m, 2H), 4.59−4.51 (m, 1H), 4.47−4.37 (m, 1H), 4.16 (d, J = 9.6 Hz, 1H), 4.02−3.84 (m, 2H), 1.36 (d, J = 7.3 Hz, 3H), 1.31 (d, J = 6.3 Hz, 6H), 1.30 (d, J = 22.3 Hz, 3H), 1.21 (d, J = 6.4 Hz, 6H). 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C26H36N4O10FPNa [M + Na]+ m/z 637.2051; found 637.2069. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Butoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ad). According to general procedures C and D described above, 4ad (26.8 mg, two steps, 21%) was obtained as a white foam. 1H NMR (500 MHz, MeOD) δ 8.00 (d, J = 7.6 Hz, 1H), 7.39−7.35 (m, 2H), 7.28 (d, J = 8.5 Hz, 3H), 7.23−7.17 (m, 1H), 6.25 (d, J = 18.8 Hz, 1H), 5.04−4.91 (m, 1H), 4.60−4.52 (m, 1H), 4.48− 4.38 (m, 1H), 4.20 (t, J = 6.6 Hz, 2H), 4.18−4.12 (m, 1H), 4.00−3.86 (m, 2H), 1.73−1.62 (m, 2H), 1.52−1.40 (m, 2H), 1.36 (d, J = 7.1 Hz, 3H), 1.31 (d, J = 22.3 Hz, 3H), 1.21 (dd, J = 6.3, 1.9 Hz, 6H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 174.32 (d, J = 5.4 Hz), 165.16, 157.63, 154.63, 152.12 (d, J = 6.8 Hz), 145.06 (br s), 130.89, 126.25, 121.31 (d, J = 4.9 Hz), 101.55 (d, J = 182.6 Hz), 97.46, 91.98 (br s), 81.15 (d, J = 7.7 Hz), 73.00 (d, J = 18.2 Hz), 70.19, 66.92, 65.75 (br s), 51.72, 31.85, 21.90 (d, J = 6.6 Hz), 20.58 (d, J = 6.2 Hz), 19.98, 17.02, 16.85 (d, J = 25.6 Hz), 13.94. 31P NMR (121 MHz, MeOD) δ 3.96. HRMS calcd for C27H39N4O10FP [M + H]+ m/z 629.2388; found 629.2392. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Isobutoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ae). According to general procedures C and D described above, 4ae (46.5 mg, two steps, 37%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.00 (d, J = 7.6 Hz, 1H), 7.44−7.15 (m, 6H), 6.25 (d, J = 18.2 Hz, 1H), 5.11−4.90 (m, 1H), 4.62−4.49 (m, 1H), 4.47−4.36 (m, 1H), 4.16 (d, J = 9.9 Hz, 1H), 3.98 (d, J = 6.6 Hz, 2H), 3.96−3.79 (m, 2H), 2.13−1.80 (m, 1H), 1.36 (d, J = 7.6 Hz, 3H), 1.30 (d, J = 23.9 Hz, 3H), 1.21 (d, J = 6.2 Hz, 6H), 0.98 (d, J = 6.7 Hz, 6H). 31P NMR (121 MHz, MeOD) δ 3.99. HRMS calcd for C27H39N4O10FP [M + H]+ m/z 629.2398; found 629.2394. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Pentyloxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4af). According to general procedures C and D 6084



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3.96. HRMS calcd for C30H37N4O10FP [M + H]+ m/z 663.2231; found 663.2237. Inhibition of HCV Replicon and Cytotoxicity Assays. The antiHCV activity and cytotoxicity were measured in Huh7 cell lines expressing a stable subgenomic HCV genotype 1b replicon encoding the luciferase reporter gene (WuXi AppTec). All compounds were supplied as an approximately 10 μM solution in 100% DMSO. Compounds were tested in a 3-fold concentration dilution series in 96well plates, and the DMSO concentration in the final assay wells was 0.5%. All serial dilutions for each compound were performed in duplicates within the same plate. To each well was added 8000 suspended HCV replicon cells. The plates were incubated for 72 h at 37 °C with 5% CO2. In cytotoxicity assays, the Cell Titer-Fluor Cell Viability Assay (Promega, Madison WI) was used to examine toxicity of the test compounds. Cell Titer-Fluor reagent was added to cells, and the plates were incubated for 5 min at 37 °C with 5% CO2 before measuring relative fluorescence units (RFU) in an EnVision spectrophotometer (PerkinElmer, Waltham, MA). The EC50 assay was performed in the same wells as the CC50 assay. Bright-Glo reagent (Promega, Madison WI) was added to cells, and plates were incubated for 5 min before the luminescence signal was measured with a PerkinElmer Envision Plate Reader (PerkinElmer, Waltham, MA). The EC50 and CC50 were calculated using Graphpad Prism software. S282T Mutant Replicon Activity. According to the method described above, the Huh7 cell lines expressing wild-type and S282T mutant HCV genotype 1b replicon (WuXi AppTec) were used to evaluate resistant profiles of the tested compounds. Metabolic Stability of Compounds in Human Liver S9 Fractions. Working solution (10 μM, 1% DMSO, 5% MeOH) was prepared by diluting a 40 μL compound solution in DMSO (1 mM) with 360 μL of 100 mM Tris buffer. The compound or control working solution (10 μL/well) was added to all plates (T0, T5, T10, T20, T30, T60) except matrix blank. S9 solution was dispensed (440 μL/well, final concentration of 1 mg of protein/mL) to a 96-well plate as reservoir according to the plate map and added to every plate (50 μL/well) by Apricot. The compound and S9 solution were incubated at 37 °C. At the desired times (0, 5, 10, 20, 30, and 60 min), cold ACN (300 μL/ well, including 100 ng/mL of Tolbutamide as internal standard) were added to terminate the reaction. The sampling plates were shaken for 5 min and then centrifuged at 4000 rpm for 20 min. While centrifuging, a new 96-well plate was loaded with 300 μL of HPLC water and 100 μL of supernatant. The mixture was transferred for LC/ MS/MS. Each compound (10 μL inject volume) was separated using a Phenomenex Synergi Hydro-RP 80A 4 μm C18 column (2.0 × 30 mm, part no.: 00A-4375-B0) with the LC/MS/MS (LC Shimadzu LC 20-AD; MS: API 4000). The elution liquid gradient was generated as the following steps using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at 800 μL/min; 0−1.1 min, a liner gradient of solvent B from 5 to 90%; 1.1−1.3 min, 90% solvent B; 1.3−1.5 min, 5% solvent B. Analysis was performed in Multiple Reaction Monitoring (MRM) mode. The ESI ion source parameters were as follows: curtain gas (CUR), 20 psi; collision gas (CAD), 10 psi; ion spray voltage (ISV), −5500 V; nebulizer gas (GS1), 55 psi; turbo gas (GS2), 60 psi; and temperature, 600 °C. Area ratio of analyte peak to internal standard peak was tested for the amount of parent compound, and the percentage remaining was calculated on the basis of the initial ratio measured at 0 min. The half-life of each compound was calculated using Graph-Pad Prism software. Metabolic Stability of Compounds in Human Plasma. Prior to the experiments, the pooled frozen plasma was thawed in a water bath at 37 °C for compound dilution and then centrifuged at 4000 rpm for 5 min. The value of pH was measured and adjusted to 7.4. The plasma was prewarmed before adding compounds. The plasma samples (2 μM) were prepared by diluting 4 μL of the working solution (200 μM in DMSO) with 796 μL of diluted plasma in duplicates. Samples for each time point were taken at 0, 10, 30, 60, and 120 min in a volume of 100 μL and quenched with 500 μL stop solutions (100% ACN containing 200 ng/mL of Tolbutamide plus 20 ng/mL of Buspirone). Each plate was centrifuged at 4000 rpm for 20 min. The resulting supernatant (100 μL) was transferred from each well to the sample

described above, 4af (25.4 mg, two steps, 20%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.00 (d, J = 7.6 Hz, 1H), 7.51−7.14 (m, 6H), 6.25 (d, J = 18.6 Hz, 1H), 4.97 (dt, J = 12.5, 6.3 Hz, 1H), 4.55 (dd, J = 10.3, 5.8 Hz, 1H), 4.49−4.36 (m, 1H), 4.18 (t, J = 6.6 Hz, 2H), 4.16 (d, J = 9.1 Hz, 1H), 4.02−3.82 (m, 2H), 1.81− 1.61 (m, 2H), 1.47−1.34 (m, 7H), 1.30 (d, J = 22.5 Hz, 3H), 1.21 (dd, J = 6.3, 0.9 Hz, 6H), 0.93 (t, J = 7.0 Hz, 3H). 31P NMR (121 MHz, MeOD) δ 3.98. HRMS calcd for C28H41N4O10FP [M + H]+ m/z 643.2544; found 643.2548. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-(((Pentan-3-yloxy) carbonyl)amino)-2-oxo-pyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ag). According to general procedures C and D described above, 4ag (30.7 mg, two steps, 24%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.00 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 6.9 Hz, 3H), 7.20 (t, J = 7.2 Hz, 1H), 6.26 (d, J = 19.1 Hz, 1H), 5.07−4.89 (m, 1H), 4.77−4.68 (m, 1H), 4.56 (dd, J = 11.2, 5.0 Hz, 1H), 4.49−4.34 (m, 1H), 4.16 (d, J = 8.5 Hz, 1H), 4.01−3.83 (m, 2H), 1.76−1.58 (m, 4H), 1.37 (d, J = 7.7 Hz, 3H), 1.32 (d, J = 23.9 Hz, 3H), 1.22 (d, J = 6.1 Hz, 6H), 0.95 (t, J = 7.4 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 175.35, 174.31, 165.29, 157.68, 154.63, 144.99, 130.91, 126.26, 121.32 (d, J = 4.9 Hz), 101.58 (d, J = 182.3 Hz), 97.51, 91.97, 81.16 (d, J = 7.5 Hz), 80.42, 73.02 (d, J = 17.6 Hz), 70.22, 65.77, 51.73, 27.61, 21.92 (d, J = 6.9 Hz), 20.59 (d, J = 6.3 Hz), 16.86 (d, J = 25.6 Hz), 9.87. 31P NMR (121 MHz, MeOD) δ 3.97. HRMS calcd for C28H40N4O10FPNa [M + Na]+ m/z 665.2364; found 665.2381. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Hexyloxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ah). According to general procedures C and D described above, 4ag (43.3 mg, two steps, 33%) was obtained as a white foam. 1H NMR (500 MHz, MeOD) δ 8.01 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.9 Hz, 2H), 7.30 (t, J = 6.5 Hz, 3H), 7.21 (t, J = 7.4 Hz, 1H), 6.27 (d, J = 18.4 Hz, 1H), 4.98 (hept, J = 6.2 Hz, 1H), 4.63−4.53 (m, 1H), 4.50−4.40 (m, 1H), 4.29−4.11 (m, 3H), 4.07−3.82 (m, 2H), 1.81−1.62 (m, 2H), 1.49−1.35 (m, 9H), 1.32 (d, J = 22.3 Hz, 3H), 1.22 (dd, J = 6.3, 1.9 Hz, 6H), 0.93 (t, J = 6.9 Hz, 3H). 31P NMR (202 MHz, MeOD) δ 3.98. HRMS calcd for C29H43N4O10FP [M + H]+ m/ z 657.2701; found 657.2708. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4((Cyclohexyloxycarbonyl)amino)-2-oxo-pyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4ai). According to general procedures C and D described above, 4ai (36.0 mg, two steps, 28%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 7.99 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.33−7.24 (m, 3H), 7.20 (t, J = 7.2 Hz, 1H), 6.25 (d, J = 19.3 Hz, 1H), 4.97 (hept, J = 6.3 Hz, 1H), 4.77−4.68 (m, 1H), 4.56 (dd, J = 11.2, 5.2 Hz, 1H), 4.48−4.37 (m, 1H), 4.16 (d, J = 8.8 Hz, 1H), 4.05−3.79 (m, 2H), 2.01−1.88 (m, J = 7.5 Hz, 2H), 1.85−1.71 (m, 2H), 1.65−1.40 (m, 6H), 1.37 (d, J = 7.2 Hz, 3H), 1.31 (d, J = 22.5 Hz, 3H), 1.22 (d, J = 6.2 Hz, 6H). 31P NMR (121 MHz, MeOD) δ 3.99. HRMS calcd for C29H41N4O10FP [M + H]+ m/z 655.2544; found 655.2548. (S)-Isopropyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-(4-((Benzyloxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4aj). According to general procedures C and D described above, 4aj (139.8 mg, two steps, 42%) was obtained as a white foam. 1H NMR (300 MHz, MeOD) δ 8.00 (d, J = 7.6 Hz, 1H), 7.45−7.25 (m, 10H), 7.19 (t, J = 7.2 Hz, 1H), 6.25 (d, J = 19.1 Hz, 1H), 5.22 (s, 2H), 5.05−4.89 (m, 1H), 4.56 (dd, J = 11.2, 5.2 Hz, 1H), 4.48−4.36 (m, 1H), 4.16 (d, J = 8.9 Hz, 1H), 4.03−3.84 (m, 2H), 1.37 (d, J = 7.1 Hz, 3H), 1.30 (d, J = 22.4 Hz, 3H), 1.20 (dd, J = 6.2, 1.4 Hz, 6H). 13C NMR (75 MHz, MeOD) δ 174.36 (d, J = 5.3 Hz), 165.13, 157.60, 154.44, 152.14 (d, J = 6.9 Hz), 145.17, 137.15, 130.93, 129.61, 129.47, 129.31, 126.28, 121.32 (d, J = 4.9 Hz), 116.84, 101.59 (d, J = 182.5 Hz), 97.51, 91.93, 81.16 (d, J = 7.7 Hz), 73.00 (d, J = 18.1 Hz), 70.24, 68.68, 65.75, 51.73, 21.94 (d, J = 6.3 Hz), 20.62 (d, J = 6.3 Hz), 16.88 (d, J = 25.6 Hz). 31P NMR (121 MHz, MeOD) δ 6085



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plate and mixed with 200 μL of ultrapure water. The plate was shaken at 800 rpm for approximately 10 min before submitting to LC/MS/ MS analysis. Each compound (10 μL inject volume) was separated using an ACE 5 Phenyl 4 μm C18 column (2.1 × 50 mm, part no.: ACE-125−0502) with LC/MS/MS (LC Shimadzu LC 20-AD; MS: API 4000). The elution liquid gradient was generated as the following steps using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at 700 μL/min; 0−0.8 min, a liner gradient of solvent B from 5 to 95%; 0.8−1.0 min, 95% solvent B; 1.0−1.2 min, 5% solvent B. Analysis was performed in Multiple Reaction Monitoring (MRM) mode. The ESI ion source parameters were as follows: curtain gas (CUR), 20 psi; collision gas (CAD), 10 psi; ion spray voltage (ISV), −5500 V; nebulizer gas (GS1), 55 psi; turbo gas (GS2), 60 psi; and temperature, 600 °C. The percentage remaining and the slope (ke min−1) of the disappearance of test compounds after incubation in plasma are calculated based on the initial linear part of the curve. The data is fitted by linear regression. The percentage remaining = 100 (PAR at appointed incubation time/PAR at time 0) where PAR was the peak area ratio of test compound versus internal standard. Half-life of test compound: t1/2 = ln 2/ke (min). In Vitro Metabolic Study. The dosing solutions were prepared by mixing 30 μL of 50 mM test compound solutions with 29.970 mL of DMEM supplied with 10% FBS. Huh-7 cells were used for metabolite semiquantification. The Huh-7 cells (2 mL/well, 0.35 million/mL in DMEM supplied with 10% FBS) were plated to a 6-well cell culture plate and incubated for 18 h at 37 °C in a 95% humidified incubator at 5% CO2. The cell density was approximately 1 million cells/well at dosing time. After 18 h of incubation, the old media was replaced with 2 mL of prewarmed dosing solution, and then the sample plate was returned to the incubator for continuing incubation. The plates were incubated at 37 °C in a 95% humidified incubator at 5% CO2 for 0, 1, 6, 8, 12, 24, and 48 h. At the indicated time (0, 1, 6, 8, 24, and 48 h), 200 μL of supernatant of each sample at each time point was transferred to an Eppendorf tube containing 600 μL of stop solution followed by a 1 min votex and 10 min of centrifugation (14000 rpm, 4 °C). The resulting supernatant was transferred into another set of prelabeled 96 deep-well plates containing dilution solution. The plates were sealed and stored at 4 °C until LC/MS/MS analysis. At each time point, the supernatant was aspirated and washed with 2 mL of cold 0.9% NaCl twice. The washing buffer was vacuumed away without touching the cells. Then, 800 μL of 100% MeOH containing internal standard was added to lyse the cells. The cells were lifted from the plate (using Corning Costar Cell Lifter to scratch the cells off the plate). The cell lysate (both liquid and solid) was transferred into a clean Eppendorf tube. Each well was rinsed with 800 μL of 100% MeOH containing internal standard, and then the media was transferred into the same tube. The samples were placed at −80 °C for at least 3 h. The samples were warmed to room temperature and centrifuged for 10 min at 14000 rpm at 4 °C. The supernatant was transferred into a clean Eppendorf tube. The samples were dried in a Speed-Vac and reconstituted with 1.2 mL of 0.18 mM DBAA and 0.0005% NH3·H2O in 100% H2O and centrifuged for 1 min at 13000 rpm; then, the samples were injected into LC/MS/MS. Each compound (10 μL inject volume) was separated using an ACE 5 Phenyl 4 μm C18 column (2.1 × 50 mm, part no.: ACE-125−0502) with the LC/MS/MS (LC Shimadzu LC 20-AD; MS: API 4000). The elution liquid gradient was generated as the following steps using solvent A (1.2 mL of 0.18 mM DBAA and 0.0005% NH3·H2O in 100% H2O) and solvent B (3 mM NH4OAc and 10 mM DMHA in 50% ACN/H2O) at 700 μL/min; 0−0.5 min, 100% solvent A; 0.5−4.0 min, a liner gradient of solvent B from 0 to 25%; 4.0−8.0 min, a liner gradient of solvent B from 25 to 80%; 8.0−9.5 min, 80% solvent B; 9.5−13.0 min, 100% solvent A. Analysis was performed in Multiple Reaction Monitoring (MRM) mode. The ESI ion source parameters were as follows: curtain gas (CUR), 20 psi; collision gas (CAD), 10 psi; ion spray voltage (ISV), −4500 V; nebulizer gas (GS1), 55 psi; turbo gas (GS2), 60 psi; and temperature, 600 °C. Methods for Metabolite Semiquantification. The amount of metabolite was determined on the basis of the Δ mean peak area ratio (ΔMPAR). ΔMPAR was the MPAR at each time point minus the

MPAR at t0, where the MPAR was the mean value of the PAR (the PAR was the peak area ratio of test compound versus that of the internal standard). In Vivo Pharmacokinetic Study. Pharmacokinetic studies were performed in Beagle dogs (3 sex−1 group−1). Oral dosing was administered by gavage at a dose of 150 mg/kg in hydroxypropyl-βcyclodextrin in water (20% w/v). Dose volume was 2.5 mL/kg. Blood samples were collected at 0.5, 0.75, 1, 2, 4, 6, 8, 12, and 24 h post dose via venipuncture into Eppendorf tubes containing K2EDTA. Plasma was isolated by centrifugation and kept on ice for processing. Then, 50 μL of supernatant of each sample at each time point was transferred to an Eppendorf tube containing 450 μL of ice acetonitrile, followed by 1 min of vortexing and 10 min of centrifugation (14000 rpm, 4 °C). All of the resulting samples were kept at −80 °C until LC/MS/MS analysis. An AB SCIEX API 4000 mass spectrometer was equipped with a Shimadzu HPLC system (LC-20A), the US AB MS system (API4000), electrospray ionization, and an Analyst 1.5.1 workstation. LC conditions were as follows: column, Waters XSelect HSS T3 XP column (2.5 μm, 3.0 mm × 50 mm); mobile phase of (A) 0.1% formic acid in water, (B) 100% acetonitrile; flow rate: 0.7 mL/min; gradient within 0−5 min; 1.5 min at 99% A, 2.5 min at 10% A and 90% B, 5 min at 99% A. Mass spectometer conditions were as follows: positive ion mode was chosen under Multiple Reaction Monitoring (MRM) mode. MRM parameters of compound 4c: parent ion (Q1Mass), 599.1 Da; product ion (Q3Mass), 418.2 Da; declustering voltage (DP), 100 V; collision voltage (CE), 23 eV. MRM parameters of zolpidem (IS): parent ion (Q1Mass), 308.2 Da; product ion (Q3Mass), 235.2 Da; declustering voltage (DP), 80 V; collision voltage (CE), 45 eV. The acquisition and processing of data were performed using an Analyst 1.5.1 workstation. Noncompartmental PK analysis was performed on plasma concentration data to calculate PK parameters using WinNonlin (version 6.1; Pharsight, Mountain View, CA, USA). Acute Toxicity Study. ICR mice (5 sex−1 group−1; Vital River Laboratory Animal Technology Co., Ltd., Beijing. China) weighing 22−25 g were used. Compound 4c and vehicle (30% PEG400/30% Tween 20/20% corn oil/20% water) were orally administrated as a single dose via gavage at four dose levels: 150, 500, 1200, and 1800 mg/kg of body weight. All mice were observed for mortality and morbidity once a day over a period of 14 days. Clinical signs of toxicity and body weight gain changes were recorded once per day. Subacute Toxicity Study. ICR mice (5 sex−1 group−1; Vital River Laboratory Animal Technology Co., Ltd., Beijing. China) weighing 22−25 g were used in subacute toxicity studies. Compound 4c and vehicle (30% PEG400/30% Tween 20/20% corn oil/20% water) were orally administrated via gavage at three dose levels: 100, 500, and 1000 mg/kg of body weight. Administration was performed once per day for 14 days. All mice were observed daily to detect any signs of toxicity such as body weight gain changes, skin and fur quality, inflammation, and any necrosis over a period of 14 days. At day 15, each animal was anesthetized. Organ weight gain changes were detected. The blood samples were placed separately in sterilized heparinized tubes for further biochemical evaluations. Hematological parameters, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) were estimated. For histopathological examinations, organs were fixed in 10% formamide before being embedded in paraffin. After routine processing, 5 μm paraffin sections were prepared and stained with hematoxylin and eosin before microscopic examination.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00262. Methods for the general syntheses and experimental information on key compounds, 1H and 13C NMR and mass spectral data for compounds, molecular formula 6086



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strings (SMILES), and details of the biological assays (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xiaoan Wen: 0000-0001-7852-1154 Haoliang Yuan: 0000-0003-0248-4347 Hongbin Sun: 0000-0002-3452-7674 Author Contributions §

L.Z., L.D., and X.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from National Natural Science Foundation of China (Grants 81602966, 81573299, 81473080, and 81373303) is gratefully acknowledged. This project is also supported by the “111 Project” from the Ministry of Education of China, the State Administration of Foreign Expert Affairs of China (No. 111-2-07), program for Changjiang Scholars, Innovative Research Team in University (IRT1193), and the Jiangsu Province Natural Science Foundation (BK20160759).

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ABBREVIATIONS USED HCV, hepatitis C virus; NS5B, nonstructural protein 5B polymerase; WT, wild-type; EDCI, 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine; TMSCl, trimethyl chlorosilane; RFU, relative fluorescence units

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REFERENCES

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