Evaluation of Novel Phosphoramidate ProTides of the 2′-Fluoro Derivatives of a Potent Anti-Varicella Zoster Virus Bicyclic Nucleoside Analogue

Abstract

Background:

Recently, the synthesis and antiviral activity of a series of 2′-fluoro derivatives of the most potent anti-varicella zoster virus (VZV) agent reported to date, the bicyclic nucleoside analogue Cf1743, have been reported.

Methods:

Here, we present molecular modelling studies for the interaction of these compounds with VZV-encoded thymidine kinase (TK) and we report the synthesis of a series of phosphoramidate ProTides of these compounds designed to bypass the nucleoside kinase dependence of the parent nucleoside analogues.

Results:

The phosphoramidate prodrugs were equally effective as their parent compounds against VZV in cell culture, but lost antiviral potency against TK-deficient VZV strains.

Conclusions:

ProTide-based kinase bypass is not successful in this case.

Introduction

The pentylphenyl bicyclic nucleoside analogue (BCNA) Cf1743 (1; Figure 1) is the most potent anti-varicella zoster virus (VZV) agent reported to date, and is inhibitory in the lower to subnanomolar range [1]. Its 5′-valyl ester prodrug, FV100 (2), is now in Phase II clinical trials for VZV/shingles [2]. After our discovery of this new class of anti-VZV agents, a significant structure–activity relationship study has been undertaken, including modifications of the side chain [3 ,–7], of the phenyl ring [8], at the 7-position [9 –11] and of the sugar [12 ,,–19]. The mechanism of action of the BCNAs is not completely understood, but probably involves selective VZV thymidine kinase (VZV-TK)-mediated phosphorylation to an active phosphorylated form, which is not yet completely understood [20].

Recently, we reported the synthesis and the biological activity of a series of 2′-fluoro BCNA derivatives of 1, including 2′-α-fluoro (3), 2′-β-fluoro (4) and 2′,2′-difluoro (5) BCNA derivatives (Figure 2). Only compound 4 showed full retention of antiviral activity, whereas 2′-α-fluoro and 2′,2′-difluoro BCNA derivatives showed a considerable loss of activity, approximately 80-fold and 150-fold, respectively, compared with the parent compound. All compounds showed no activity against VZV-TK-deficient strains, supporting previous data showing that they need to be phosphorylated by this viral kinase to exert antiviral activity [21].

With regard to the inhibitory activity versus the VZV-TK-catalysed phosphorylation of thymidine, the α-fluoro derivative (3) retains low inhibitory activity, comparable to the parent drug. The β-fluoro derivative (4) showed a loss of potency (10-fold), whereas the 2′,2′-difluoro derivative (5) showed an inhibitory activity midway between the other two compounds and was approximately fourfold less potent than the parent compound [21].

It is interesting to note that, despite being a good competitive inhibitor of the VZV-TK-catalysed thymidine phosphorylation (that is, an alternative substrate), compound 3 did not show the same potency in the antiviral assay. This aspect has already been noted in other BCNA derivatives and points to different structure–activity relationships for the activating (phosphorylation) enzyme and their eventual antiviral target protein [22]. Moreover, compound 4 is thus far the only sugar-modified derivative of Cf1743, which fully retains the antiviral activity of the parent compound.

Figure 1.

Structures of bicyclic nucleoside analogue lead compound (1) and its prodrug FV100 (2)

Figure 2.

Structure of the 2′-fluoro derivatives of compound 1

It has been reported that the conformation of the sugar moiety (north or south) is fundamental for the interaction with the active site of target enzymes, such as kinases and DNA polymerases (viral or cellular), and that usually one conformation is preferred to the other [23]. Sugars are usually in dynamic equilibrium with both conformations, as the energy barrier is quite low. To block the conformation, either to the north or to the south conformation, locked nucleoside analogues (LNAs) have been designed [24]. According to these studies, which used herpes simplex virus (HSV)-TK, it appears that during the first step of phosphorylation, the LNA in the north conformation is a weak substrate for HSV-TK, whereas the one in the south does not bind. However, during the second step of phosphorylation, which is mediated by HSV-TK, it has been found that the south conformer is a better substrate than the north. During the incorporation of both conformers into the DNA, the north was preferred to the south. This has also been demonstrated by antiviral testing, wherein the north conformer was found to be active and the south conformer was inactive [24].

In order to further investigate the results obtained with these compounds, molecular modelling studies and the synthesis of novel phosphoramidate ProTides of compounds 3, 4 and 5 were performed in the present study.

Methods

Solvents and reagents

The following anhydrous solvents were bought from Sigma–Aldrich Co. (St Louis, MO, USA) with a sure seal stopper: dichloromethane (DCM), diethyl ether, N-methylimidazole (NMI), pyridine, tetrahydrofuran (THF) and triethylamine (TEA). All reagents that were commercially available were used without further purification.

Thin-layer chromatography

Pre-coated aluminium-backed plates (60 F₂₅₄, 0.2 mm thickness; Merck, Darmsted, Germany) were visualized under both short- and long-wave ultraviolet light (254 nm and 366 nm, respectively). Preparative thin-layer chromatography (TLC) plates (20×20 cm, 500–2,000 μm) were purchased from Merck.

Column chromatography

Column chromatography (CC) processes were carried out using silica gel supplied by Fisher Scientific (Hampton, NH, USA; 60 A, 35–70 μm). Glass columns were slurry packed using the appropriate eluent and samples were applied either as a concentrated solution in the same eluent or preadsorbed on silica gel.

HPLC

Analytical and semi-preparative HPLC were conducted using Varian ProStar (LC Work Station, Varian ProStar 335 Diode Array Detector, Varian 701 HPLC Fraction Collector and ProStar 210 Solvent Delivery Systems; Varian, Inc., Palo Alto, CA, USA), with Varian Polaris C18-A (10 μm) as an analytic column and Varian Polaris C18-A (10 μm) as a semi-preparative column. The software used was the Galaxie Chromatography Data System.

Nuclear magnetic resonance

¹H-nuclear magnetic resonance (NMR; 500 MHz), ¹³C-NMR (125 MHz), ³¹P-NMR (202 MHz) and ¹⁹F-NMR (471 MHz) were recorded on a Bruker Avance 500 MHz Spectrometer (Bruker Instruments, Inc., Billerica, MA, USA) at 25°C. Spectra were calibrated to the residual signal of the deuterated solvent used. Chemical shifts are given in parts per million and coupling constants (J) in Hertz.

The following abbreviations are used in the assignment of NMR signals: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad singlet (bs), doublet of doublet (dd) and doublet of triplet (dt).

Mass spectroscopy

High-resolution mass spectroscopy (MS) was performed as a service by Cardiff University (Cardiff, UK) using electrospray.

Elemental analyses

Elemental analysis (CHN) microanalyses were performed as a service by Medac, Ltd (Surrey, UK).

Standard procedure A: Synthesis of phosphorochloridates

To a stirred solution of the appropriate aryl dichlorophosphate (1.00 mol/eq) and the appropriate amino acid ester salt (1.00 mol/eq) in anhydrous DCM, anhydrous TEA (2.00 mol/eq) was added dropwise at −78°C under an argon atmosphere. Following the addition of TEA, the reaction mixture was stirred at −78°C for 0.5–1 h, then at room temperature (rt) for 2–3.5 h. Formation of the desired compound was monitored by ³¹P-NMR. After this period, the solvent was removed under reduced pressure and the residue triturated with anhydrous diethyl ether. The precipitate was filtered under nitrogen and the solution was concentrated to produce an oil. Most of the aryl phosphorochloridates synthesized were purified by flash CC (eluting with ethyl acetate/petroleum ether =60/40).

Standard procedure B: Synthesis of phosphoramidates

To a stirring solution of the appropriate nucleoside (1.00 mol/eq) and the appropriate phosphorochloridate (8.00–10.00 mol/eq) in anhydrous THF, NMI (8.00–13.00 mol/eq) was added dropwise and the reaction was stirred at rt for 1 day. After this period, the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with water (twice) and with 0.5 N HCl (twice). The organic phase was dried over MgSO₄, filtered, reduced to dryness, and the crude product was purified by column chromatography eluting with DCM/MeOH in different proportions.

Synthesis of 5-iodo-2′-deoxy-2′-α-fluorouridine (7)

To a solution of 2′-α-fluoro-2′deoxyuridine (6; 2.00 g, 8.12 mmol) in anhydrous acetonitrile (50 ml), iodine (1.24 g, 4.87 mmol) and ceric ammonium nitrate (CAN; 2.23 g, 4.06 mmol) were added, and the reaction mixture was stirred under reflux for 1 h. After this period, the reaction mixture was quenched with a saturated solution of Na₂S₂O₃ and then concentrated. The residue was dissolved in ethyl acetate and washed with brine (twice). The organic phase was dried over MgSO₄, concentrated to give a pale yellow solid (79%, 2.40 g). ¹⁹F-NMR (DMSO, 471 MHz): δ −202.14. ¹H-NMR (DMSO, 500 MHz): δ 11.71 (1H, s, NH), 8.52 (1H, s, H-6), 5.86 (1H, d, J=16.9, H-1′), 5.58 (1H, d, J=6.3, 3′-OH), 5.36 (1H, s, 5′-OH), 5.08 (0.5H, s, H-2′), 4.98 (0.5H, s, H-2′), 4.17 (1H, d, H-3′), 3.89 (1H, d, J=7.5, H-4′), 3.81 (1H, d, J=10.5, H-5′), 3.60 (1H, d, J=10.5, H-5′). ¹³C-NMR (DMSO, 126 MHz): δ 58.50 (C-5′), 66.73 (d, J_C–F=16.3, C-3′), 69.04 (C-5), 83.00 (C-4′), 87.39 (d, J_C–F=34.1, C-1′), 95.12 (d, J_C–F=184.3, C-2′), 144.66 (C-6), 149.94 (C-2), 160.53 (C-4).

Synthesis of 3-(2′-α-fluoro-2′-deoxy-β-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (3)

To a solution of 7 (1.50 g, 4.03 mmol) in anhydrous dimethyl formamide (DMF; 20 ml) 4-n-pentylphenylacetylene (2.36 ml, 12.09 mmol), tetrakis (0.47 g, 0.40 mmol), copper (I) iodide (0.15 g, 0.81 mmol) and DIPEA (1.40 ml, 8.06 mmol) were added, and the reaction mixture was stirred at rt under an argon atmosphere overnight. After this period, copper (I) iodide (0.15 g, 0.81 mmol) and anhydrous TEA (20 ml) were added and the reaction mixture was stirred at 85°C for 7.5 h. The solvent was then removed in vacuo, and the resulting residue was triturated with methanol, filtered and washed with methanol and DCM to obtain a black solid (pure by NMR). The organic phase was concentrated and the residue purified by CC, eluting with DCM/MeOH=96/4. The compound was obtained as a dark solid, which was combined with the previous precipitate and filtered through a silica gel column, eluting with DCM/MeOH=96/4, to give a black solid. The solid obtained was washed with acetone, filtered and the solid was further washed with petroleum ether to give a white-grey solid (42%, 0.70 g). ¹⁹F-NMR (DMSO, 471 MHz): δ −201.19. ¹H-NMR (DMSO, 500 MHz): δ 8.94 (1H, s, H-4), 7.75 (2H, d, J=8.1, H_a), 7.32 (2H, d, J=8.1, H_b), 7.22 (1H, s, H-5), 6.02 (1H, d, J=17.0, H-1′), 5.60 (1H, d, J=6.6, 3′-OH), 5.42 (1H, t, J=4.9, 5′-OH), 5.00 (1H, dd, J=3.7, J_H-F=52.6, H-2′), 4.26–4.20 (1H, m, H-3′), 3.95 (1H, dd, J=12.4, 2.9, H-4′), 3.77–3.67 (2H, m, H-5′), 2.61 (2H, t, J=7.6, α-CH₂), 1.65–1.61 (2H, m, β-CH₂), 1.42–1.18 (4H, m, γ-CH₂, δ-CH₂), 0.86 (3H, t, J=6.9, ω-CH₃). ¹³C-NMR (DMSO, 126 MHz): δ 13.84 (CH₃), 21.88, 30.33, 30.79, 34.88 (C₄H₈), 58.42 (C-5′), 66.31 (d, J_C–F=16.3, C-3′), 83.04 (C-4′), 89.54 (d, J_C–F=34.0, C-1′), 94.16 (d, J_C–F=185.7, C-2′), 98.56 (C-5), 107.19 (C-4a), 124.58 (C-H_b), 125.77 (ipso-C), 128.96 (C-H_a), 137.77 (C-4), 144.14 (para-C), 153.69 (C-6), 154.12 (C-2), 171.27 (C-7a). EI MS=416.1738 (M⁺). Anal. Calcd for C₂₂H₂₅FN₂O₅·0.5H₂O: C, 62.11; H, 6.16; N, 6.58. Found: C, 61.73; H, 6.15; N, 6.41.

Synthesis of 2-deoxy-1-α-bromo-2-β-fluoro-3,5-di-O-benzoyl-d-ribofuranose (9)

To a solution of 2-β-fluoro-2-deoxy-1,3,5-tribenzoylribose (8; 2.00 g, 4.31 mmol) in anhydrous DCM (9 ml), a 33% by weight solution of HBr in acetic acid (1.54 ml, 9.04 mmol) was added dropwise under an argon atmosphere, and the reaction mixture was stirred at rt overnight. After this period, a 33% by weight solution of HBr in acetic acid (0.77 ml, 4.50 mmol) was added and the reaction mixture was stirred at rt for a further 7 h. Next, the reaction mixture was quenched with saturated solution of NaHCO₃. The organic phase was washed with a saturated solution of NaHCO₃ (twice), dried over MgSO₄, then concentrated to give a colourless oil (95%, 1.73g). ¹⁹F-NMR (CDCl₃, 471 MHz): δ −165.93. ¹H-NMR (CDCl₃, 500 MHz): δ 8.18–8.04 (4H, m, Bz), 7.69–7.43 (6H, m, Bz), 6.66 (1H, d, J=12.2, H-1′), 5.57 (1H, dd, J=3.2, J_H-F=22.0, H-2′), 4.87–4.72 (4H, m, H-3′, H-4′, H-5′).

Synthesis of 2,4-bis-O-(trimethylsilyl)-5-iodouracil (11)

To a suspension of 10 (0.49 g, 2.04 mmol) in anhydrous acetonitrile (10 ml), ammonium sulphate (0.027 g, 0.2 mmol) and hexamethyldisilazane (0.48 ml, 2.30 mmol) were added, and the reaction mixture was stirred at reflux for 22 h. After this period, hexamethyldisilazane (0.96 ml, 4.08 mmol) was added, and the reaction mixture was stirred at reflux for a further 4 h and then concentrated. The crude product was used in the next step without purification.

Synthesis of 1-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-D-arabino-furanosyl)-5-iodouracil (12)

To a solution of 9 (3.50 g, 8.27 mmol) in DCM (65 ml) and 11 (8.27 mmol) in acetonitrile (15 ml), NaI (0.99 g, 6.62 mmol) was added, and the reaction mixture was stirred at rt for 6 days. After this period, the suspension was filtered and the solid was washed with water and DCM to give the desired compound as a white solid (51%, 2.45 g). ¹⁹F-NMR (DMSO, 471 MHz): δ −199.13. ¹H-NMR (DMSO, 500 MHz): δ 8.08–7.99 (5H, m, H-6, Bz), 7.74–7.66 (2H, m, Bz), 7.60–7.52 (4H, m, Bz), 6.31 (1H, dd, J=3.7, J_H-F=19.4, H-1′), 5.71 (1H, ddd, J=4.8, 1.3, J_H-F=20.3, H-3′), 5.54 (1H, ddd, J=3.8, 1.4, J_H-F=50.7, H-2′), 4.81–4.71 (2H, m, H-5′), 4.63 (1H, dd, J=8.3, 4.3, H-4′).

Synthesis of 5-iodo-2′-β-fluoro-2′-deoxyuridine (13)

To a stirring solution of 12 (2.40 g, 4.14 mmol) in anhydrous methanol (60 ml), sodium methoxide (0.49 g, 9.70 mmol) was added, and the reaction mixture was stirred at rt for 1 h. After this period, the reaction was neutralized with amberlite, filtered and concentrated to give the desired product, which was used in the following step without further purification. ¹⁹F-NMR (DMSO, 471 MHz): δ −198.65. ¹H-NMR (DMSO, 500 MHz): δ 8.21 (1H, s, H-6), 6.08 (1H, dd, J=4.5, J_H-F=13.9, H-1′), 5.93 (1H, bs, 3′-OH), 5.27–5.25 (1H, m, 5′-OH), 5.14–5.11 (1H, m, H-2′), 5.04–4.99 (1H, m, H-2′), 4.29–4.19 (1H, m, H-3′), 3.81–3.78 (1H, m, H-4′), 3.71–3.65 (1H, m, H-5′), 3.61–3.56 (1H, m, H-5′).

Synthesis of 3-(2′-α-fluoro-2′-deoxy-β-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (4)

To a solution of 13 (1.54 g, 4.14 mmol) in anhydrous DMF (20 ml), 4-n-pentylphenylacetylene (2.40 ml, 12.41 mmol), tetrakis (0.48 g, 0.41 mmol), copper (I) iodide (0.16 g, 0.83 mmol) and anhydrous DIPEA (1.44 ml, 8.27 mmol) were added, and the reaction mixture was stirred at rt under an argon atmosphere overnight. After this period, copper (I) iodide (0.16 g, 0.83 mmol) and anhydrous TEA (20ml) were added, and the reaction mixture was stirred at 85°C for 8 h. The solvent was then removed in vacuo and the residue was triturated with DCM, stirred at rt for 2 h, and then filtered and washed with DCM to give a white solid (45%, 0.77 g). A sample was filtered through silica gel for biological testing. ¹⁹F-NMR (DMSO, 471 MHz): δ −197.85. ¹H-NMR (DMSO, 500 MHz): δ 8.73 (1H, s, H-4), 7.74 (2H, d, J=8.3, Ph), 7.33 (2H, d, J=8.3, Ph), 7.22 (1H, s, H-5), 6.25, 6.22 (1H, 2d, J=3.7, J_H-F=17.1, H-1′), 6.07 (1H, d, J=4.6, 3′-OH), 5.28 (1H, t, J=5.8, 5′-OH), 5.24, 5.14 (1H, 2dd, J=3.6, 2.3, J_H-F=52.0, H-2′), 4.29, 4.26 (1H, 2dd, J=6.1, 4.3, J_H-F=18.2, H-3′), 3.99 (1H, q, H-4′), 3.71–3.63 (2H, m, H-5′), 2.62 (2H, t, α-CH₂), 1.59 (2H, quintet, β-CH₂), 1.35–1.24 (4H, m, γ-CH₂, δ-CH₂), 0.86 (3H, t, CH₃). ¹³C-NMR (DMSO, 126 MHz): δ 13.85 (CH₃), 21.87 (CH₂), 30.32 (β-CH₂), 30.78 (CH₂), 34.87 (α-CH₂), 60.13 (C-5′), 73.08 (d, J_C-F=24.5 Hz, C-3′), 85.22 (C-4′), 86.15 (d, J_C-F=16.6 Hz, C-1′), 94.66 (d, J_C-F=191.5 Hz, C-2′) 98.60 (C-5), 107.14 (C-4a), 124.61 (Ph), 125.70 (ipso-C), 128.99 (Ph), 138.70 (C-4), 144.20 (para-C), 153.47 (C-6), 154.20 (C-2), 171.30 (C-7a). EI MS=416.1749 (M⁺). HPLC=H₂O/AcCN from 100/0 to 0/100 in 30 min = retention time 23.81 min.

Synthesis of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate (15)

A solution of 14 (5.0 g, 0.013 mmol) in anhydrous THF (40 ml) and anhydrous diethyl ether (10 ml) was cooled to −78°C under an argon atmosphere and lithium tri(tert-butoxy)aluminium hydride (14.58 ml, 1.0 M in THF) was added dropwise. The reaction mixture was stirred for 1 h at −78°C and quenched by the slow addition of methanol (3.2 ml). The reaction mixture was allowed to warm to rt and then ethyl acetate (162 ml) was added. The organic phase was washed with equal volumes of saturated NaHCO₃ solution and brine, and the organic layer was dried over Na₂SO₄ and concentrated to give a thick oil as a mixture of anomers (84%, 4.14 g). ¹⁹F-NMR (CDCl₃, 471 MHz): δ −108.97, −109.50, −123.19, −123.70, −124.93, −125.46. ¹H-NMR of major (approximately 55%) anomer (CDCl₃, 500 MHZ): δ 8.14–7.99 (4H, m, Bz), 7.66–7.55 (2H, m, Bz), 7.51–7.39 (4H, m, Bz), 5.81–5.73 (1H, m, H-3), 5.52–5.49 (1H, m, H-1), 4.82–4.58 (3H, m, H-4, H-5), 3.66 (1H, s, OH). ¹H-NMR of minor (approximately 45%) anomer (CDCl₃, 500 MHZ): δ 8.14–7.99 (4H, m, Bz), 7.66–7.55 (2H, m, Bz), 7.51–7.39 (4H, m, Bz), 5.55–5.49 (1H, m, H-3), 5.40–5.35 (1H, m, H-1), 4.77–4.60 (2H, m, H-5), 4.51–4.45 (1H, m, H-4), 3.93 (1H, s, OH).

Synthesis of 2-deoxy-D-erythro-2,2-difluoro-ribofuranose-3,5-dibenzoate-1-methanesulfonate (16)

A solution of 15 (4.14 g, 10.9 mmol) in anhydrous DCM (52 ml) and anhydrous TEA (2.4 ml) was cooled to 0°C and methane sulfonylchloride (1.23 ml, 15.8 mmol) was added dropwise. The reaction was stirred at rt for 18 h. After this period, the reaction mixture was partitioned between DCM (140 ml) and a saturated solution of NaHCO₃ (56 ml). The organic phase was dried over Na₂SO₄ and concentrated to give an oil as a mixture of anomers (quantitative, 5.03 g). ¹⁹F-NMR (CDCl₃, 471 MHz): δ −107.70, −108.22, −120.65, −121.17, −122.21, −122.73, −123.76, −124.45. ¹H-NMR of major (approximately 60%) anomer (CDCl₃, 500 MHZ): δ 8.13–8.04 (4H, m, Bz), 7.65–7.54 (2H, m, Bz), 7.50–7.41 (4H, m, Bz), 6.20–6.15 (1H, d, J=5.62 Hz, H-1), 5.65–5.58 (1H, dd, J₁=4.25 Hz, J₂=16.45 Hz, H-3), 4.93–4.89 (1H, m, H-4), 4.81–4.61 (2H, m, H-5), 3.17 (3H, s, CH₃). ¹H-NMR of minor (approximately 40%) anomer (CDCl₃, 500 MHZ): δ 8.13–8.04 (4H, m, Bz), 7.65–7.54 (2H, m, Bz), 7.50–7.41 (4H, m, Bz), 6.11–6.07 (1H, d, J=6.37 Hz, H-1), 6.03–5.94 (1H, m, H-3), 4.81–4.61 (3H, m, H-4, H-5), 3.03 (3H, s, CH₃).

Synthesis of 1-(3,5-Di-O-benzoyl-2-deoxy-2,2-difluoro-β-D-erythro-pentofuranos-1-yl)-5-iodouracil (17)

Compound 10 (3.61 g, 15.2 mmol) was treated with an excess of hexamethyldisilazane (100 ml) in the presence of (NH₄)₂SO₄ (0.10 g, 0.76 mmol) under an argon atmosphere and refluxed at 125–130°C for 4 h. The solvent was removed under reduced pressure and the resulting syrup was dissolved in anhydrous dichloroethane (57 ml). A solution of 16 (3.46 g, 7.59 mmol) in anhydrous dichloroethane (86 ml) was added. The reaction mixture was stirred for 10 min, then trimethylsilyl trifluoromethanesulfonate (2.95 ml, 16.30 mmol) was added slowly and the reaction mixture was refluxed at 90–100°C for 10 h. The reaction mixture was cooled to rt and washed with equal volumes of saturated solution of NaHCO₃ and brine. The β-anomer was obtained by precipitation from the organic solvent and washed with methanol to remove traces of α-anomer as a white solid (1.00 g, 22%). ¹⁹F-NMR (DMSO, 471 MHz): δ −111.37, −111.90, −114.17 (broad). ¹H-NMR (DMSO, 500 MHZ): δ 12.03 (1H, s, NH), 8.17 (1H, s, H-6), 8.08–7.94 (4H, m, Bz), 7.77–7.63 (2H, m, Bz), 7.61–7.46 (4H, m, Bz), 6.36 (1H, t, J=9.06 Hz, H-1′), 5.93–5.84 (1H, m, H-3′), 4.83–4.73 (3H, m, H-4′, H-5′).

Synthesis of 1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranos-1-yl)-5-iodouracil (18)

To a stirring solution of 17 (0.93 g, 1.56 mmol) in anhydrous methanol (30 ml), sodium methoxide (0.25 g, 4.67 mmol) was added, and the reaction mixture was stirred at rt overnight. The reaction was neutralized with amberlite, filtered and concentrated. The residue was purified by CC, eluting with chloroform/methanol 90/10 to give a white solid (0.56 g, 92%). ¹⁹F-NMR (DMSO, 471 MHz): δ −117.16. ¹H-NMR (DMSO, 500 MHZ): δ 11.89 (1H, s, NH), 8.37 (1H, s, H-6), 6.30 (1H, d, J=6.51 Hz, 3′-OH), 6.02 (1H, t, J=6.78 Hz, H-1′), 5.45 (1H, m, 5′-OH), 4.29–4.17 (1H, m, H-3′), 3.91–3.84 (1H, m, H-4′), 3.84–3.60 (2H, 2m, H-5′).

Synthesis of 3-(2′-difluoro-2′-deoxy-β-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (5)

To a solution of 18 (0.18 g, 0.47 mmol) in anhydrous DMF (5 ml), 4-n-pentylphenylacetylene (0.27 ml, 1.41 mmol), tetrakis (0.055 g, 0.047 mmol), copper (I) iodide (0.018 g, 0.047 mmol) and DIPEA (0.16 ml, 0.94 mmol) were added, and the reaction mixture was stirred at rt under an argon atmosphere overnight. After this period, copper (I) iodide (0.018 g, 0.047 mmol) and anhydrous TEA (5 ml) were added and the reaction mixture was stirred at 85°C for 7.5 h. The solvent was then removed in vacuo and the residue was purified by CC, with gradient elution of DCM, then DCM/MeOH=98/2, then 96/4. The compound was obtained as a brown-dark solid, which was triturated with acetone and petroleum ether, then filtered. The solid was washed with acetone and petroleum ether to give a white-pale-yellow solid (60%, 0.075 g). ¹⁹F-NMR (DMSO, 471 MHz): δ −116.84. ¹H-NMR (DMSO, 500 MHz): δ 8.75 (1H, s, H-4), 7.76 (2H, d, J=8.2, H_a), 7.34 (2H, d, J=8.2, H_b), 7.23 (1H, s, H-5), 6.35 (1H, d, J=6.5, H-1′), 6.33–6.30 (1H, m, 3′-OH), 5.43 (1H, t, J=5.3, 5′-OH), 4.36–4.18 (1H, m, H-3′), 3.99–3.95 (1H, m, H-4′), 3.91–3.85 (1H, m, H-5′), 3.75–3.69 (1H, m, H-5′), 2.63 (2H, t, J=7.6, α-CH₂), 1.68–1.52 (2H, m, β-CH₂), 1.40–1.21 (4H, m, γ-CH₂, δ-CH₂), 0.87 (3H, t, J=7.0, CH₃). ¹³C-NMR (DMSO, 126 MHz): δ 13.85 (CH₃), 21.88, 30.32, 30.79, 34.90 (C₄H₈), 58.62 (C-5′), 68.14 (t, J_C–F=22.4, C-3′), 81.11 (C-4′), 85.14 (t, J_C–F=31.2, C-1′), 98.37 (C-5), 107.94 (C-4a), 120.84, 122.91 (C-2′), 124.75 (C-H_b), 125.56 (ipso-C), 129.01 (C-H_a), 137.26 (C-4), 144.44 (para-C), 153.68 (C-6), 154.87 (C-2), 171.53 (C-7a). EI MS=435.1731 (M+H). Anal. Calcd for C₂₂H₂₄F₂N₂O₅·0.5H₂O: C, 59.59; H, 5.68; N, 6.32. Found: C, 59.38; H, 5.59; N, 6.25.

Synthesis of phenyl-(methoxy-L-alaninyl)-phosphorochloridate (21a)

Compound 21a was prepared according to standard procedure A, from phenyldichlorophosphate (19; 2.24 ml, 15.00 mmol), L-alanine methyl ester HCl (20a; 2.09 g, 15.00 mmol), anhydrous TEA (4.20 ml, 30.00 mmol) and anhydrous DCM (80 ml). The reaction mixture was stirred at −78°C for 30 min, then at rt for 2.5 h. The crude product was purified by CC, eluting with ethyl acetate/hexane =6/4, to give an oil (81%, 3.35 g). ³¹P-NMR (CDCl₃, 202 MHz): δ 7.95, 7.66. ¹H-NMR (CDCl₃, 500 MHz): δ 7.32–7.15 (5H, m, PhO), 4.42–4.34 (1H, m, NH), 4.17–4.08 (H, m, CHNH), 3.72, 3.70 (3H, 2s, CH₃O), 1.45–1.43 (3H, m, CHCH₃).

Synthesis of phenyl-(benzoxy-L-alaninyl)-phosphorochloridate (21b)

Compound 21b was prepared according to standard procedure A, using 19 (0.30 ml, 2.00 mmol), L-alanine benzyl ester tosylate (20b; 0.43 g, 2.00 mmol) and anhydrous TEA (0.56 ml, 4.00 mmol) in anhydrous DCM (15 ml). The reaction mixture was stirred at −78°C for 30 min, then at rt for 3.5 h. The crude product was obtained as an oil (87%, 0.62 g). ³¹P-NMR (CDCl₃, 202 MHz): δ 7.86, 7.52. ¹H-NMR (CDCl₃, 500 MHz): δ 7.33–7.28 (10H, m, PhO, OCH₂Ph), 5.15–5.13 (2H, m, OCH2Ph), 4.18–4.13 (1H, m, CHNH), 1.46–1.44 (3H, m, CH₃).

Synthesis of phenyl-(methoxy-glycinyl)-phosphorochloridate (21c)

Compound 21c was prepared according to standard procedure A, from 19 (2.24 ml, 15.00 mmol), glycine methyl ester HCl (20c; 1.88 g, 15.00 mmol), anhydrous TEA (4.20 ml, 30.00 mmol) and anhydrous DCM (80 ml). The reaction mixture was stirred at −78°C for 30 min, then at rt for 2 h. The crude product was obtained as an oil (93%, 3.70 g) and used in the next step without further purification. ³¹P-NMR (CDCl₃, 202 MHz): δ 9.03. ¹H-NMR (CDCl₃, 500 MHz): δ 7.28–7.12 (5H, m, PhO), 4.43 (1H, bs, NHCH₂), 3.84 (2H, d, NHCH₂), 3.72 (3H, s, OCH₃).

Synthesis of 3-(2′-α-fluoro-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(methoxy-l-alaninyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (22a)

Compound 22a was prepared according to standard procedure B, from 4 (0.20 g, 0.48 mmol) in anhydrous THF (5 ml) and anhydrous pyridine (5 ml), 21a (0.67 g, 2.40 mmol) in anhydrous THF (2 ml)and NMI (0.19 ml, 2.40 mmol), and the reaction mixture was stirred at rt overnight. After this period, 21a (0.40 g, 1.44 mmol) in anhydrous THF (2 ml) and NMI (0.11 ml, 1.44 mmol) were added, and the reaction mixture was stirred at rt for a further 7 h. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of citric acid (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=98/2, then 97/3, to give a white solid that was further purified by preparative TLC (gradient elution of DCM/MeOH=99/1, then 98/2, then 96/4) to give a white solid (5%, 0.015 g). ³¹P-NMR (MeOD, 202 MHz): δ 3.90, 3.83. ¹⁹F-NMR (DMSO, 471 MHz): δ −203.40, −203.72. ¹H-NMR (MeOD, 500 MHz): δ 8.72, 8.71 (1H, 2s, H-4), 7.66–7.59 (2H, m, Ph), 7.43–7.17 (7H, m, Ph), 6.87, 6.75 (1H, 2s, H-5), 6.16 (0.5H, d, J=8.3, H-1′), 6.13 (0.5H, d, J=8.5, H-1′), 5.09 (1H, dd, J=3.1, J_H-F=52.1, H-2′), 4.79–4.64 (1H, m, H-5′), 4.57–4.42 (1H, m, H-4′), 4.40–4.27 (2H, m, H-3′, H-5′), 4.08–3.99 (1H, m, CHCH₃), 3.68 (3H, s, OCH₃), 2.66 (2H, t, J=7.7, α-CH₂), 1.73–1.60 (2H, m, β-CH₂), 1.44–1.28 (7H, m, γ-CH₂, δ-CH₂, CHCH₃), 0.93 (3H, t, J=7.0, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.35 (CH₃), 20.34 (d, J_C–P=7.4, CHCH₃), 20.53 (d, J_C–P=6.4, CHCH₃), 23.56, 32.14, 32.59, 36.74 (C₄H₈), 51.55, 51.77 (CHCH₃), 52.81, 52.85 (2s, OCH₃), 65.64 (d, J_C–P=2.8, C-5′), 65.68 (d, J_C–P=2.0, C-5′), 68.80 (d, J_C–P=16.5, C-4′), 68.93 (d, J_C–P=16.2, C-4′), 82.56, (d, J_C–P=8.3, C-3′) 82.63 (d, J_C–P=8.3, C-3′), 92.26, 92.54 (2d, J_C–F=34.8, C-1′), 94.30, 95.80 (2d, J_C–F=187.7, C-2′), 98.96, 98.99 (2s, C-5), 110.45 (C-4a), 121.42, 121.50, 121.51, 121.53, 121.55, 123.94, 125.90, 126.02, 126.40, 127.12, 127.14, 130.09, 130.17, 130.20, 130.59, 130.72, 130.93, 131.00, (PhO, Ph), 138.52, 138.54 (C-4), 146.49, 146.50 (2s, para-C), 152.00, 152.05 (2s, ‘ipso’ PhO), 156.59 (C-6), 157.45, 157.49 (C-2), 173.36 (C-7a), 175.31 (d, J_C–P=5.2, COOCH₃), 175.58 (d, J_C–P=4.5, COOCH₃). EI MS=658.2330 (M+H). HPLC = H₂O/AcCN from 100/0 to 0/100 in 30 min = retention time 20.43 min.

Synthesis of 3-(2′-α-fluoro-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(benzoxy-L-alaninyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (22b)

Compound 22b was prepared according to standard procedure B, from 4 (0.17 g, 0.40 mmol) in anhydrous THF (5 ml) and anhydrous pyridine (5 ml), 21b (0.70 g, 2.00 mmol) in anhydrous THF (2 ml) and NMI (0.16 ml, 2.00 mmol), and the reaction mixture was stirred at rt overnight. After this period, 21b (0.42 g, 1.20 mmol) in anhydrous THF (2 ml) and NMI (0.09 ml, 1.20 mmol) were added, and the reaction mixture was stirred at rt for a further 24 h. NMI (0.09 ml, 1.20 mmol) was then added and the reaction mixture stirred at rt for a further 7 h. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of citric acid (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=98/2, then 97/3, to give a white solid that was further purified by preparative TLC (gradient elution of DCM/MeOH=99/1, then 98/2, then 96/4) to give another white solid (7%, 0.020 g). ³¹P-NMR (MeOD, 202 MHz): δ 3.94, 3.71. ¹⁹F-NMR (DMSO, 471 MHz): δ −200.55, −200.94. ¹H-NMR (MeOD, 500 MHz): δ 8.70, 8.67 (1H, 2s, H-4), 7.70–7.60 (2H, m, Ph), 7.47–7.18 (12H, m, Ph, PhO, CH₂Ph), 6.86, 6.79 (1H, 2s, H-5), 6.14 (0.5H, d, J=9.3, H-1′), 6.11 (0.5H, d, J=10.0, H-1′), 5.18–5.06 (2H, m, CH₂Ph), 5.00–4.93 (1H, m, H-2′), 4.71–4.51 (1H, m, H-5′), 4.50–4.41 (1H, m, H-5′), 4.36–4.23 (2H, m, H-3′, H-4′), 4.16–4.02 (1H, m, CHCH₃), 2.68 (2H, t, J=7.4, α-CH₂), 1.73–1.55 (2H, m, β-CH₂), 1.48–1.27 (7H, m, γ-CH₂, δ-CH₂, CHCH₃), 0.93 (3H, t, J=6.6, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.35 (CH₃), 20.28 (d, J_C–P=7.7, CHCH₃), 20.51 (d, J_C–P=6.3, CHCH₃), 23.56, 32.15, 32.58, 36.74 (C₄H₈), 51.68, 51.94 (CHCH₃), 65.59 (d, J_C–P=5.0, C-5′), 65.80 (d, J_C–P=5.4, C-5′), 68.03 (CH₂Ph), 68.71, 68.85 (C-4′), 82.52, 82.58 (2s, C-3′), 92.23, 92.51 (2d, J_C–F=24.4, C-1′), 95.27, 95.77 (2d, J_C–F=188.7, C-2′), 99.00, 99.02 (2s, C-5), 110.46 (C-4a), 121.45, 121.49, 121.53, 121.56, 125.93, 126.37, 126.42, 127.14, 129.14, 129.18, 129.32, 129.57, 130.19, 130.93, 130.98, (PhO, CH₂Ph, Ph), 137.17 (‘ipso’ OCH₂Ph), 138.53 (C-4), 146.50 (para-C), 152.03 (‘ipso’ PhO), 156.58 (C-6), 157.45 (C-2), 173.34 (C-7a), 174.95 (COOCH₃). EI MS=734.2616 (M+H) 756.2448 (M+Na). HPLC=H₂O/MeOH 20/80 isocratic = retention time 28.08, 30.03 min.

Synthesis of 3-(2′-α-fluoro-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(methoxy-glycinyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (22c)

Compound 22c was prepared according to standard procedure B, from 4 (0.20 g, 0.48 mmol) in anhydrous THF (10 ml), 21c (0.25 g, 0.96 mmol) in anhydrous THF (2 ml) and NMI (0.08 ml, 0.96 mmol), and the reaction mixture was stirred at rt overnight. After this period were added anhydrous pyridine (2 ml), 21c (0.25 g, 0.96 mmol) in anhydrous THF (2 ml) and NMI (0.08 ml, 0.96 mmol), and the reaction mixture was stirred at rt for a further 7 h. Next, 21c (0.38 g, 1.44 mmol) in anhydrous THF (2 ml) and NMI (0.11 ml, 1.44 mmol) were added, and the reaction mixture was stirred at rt for 16 h. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of citric acid (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=98/2, then 97/3, to give a white solid (15%, 0.046 g). ³¹P-NMR (MeOD, 202 MHz): δ 5.24, 4.95. ¹⁹F-NMR (MeOD, 471 MHz): δ −203.47, −203.56. ¹H-NMR (MeOD, 500 MHz): δ 8.71, 8.69 (1H, 2s, H-4), 7.61–7.56 (2H, m, Ph), 7.49–7.34 (2H, m, Ph), 7.34–7.17 (5H, m, Ph), 6.84, 6.73 (1H, 2s, H-5), 6.13 (1H, d, J=17.0, H-1′), 5.14, 5.04 (1H, 2t, J=3.8, J_H-F=52.0, H-2′), 4.78–4.68 (1H, m, H-5′), 4.59–4.47 (1H, m, H-4′), 4.41–4.28 (2H, m, H-3′, H-5′), 3.89–3.80 (2H, m, NHCH₂), 3.68 (3H, 2s, OCH₃), 2.64 (2H, t, J=7.7, α-CH₂), 1.71–1.59 (2H, m, β-CH₂), 1.46–1.28 (4H, m, γ-CH₂, δ-CH₂), 0.93 (3H, t, J=7.0, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.38 (CH₃), 23.57, 32.11, 32.62, 36.75 (C₄H₈), 43.76 (CH₂NH), 52.69, 52.71 (2s, OCH₃), 65.63 (d, J_C–P=5.0, C-5′), 65.72 (d, J_C–P=4.8, C-5′), 68.75 (d, J_C–P=11.7, C-4′), 68.89 (d, J_C–P=11.7, C-4′), 82.57, 82.64 (2s, C-3′), 92.17, 92.44 (2d, J_C–F=34.8, C-1′), 95.09 (d, J_C–F=187.8, C-2′), 98.98 (C-5), 110.42 (C-4a), 121.51, 121.55, 125.87, 125.90, 126.43, 127.08, 130.14, 130.97, 131.02 (PhO, Ph), 138.50, 138.56 (C-4), 146.41 (para-C), 152.00, 152.06 (2s, ‘ipso’ PhO), 156.77 (C-6), 157.35, 157.42 (C-2), 172.97 (d, J_C–P=4.5, COOCH₃), 173.03 (C-7a), 173.27 (d, J_C–P=1.8, COOCH₃). EI MS=644.2202 (M+H) and 667.2043 (M+Na). HPLC=H₂O/AcCN from 100/0 to 0/100 in 30 min = retention time 27.59 min, H₂O/MeOH 20/80 isocratic = retention time 5.88 min.

Synthesis of 3-(2′-β-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(methoxy-glycinyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (23)

Compound 23 was prepared according to standard procedure B, from 5 (0.20 g, 0.48 mmol) in anhydrous THF (10 ml) and anhydrous pyridine (2 ml), 21c (0.63 g, 2.40 mmol) in anhydrous THF (5 ml) and NMI (0.20 ml, 2.40 mmol), and the reaction mixture was stirred at rt overnight. After this period, 21c (0.63 g, 2.40 mmol) in anhydrous THF (5 ml) and NMI (0.20 ml, 2.40 mmol) were added, and the reaction mixture was stirred at rt for a further 24 h. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of HCl (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=98/2, then 97/3, to give another white solid, which was further purified by preparative TLC (gradient elution of DCM/MeOH=99/1, then 98/2, then 96/4) to give a white solid (4%, 0.012 g). ³¹P-NMR (MeOD, 202 MHz): δ 5.26, 5.15. ¹⁹F-NMR (DMSO, 471 MHz): δ −200.08. ¹H-NMR (MeOD, 500 MHz): δ 8.66, 8.64 (1H, 2s, H-4), 7.70–7.16 (9H, m, Ph, PhO), 6.88, 6.86 (1H, 2s, H-5), 6.41, 6.37 (1H, 2t, J=3.5, J_H-F=18.5, H-1′), 5.31–5.30 (0.5H, m, H-2′), 5.21–5.20 (0.5H, m, H-2′), 4.49–4.32 (4H, m, H-3′, H-4′, H-5′), 3.86–3.81 (2H, m, NHCH₂), 3.68 (3H, 2s, OCH₃), 2.67 (2H, t, J=8.3, α-CH₂), 1.70–1.64 (2H, m, β-CH₂), 1.42–1.28 (4H, m, γ-CH₂, δ-CH₂), 0.93 (3H, t, J=7.3, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.36 (CH₃), 23.56, 32.14, 32.59, 36.75 (C₄H₈), 43.70 (CH₂NH), 52.57, 52.66 (2s, OCH₃), 66.92 (C-5′), 75.25 (d, J_C–P=10.9, C-4′), 75.46 (d, J_C–P=10.6, C-4′), 85.24, 85.31 (2s, C-3′), 89.06 (d, J_C–F=13.7, C-1′), 89.16 (d, J_C–F=14.3, C-1′), 96.16 (d, J_C–F=194.4, C-2′), 98.89, 98.91 (C-5), 110.12 (C-4a), 121.56, 121.60, 121.64, 121.67, 121.70, 121.74, 125.78, 125.96, 126.21, 126.35, 127.17, 130.19, 130.66, 130.89, 130.90 (PhO, Ph), 139.75, 139.78 (C-4), 146.47 (para-C), 152.18 (2s, ‘ipso’ PhO), 156.46 (C-6), 157.46 (C-2), 172.94 (COOCH₃), 173.26 (C-7a), 173.28 (COOCH₃). EI MS=666.2001 (M+Na). HPLC = H₂O/AcCN from 95/5 to 0/100 in 30 min = retention time 28.60 min.

Synthesis of 3-(2′-di-fluoro-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(methoxy-L-alaninyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (24a) and (24b)

Compounds 24a and 24b were prepared according to standard procedure B from 6 (0.20 g, 0.46 mmol) in anhydrous THF (10 ml), 21a (0.64 g, 2.30 mmol) in anhydrous THF (5 ml) and NMI (0.18 ml, 2.30 mmol), and the reaction mixture was stirred at rt overnight. After this period, 21a (0.64 g, 2.30 mmol) in anhydrous THF (5 ml) and NMI (0.18 ml, 2.30 mmol) were added, and the reaction mixture was stirred at rt for a further 8 h. Next, NMI (0.09 ml, 1.20 mmol) was added and the reaction mixture stirred at rt for a further 24 h. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of HCl (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=100/0, then 98/2, then 97/3, to give a white solid that was further purified by preparative TLC (gradient elution of DCM/MeOH=99/1, then 98/2, then 96/4) to give another white solid as a mix of two diastereoisomers (0.005 g) and as a single diastereoisomer (0.004 g).

24a ³¹P-NMR (MeOD, 202 MHz): δ 3.98, 3.96. ¹⁹F-NMR (DMSO, 471 zMHz): δ −118.46, −118.83, −118.93, −119.44, −120.23. ¹H-NMR (MeOD, 500 MHz): δ 8.55, 8.51 (1H, 2s, H-4), 7.81–7.65 (2H, m, Ph), 7.48–7.16 (7H, m, Ph), 6.96, 6.88 (1H, 2s, H-5), 6.48–6.44 (1H, m, H-1′), 4.68–4.46 (2H, m, H-5′), 4.43–4.31 (1H, m, H-4′), 4.31–4.24 (1H, m, H-3′), 4.04–3.92 (1H, m, CHCH₃), 3.68, 3.67 (3H, 2s, OCH₃), 2.69 (2H, t, J=7.6, α-CH₂), 1.71–1.65 (2H, m, β-CH₂), 1.43–1.30 (7H, m, γ-CH₂, δ-CH₂, CHCH₃), 0.94 (3H, t, J=6.9, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.35 (CH₃), 21.00 (d, J_C–P=7.8, CHCH₃), 21.06 (d, J_C–P=7.0, CHCH₃), 23.55, 32.15, 32.57, 36.76 (C₄H₈), 50.04 (CHCH₃), 52.79 (OCH₃), 65.76 (C-5′), 69.82, 70.04 (2s, C-4′), 82.86, 82.92 (2s, C-3′), 94.47 (C-1′), 98.86, 98.89 (2s, C-5), 110.87 (C-4a), 121.32, 121.52, 121.56, 126.06, 126.09, 126.22, 126.44, 126.48, 127.01, 129.59, 129.83, 130.23, 130.40, 130.94, 131.03, 132.51, 132.64 (C-2′, PhO, Ph), 138.38 (C-4), 145.11, 146.70 (2s, para-C), 150.97 (‘ipso’ PhO), 156.66 (C-6), 158.01 (C-2), 173.56 (C-7a), 174.50 (COOCH₃). HPLC = H₂O/AcCN from 95/5 to 0/100 in 40 min = retention time 29.72, 33.48 min.

24b ³¹P-NMR (MeOD, 202 MHz): δ 3.89. ¹⁹F-NMR (DMSO, 471 MHz): δ −118.54. ¹H-NMR (MeOD, 500 MHz): δ 8.54 (1H, s, H-4), 7.71–7.69 (2H, m, Ph), 7.45–7.19 (7H, m, Ph), 6.83 (1H, 2s, H-5), 6.56–6.38 (1H, m, H-1′), 4.66–4.51 (1H, m, H-5′), 4.50–4.41 (1H, m, H-5′), 4.40–4.29 (1H, m, H-4′), 4.29–4.20 (1H, m, H-3′), 4.08–3.98 (1H, m, CHCH₃), 3.69 (3H, s, OCH₃), 2.69 (2H, t, J=7.7, α-CH₂), 1.73–1.58 (2H, m, β-CH₂), 1.46–1.26 (7H, m, γ-CH₂, δ-CH₂, CHCH₃), 0.94 (3H, t, J=7.0, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.35 (CH₃), 20.45 (d, J_C–P=6.6, CHCH₃), 23.56, 32.15, 32.58, 36.75 (C₄H₈), 50.04 (CHCH₃), 52.78 (OCH₃), 65.56 (C-5′), 68.87 (C-4′), 81.04 (C-3′), 94.47 (C-1′), 98.72 (C-5), 110.81 (C-4a), 121.44, 121.48, 121.53, 121.57, 126.05, 126.24, 127.00, 129.55, 129.83, 130.23, 130.82, 130.98, 132.63 (C-2′, PhO, Ph), 138.38 (C-4), 146.72 (para-C), 156.66 (C-6), 158.02 (C-2), 173.56 (C-7a), 174.50 (COOCH₃). HPLC=H₂O/AcCN from 95/5 to 0/100 in 40 min = retention time 34.41 min; H₂O/MeOH 20/80 isocratic =23.53.

Synthesis of 3-(2′-di-fluoro-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(benzoxy-L-alaninyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (24c)

Compound 24c was prepared according to standard procedure B from 6 (0.20 g, 0.46 mmol) in anhydrous THF (12 ml), 21b (0.81 g, 2.30 mmol) in anhydrous THF (5 ml) and NMI (0.18 ml, 2.30 mmol), and the reaction mixture was stirred at rt overnight. After this period, 21b (0.81 g, 2.30 mmol) in anhydrous THF (5 ml) and NMI (0.18 ml, 2.30 mmol) were added, and the reaction mixture was stirred at rt for a further 8 h. Next, NMI (0.09 ml, 1.20 mmol) was added and the reaction mixture was stirred at rt overnight. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of citric acid (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=100/0, then 98/2, then 96/4, to give a white solid that was further purified by preparative HPLC (isocratic MeOH/H₂O=80/20) to give another white solid (4%, 0.012 g). ³¹P-NMR (MeOD, 202 MHz): δ 3.99, 3.79. ¹⁹F-NMR (DMSO, 471 MHz): δ −117.93, −118.23, −118.44, −118.74, −119.61. ¹H-NMR (MeOD, 500 MHz): δ 8.52, 8.49 (1H, 2s, H-4), 7.66 (2H, d, J=7.9, Ph), 7.40–7.17 (12H, m, Ph, PhO, CH₂Ph), 6.85, 6.79 (1H, 2s, H-5), 6.44 (1H, dd, J=14.8, 7.4, H-1′), 5.21–5.10 (2H, m, CH₂Ph), 4.65–4.52 (1H, m, H-5′), 4.51–4.38 (1H, m, H-5′), 4.39–4.29 (1H, m, H-4′), 4.27–4.16 (1H, m, H-3′), 4.16–4.04 (1H, m, CHCH₃), 2.67 (2H, t, J=7.5, α-CH₂), 1.72–1.60 (2H, m, β-CH₂), 1.47–1.28 (7H, m, γ-CH₂, δ-CH₂, CHCH₃), 0.93 (3H, t, J=7.0, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.36 (CH₃), 20.26 (d, J_C–P=7.7, CHCH₃), 20.43 (d, J_C–P=6.9, CHCH₃), 23.56, 32.14, 32.58, 36.75 (C₄H₈), 51.71, 51.93 (CHCH₃), 65.55 (d, J_C–P=6.1, C-5′), 65.62 (d, J_C–P=5.8, C-5′), 68.02, 68.05 (2s, CH₂Ph), 70.75, 70.91 (2s, C-4′), 81.03 (C-3′), 87.68 (C-1′), 98.75, 98.78 (2s, C-5), 110.80 (C-4a), 121.51, 121.54, 121.55, 121.59, 126.05, 126.41, 126.98, 129.12, 129.20, 129.35, 129.58, 130.21, 130.91, 130.97, (C-2′, PhO, CH₂Ph, Ph), 137.17 (‘ipso’ OCH₂Ph), 138.25, 138.35 (2s, C-4), 146.68 (para-C), 152.07 (‘ipso’ PhO), 156.62 (C-6), 157.94 (C-2), 173.49 (C-7a), 174.66, 174.96 (COOCH₂Ph). EI MS=752.2555 (M+H), 774.2372 (M+Na). HPLC=H₂O/AcCN from 95/5 to 0/100 in 40 min = retention time 38.96 min; H₂O/MeOH 20/80 isocratic = retention time 33.48, 35.43 min.

Synthesis of 3-(2′-di-fluoro-2′-deoxy-β-D-ribofuranosyl-5-[phenyl-(methoxy-glycinyl)]-phosphate)-6-(4-n-pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (24d)

Compound 24d was prepared according to standard procedure B from 6 (0.20 g, 0.46 mmol) in anhydrous THF (10 ml), 21c (0.61 g, 2.30 mmol) in anhydrous THF (5 ml) and NMI (0.18 ml, 2.30 mmol), and the reaction mixture was stirred at rt overnight. After this period, 21c (0.61 g, 2.30 mmol) in anhydrous THF (5 ml) and NMI (0.18 ml, 2.30 mmol) were added, and the reaction mixture was stirred at rt for further 8 h. Next, NMI (0.09 ml, 1.20 mmol) was added, and the reaction mixture stirred at rt for a further 24 h. After this period, the solvent was removed and the residue dissolved in DCM. The organic phase was washed with a 0.5 M aqueous solution of HCl (twice) and water (twice), dried over MgSO₄ and concentrated. The residue was purified by CC gradient elution of DCM/MeOH=98/2, then 97/3, to give a white solid that was further purified by preparative TLC (gradient elution of DCM/MeOH =99/1, then 98/2, then 96/4) to give another white solid (14%, 0.042 g). ³¹P-NMR (MeOD, 202 MHz): δ 5.42, 4.92. ¹⁹F-NMR (DMSO, 471 MHz): δ −118.13, −118.65. ¹H-NMR (MeOD, 500 MHz): δ 8.55, 8.52 (1H, 2s, H-4), 7.70–7.59 (2H, m, Ph), 7.48–7.17 (7H, m, Ph, PhO), 6.88, 6.79 (1H, 2s, H-5), 6.45 (1H, d, J=7.0, H-1′), 4.73–4.63 (1H, m, H-5′), 4.60–4.48 (1H, m, H-5′), 4.47–4.30 (1H, m, H-4′), 4.30–4.23 (1H, m, H-3′), 3.89–3.82 (2H, m, NHCH₂), 3.70 (3H, 2s, OCH₃), 2.64 (2H, t, J=7.6, α-CH₂), 1.69–1.59 (2H, m, β-CH₂), 1.45–1.26 (4H, m, γ-CH₂, δ-CH₂), 0.92 (3H, t, J=7.0, CH₃). ¹³C-NMR (MeOD, 126 MHz): δ 14.39 (CH₃), 23.57, 32.10, 32.61, 36.76 (C₄H₈), 43.72 (CH₂NH), 52.72 (OCH₃), 65.55 (C-5′), 70.90 (C-4′), 80.97 (C-3′), 87.40, 87.52 (C-1′), 98.76, 98.79 (2s, C-5), 110.80, 110.83 (2s, C-4a), 121.53, 121.56, 121.60, 123.49, 126.01, 126.03, 126.44, 126.47, 126.92, 126.94, 129.57, 130.18, 130.86, 130.96, 131.00, 132.61 (C-2′, PhO, Ph), 138.32, 138.44 (C-4), 146.63 (para-C), 152.03, 152.09 (2s, ‘ipso’ PhO), 156.64 (C-6), 157.88, 157.94 (2s, C-2), 173.04, 173.08 (2s, C-7a), 173.47 (COOCH₃). EI MS=662.2073 (M+H), 684.1885 (M+Na). HPLC = H₂O/AcCN from 100/0 to 0/100 in 40 min = retention time 35.12 min; H₂O/MeOH 20/80 isocratic = retention time 21.44 min.

Procedures for the anti-VZV experiments in HEL cell cultures

The laboratory wild-type VZV strains Oka and Davis and the TK-deficient VZV strains 07/1 and YS were used. Confluent HEL cell cultures grown in 96-well microtitre plates were inoculated with VZV at an input of 20 plaque-forming units per well. After a 2 h incubation period, residual virus was removed and varying concentrations of the test compounds were added (in duplicate). Antiviral activity was expressed as the effective concentration required to reduce viral plaque formation after 5 days by 50% (EC₅₀) as compared with untreated controls. Cytotoxicity was expressed as the minimum cytotoxic concentration or the compound concentration that causes a microscopically detectable alteration of cell morphology.

Molecular modelling

All molecular modelling studies were performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 8 using Molecular Operating Environment 2008.10 (MOE; Chemical Computing Group, Inc., Montreal, QC, Canada) and Plants 1.0 [25]. Hydrogen atoms were added to the crystal structure of proteins and minimised with MOE until a gradient of 0.05 kcal/mol/Å was reached, using the MMFF94x forcefield. The partial charges were automatically calculated. Docking experiments were carried out using Plants 1.0 with the default parameters. The results were examined with MOE.

Results

Compound 3 was synthesized (Figure 3) starting from the commercially available 2′-fluoro-2′-deoxyuridine 6, which was selectively iodinated at the 5-position using iodine and CAN in acetonitrile under reflux giving 7 in good yield [26]. Compound 7 was then coupled with (4-pentylphenyl)acetylene under Sonogashira conditions to give the desired compound.

Compound 4 was synthesized (Figure 4), under the same conditions as shown previously, starting from the intermediate (13), which was prepared according to the procedure already described in the literature [27].

The commercially available compound 8 was converted to the 1-bromo derivative (9) using hydrobromic acid in acetic acid giving the pure compound without purification, which was then coupled in the presence of sodium iodide with the activated base (11), obtained by silylation of 5-iodouracil (10) using hexamethyldisilazane in the presence of ammonium sulphate. The desired β-fluoro BCNA anomer (12) was obtained by precipitation during the reaction to give a white solid. However, from the filtrate, another solid was obtained as a mixture of two anomers (α and β). The β-anomer was characterized by NMR, comparing the results with the data in the literature. The deprotection of the benzoyl group was performed using sodium methoxide in anhydrous methanol to give 13, which was used without further purification in the coupling step with (4-pentylphenyl)acetylene under standard conditions to give the desired compound 4.

Figure 3.

Synthesis of the 2′-α-fluoro derivative of compound 1

Figure 4.

Synthesis of the 2′-β-fluoro derivative of compound 1

Figure 5.

Synthesis of the 2′-gem-fluoro derivative of compound 1

The 2′,2′-difluoro BCNA derivative (5; Figure 5) was obtained by coupling under Sonogashira conditions starting from compound 18, which was prepared according to the literature [28]. The commercially available compound 14 was reduced using lithium tri(tert-butoxy)alluminium hydride to give compound 15 in good yield, which was then mesylated to 16 and coupled with the silylated 5-iodouracil to give a mixture of the α- and β-anomers 17. The desired β-anomer was obtained by precipitation from dichloroethane and the corresponding solid formed was filtered and washed with methanol to remove the trace of the α-anomer. The final deprotection was carried out using sodium methoxide in methanol to give 18 as a white solid.

The phosphorochloridates (21a–c) were synthesised (Figure 6) according to standard procedures by coupling the phenyl dichlorophosphate (19) with the appropriate amino acid ester salt (20a–c) using anhydrous TEA as a base to give the desired compounds as oils.

The synthesis of the phosphoramidates (Figure 7) was performed by coupling the appropriate nucleosides (3–5) with the appropriate phosphorochloridates (21a–c) using NMI as coupling reagent following the Van Boom procedure [29].

The coupling reaction proceeded well in the case of the glycinyl derivatives, for which all the compounds have been isolated. In the case of the β-fluoro BCNA derivative, only one compound, the glycinyl derivative, was isolated. By contrast, for the other two coupling reactions, it was not possible to isolate the desired compounds because the reaction progressed poorly, leaving starting material and only trace amount of final product on the TLC. A possible explanation for these results could be the conformation of the sugar of the β-fluoro BCNA derivative, which might be inaccessible for phosphorylation as supported by the computational studies previously reported, showing the difference in the sugar conformation between the β-fluoro BCNA and the other two derivatives.

Molecular modelling studies

In order to understand the differences in the inhibitory activity of the compounds versus VZV-TK for the fluoro BCNA derivatives, the affinity with the enzyme has been probed using docking techniques. Firstly, stochastic and systematic searches have been performed, with the aim to find the lowest energy conformation for these compounds. From these studies (Figure 8), it has been found that the north conformation of the sugar is slightly more stable for the α-fluoro derivative (3) and for the difluoro derivative (5) compared with the south conformation by 1.8 and 1.0 kcal, respectively. By contrast, for the β-fluoro derivative (4), the south conformation was more stable than the north conformation by 1.2 kcal. The lowest energy minimized structures of both north and south conformations for these compounds have been docked into VZV-TK (protein data bank 1OSN). In the case of compound 3 (Figure 9), the north conformation displayed a better docking compared with the south, because of the formation of a hydrogen bond between the fluorine and the hydroxyl of the tyrosine 59 of the enzyme, which was 2.78 Å in the case of the north conformation and 3.87 Å for the south conformation.

Figure 6.

General synthesis of phosphorochloridates

Figure 7.

General synthesis of the phosphoramidates of 2′-fluoro derivative of 1

Figure 8.

Lowest energy conformation for compounds 3, 4 and 5

Figure 9.

Varicella zoster virus thymidine kinase docking for compound 3

In the case of the β-fluoro BCNA derivative (4; Figure 10), the south conformation showed a better docking compared to the north. However, the fluorine in the β-position was not able to interact with the tyrosine 59 of the enzyme.

In the case of the difluoro BCNA derivative (5; Figure 11), both north and south conformations showed the same interaction with the active site of the enzyme. Indeed, the formation of the hydrogen bond between the fluorine and the hydroxyl of the tyrosine 59, with a distance of 2.82 Å for the north conformation and 2.52 Å for the south, was detected.

These studies support the results of the enzymatic experiment, which showed good affinity for compound 3 and poor affinity for 4, whereas 5 showed an intermediate affinity for the enzyme. These results are also in agreement with those obtained using the LNA, for which the north conformation is preferred to the south in the case of the first step of phosphorylation, whereas the south is preferred for the second phosphorylation. Finally, the results are in agreement with both enzymatic and biological results previously reported [21].

Figure 10.

Varicella zoster virus thymidine kinase docking for compound 4

Figure 11.

Varicella zoster virus thymidine kinase docking for compound 5

Biology

The synthesised compounds were previously evaluated for their ability to inhibit VZV-encoded TK (Table 1). Although compounds 1 and 3 were comparably inhibitory to VZV-TK-catalysed thymidine phosphorylation (50% inhibitory concentration 2.8–3.6 μM), 5 and 4 were less inhibitory (50% inhibitory concentration 12 and 37 μM, respectively). None of these compounds were inhibitory against HSV type-1 TK and cytosolic TK-1, attesting again to the high specificity of the BCNAs for VZV-TK.

Table 1.

Anti-VZV activity for the target compounds

	Antiviral activity, EC₅₀ (μM)
	TK-positive VZV		TK-negative VZV		Cytotoxicity, μM
Compound	YS strain	Oka strain	07/1 strain	YS strain	MCC	CC₅₀

22a	3.8	0.55	>5	>5	20	10
22b	7.4	0.69	≥20	11.1	50	39.3
22c	≥5	1.1	>5	>5	35	40.2
23	0.087	0.03	>20	9.9	50	33.9
24d	7.8	3.0	9.3	8.0	20	32.7
3	≥50	1.16	>50	>50	≥50	>100
4	0.03	0.0082	>50	>50	35	>100
5	14.9	8.7	>50	>50	>50	>100
1	0.014	0.006	>2	>2	>2	>100

CC₅₀, cytotoxic concentration required to reduce cell growth by 50%; EC₅₀, effective concentration required to reduce virus plaque formation by 50% (virus input was 20 plaque-forming units); MCC, minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology; TK, thymidine kinase; VZV, varicella zoster virus.

In addition, these compounds were evaluated for their ability to inhibit two different TK-positive VZV strains and two TK-negative VZV strains. Compound 1 and the parent fluoro derivatives 3, 4 and 5 are included as a reference. From Table 1, it is clear that the fluoro BCNA ProTide derivatives retain the same activity as their corresponding parent compounds. Whereas the β-fluoro BCNA (4; EC₅₀ 0.0082 μM) has an anti-VZV activity close to that of the prototype compound 1 (EC₅₀ 0.006 μM), its phosphoramidate derivative (23) had also a similar (or slightly higher) antiviral activity (EC₅₀ 0.03 μM). Although the α-fluoro (3) and difluoro (5) BCNAs were markedly less active (EC₅₀ 1.16 and 8.7 μM, respectively) than the prototype compound 1, the corresponding phosphoramidate prodrugs of 3 and 5 (that is, 22a–c and 23) had a comparable anti-VZV activity (EC₅₀ 0.55–1.1 μM and 3.0 μM, respectively). When evaluated against TK-deficient VZV strains, all the prodrugs showed some antiviral activity (EC₅₀ approximately 10 μM), but were clearly less potent compared with the TK-positive VZV strains. Interestingly, all the compounds showed a moderate cytostatic activity, and possibly the apparent antiviral activity found against the TK-deficient VZV strains might be related to this increased toxicity.

From these results it is clear that the phosphoramidate ProTide technology did not significantly improve the activity of the parent compounds. The decreased antiviral activity against TK-deficient VZV strains suggests that the 5′-monophosphate derivatives are not efficiently released intracellularly, or, alternatively, the further phosphorylation to the 5′-diphosphate (which can be carried out by the VZV-TK) does not occur because of the TK deficiency. In the latter case, it is assumed that the antivirally active BCNA metabolite is the 5′-diphosphate derivative or a metabolite derived thereof (that is, BCNA-5′-triphosphate).

Discussion

To summarize, molecular modelling studies using VZV-TK showed an agreement between the conformation of the sugar and the enzymatic results already presented, indicating that compound 3 (α-fluoro BCNA) is preferred over the other derivatives. The application of the phosphoramidate ProTide technology to the 2′-fluoro derivatives of the anti-VZV compounds was unsuccessful, in that none of these compounds showed an increase of the antiviral activity. The VZV-TK dependence of the BCNAs appears to confer a significant antiviral selectivity.

Footnotes

Acknowledgements

We thank Lizette van Berckelaer, Anita Camps, Lies Van den Heurck and Steven Carmans for their technical assistance and the Concerted Actions of the KU Leuven (GOA 10/014) for financial support.

JB and CM are coinventors of Cf1743. All other authors declare no competing interests.

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