Synthesis of 5,5-difluoro-5-phosphono-pent-2-en-1-yl nucleosides as potential antiviral agents

Abstract

A series of hitherto unknown acyclic 5,5-difluoro-5-phosphono-pent-2-en-1-yl-pyrimidines (9a, b, 13a, b), -purines (16a, b) and -(1,2,4)-triazolo-3-carboxamide (19) were successfully synthesized from (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene in a stereoselective manner. All the synthetized compounds were assayed for antiviral activity against various viruses, but were found to be neither active nor toxic.

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Introduction

Viruses are infectious agents that can replicate their genome within host cells. Many antiviral drugs are nucleoside or nucleotide analogs. Acyclic nucleoside phosphonates (ANPs)¹ are a class of nucleotide analogs, originally developed by A. Holy's group,² which exhibit a broad spectrum of antiviral activities. ANPs possess a common structure, a nucleobase attached to an aliphatic side chain containing a phosphonate moiety (C–P), and have an increased metabolic stability and resistance to chemical and biological degradation.³ Their activities are reliant on their diphosphorylation by NDP and NTP kinases, and further incorporation into the viral DNA where they can act as chain terminators. Three ANPs are in current clinical use for the treatment of serious viral infections, adefovir (PMEA), tenofovir [(R)-PMPA] and cidofovir [(S)-CDV] against hepatits B virus (HBV), human immunodeficiency virus (HIV) and cytomegalovirus (CMV), respectively, (Fig. 1).

Fig. 1 Structure of approved antiviral acyclic nucleoside phosphonates.

Due to drug-resistant viruses and emerging viruses, in an effort to identify new nucleoside inhibitors of viral enzymes, new generations of ANPs, including fluorinated ANPs, were prepared and evaluated for their antiviral activity.^4,5 Over the last decade, our laboratory has developed a new family of ANPs based on the (E)-but-2-enyl linker between the phosphonate moiety and the nucleobase. Several of them exhibited antiviral activity against DNA and RNA viruses in submicromolar concentrations.⁶ During our investigations, we have demonstrated that the N¹-[(E)-4-phosphono-but-2-en-1-yl]-thymine is a substrate of human TMPK and that the (E)-but-2-enyl moiety mimics the conformation of the C1′–O4′–C4′–C5′ atoms from the natural substrate, the thymidine 5′-monophosphate, (Fig. 2).⁶ However, unlike natural nucleotide, our molecule missed the oxygen of the phosphate group (e.g., –O–P). Thus, we turn our attention to the introduction of a gem-difluoromethylphosphonate moiety (e.g., –CF₂–P), which is isopolar and isosteric to the phosphate group.⁷

Fig. 2 Structure of newly synthesized 5,5-difluoro-5-phosphono-pent-2-en-1-yl nucleosides as potent mimics of dNMP.

In fact, due to specific properties of fluorine (high electronegativity, small steric size, hydrogen bond acceptor, …), its introduction into biologically active molecules could lead to major changes in their biological properties, such as reported by Halazy et al.⁸ for the 9-(5,5-difluoro-5-phosphonopentyl)-guanine, an inhibitor of purine nucleoside phosphorylase (PNP), a key enzyme in the purine metabolism.⁹ Therefore, based on these findings, it was interesting to design and synthesize a new type of acyclic nucleoside phosphonate, the 5,5-difluoro-5-phosphono-pent-2-en-1-yl-pyrimidines, purines and -triazole, and to evaluate their inhibitory activity against several viruses.

Results and discussion

Based on our previous work on the preparation of unsaturated acyclic nucleoside phosphonate using olefin cross-metathesis as key step,^10–12 we decided to utilize this reaction between the unsaturated gem-difluorophosphonate 3 and N¹-crotylated N³-protected thymine.¹⁰ The key intermediate 3 was synthesized from (diethoxyphosphinyl)difluoromethyl zinc bromide (2), allylic iodine, under CuBr catalysis, following the procedure introduced by Burton et al.^13,14 Compound 3 was then engaged in the reaction of cross metathesis in presence of N³-benzoyl-N¹-crotylthymine 4 with Grubbs–Nolan catalyst¹⁵ in dichloromethane. If cross-metathesis reactions were reported with fluorinated substrates,¹⁶ despite all our attempts and contrary to our results with non-fluorinated phosphonate derivatives, we never obtained the desired product (Scheme 1); this could be due to the strong electron-withdrawing effect of the 2-gem-difluoro group and to the low reactivity of both cross partners with the metal alkylidene complex.

Scheme 1 Reagents and conditions: (a) Zn_act, 1,2-dibromoethane, TMSCl, THF_anh, 40 °C, 12 h; (b) CuBr, LiCl_act, allyl iodide, rt, 24 h, 35%; (c) 4 (1.5 eq.), Nolan–Grubb's II catalyst (10 mol%), CH₂Cl_2anh, reflux, 24 h.

Alternatively we decided to introduce the nucleobase moiety through direct N-alkylation of protected and unprotected nucleobases with the corresponding (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5). Starting from bromodifluoromethylphophonate (1), the previously described organo-zinc intermediate 2 was reacted with (E)-1,4-dibromobut-2-ene¹⁴ at 0 °C, to yield the desired compound 5 in 80% with no observed isomerization of the double bond (Scheme 2).

Scheme 2 Reagents and conditions: (a) Zn_act, 1,2-dibromoethane, TMSCl, THF_anh, 40 °C, 12 h; (b) CuBr, LiCl_act, trans-1,4-dibromobutene, 0 °C, 4 h, 80%.

Then uracil 6a was converted to its N³-benzoyl derivative 7a through a two steps procedure involving first the formation of N¹,N³-dibenzoyl derivative in presence of an excess of benzoyl chloride in CH₃CN/pyridine mixture, then its selective N¹-deprotection by treatment with potassium carbonate in 1,4-dioxane, (Scheme 3).¹⁷

Scheme 3 Reagents and conditions: (a) (i) BzCl, CH₃CN/pyridine, rt (ii) K₂CO₃ (0.5 M), dioxane, 70 °C, 90% (for R = H) and 96% (for R = CH₃); (b) (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5), Cs₂CO₃, dry DMF, 85% (for R = H) and 81% (for R = CH₃); (c) TMSBr, CH₂Cl₂, rt, 72 h, 90% (for R = H) and 96% (for R = CH₃).

Similarly, thymine 6b was converted to its N³-protected derivative bromide their 7b. Finally, the successful N¹-alkylation of 7a and 7b on 5 in the presence of cesium carbonate in DMF proceeded in good yields and excellent regioselectivities and afforded 8a and 8b, in 86% and 75% yields, respectively. Simultaneous deprotection of the N³-benzoyl group and phosphonic esters with TMSBr/CH₂Cl₂ afforded analogues 9a and 9b, in good yields, respectively. These coupling conditions were extended to other nucleic bases. Cytosine 10a and its fluorinated analogue 10b were converted to N⁴-bis(Boc)-cytosine derivatives 11a and 11b, respectively, in good yileds, through N¹,N³,N⁴-tris-Boc forms followed by regioselective N¹ deprotection with saturated solution of NaHCO₃ in methanol.¹⁸ Alkylation at N¹ position of 11a and 11b in presence of derivative 5, according to the same previous conditions using Cs₂CO₃, afford 12a and 12b, in 85% and 77% yield, respectively.¹⁹ Deprotection with TMSBr afforded the expected free phosphonates 13a and 13b, respectively, in quantitative yield (Scheme 4).

Scheme 4 Reagents and conditions: (a) (i) Boc₂O, DMAP, dry THF (ii) saturated NaHCO₃, MeOH 50 °C, 62%; (b) (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5), Cs₂CO₃, dry DMF, 85% (for R = H) and 77% (for R = F); (c) TMSBr, CH₂Cl₂, rt, 72 h, quantitative for R = H and for R = F.

Direct coupling of the purines 14a and 14b with difluorophosphonate derivative 5 in DMF with Cs₂CO₃ during 20 h at rt provided the desired N⁹-alkylated purine nucleotides 15a and 15b in 50 and 59% yields, respectively.²⁰ Then, 6-chloropurine derivative 15a was converted to its hypoxanthine analogue 16a by treatment with TMSBr to release phosphonic acid followed by refluxing an aqueous solution of hydrochloric acid in 54% overall yield, (Scheme 5).²¹ Similarly, the 2-amino-6-chloropurine derivatives 15b gave the guanine 16b in 60% overall yields.

Scheme 5 Reagents and conditions: (a) (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5), Cs₂CO₃, dry DMF, rt, 24 h, 50% (for R = H) and 59% (for R = NH₂); (b) (i) TMSBr, CH₂Cl₂, rt then (ii) HCl (1 M), reflux, 54% (for R = H) and 60% (for R = NH₂).

Finally, with respect to the broad-spectrum antiviral drug ribavirin, which possess a 1,2,4-triazole-3-carboxamide nucleobase, its acyclic difluorinated phosphonate 19 was synthesized from 17, in a similar pathway, (Scheme 6).

Scheme 6 Reagents and conditions: (a) (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5), Cs₂CO₃, dry DMF, 42%; (b) TMSBr, CH₂Cl₂, rt, 72 h, 96%.

All the synthesized 5,5-difluoro-5-phosphono-pent-2-en-1-yl nucleosides, 9a, b, 13a, b, 16a, b and 19, were evaluated against a wide variety of viruses, to determine their antiviral activity (EC₅₀) in HEL, MDCK, Vero and HeLa cell lines, as the effective concentration required to reduce virus-induced cytopathicity or plaque formation by 50%. Compounds were evaluated against vaccinia virus (VV), herpes simplex virus 1 (HSV-1) (KOS strain), herpes simplex virus 2 (HSV-2) (G strain), thymidine kinase deficient (TK-) HSV-1, vesicular stomatis virus (VSV), varicella-zoster virus (VZV) (TK⁺ and TK⁻ strains), human cytomegalovirus (HCMV) (AD-169 and Davis strains) in HEL, vesicular stomatitis virus (VSV), Coxsackie B4, respiratory syncytial virus in HeLa cell cultures, parainfluenza-3, reovirus-1, Sindbis virus and Coxsackie B4 in Vero cells and influenza virus in MDCK cells. All of the synthesized compounds did not exhibit promising antiviral activity.

Conclusions

In summary, a series of hitherto unknown acyclic 5,5-difluoro-5-phosphono-pent-2-en-1-yl-pyrimidines (9a, b, 13a, b), -purines (16a, b) and -(1,2,4)-triazolo-3-carboxamide (19) were successfully synthesized from (E)-1-bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5) in a convergent stereoselective manner. Surprisingly, it was discovered that cross-metathesis, in our hand, cannot afford the desired difluorinated phosphonate compounds. However, the final nucleosides were obtained, in good yields, by N-alkylation of various nucleobases with (E)-diethyl-5-bromo-1,1-difluoropent-3-enylphosphonate.

However, none of the synthesized compounds showed significant antiviral activities. One plausible explanation could be due to a poor penetration to the cell and to the lack of next phosphorylation steps which could be dependent to the length of the acyclic chain.

Experimental section

General methods

Commercially available chemicals were of reagent grade and used as received. The reactions were monitored by thin layer chromatography (TLC) analysis using silica gel plates (Kieselgel 60F254, E. Merck). Column chromatography was performed on Silica Gel 60 M (0.040–0.063 mm, E. Merck). The ¹H and ¹³C NMR spectra were recorded on a Varian InovaUnity 400 spectrometer (400 MHz) in (d₄) methanol, CDCl₃, shift values in parts per million relative to SiMe₄ as internal reference. High resolution mass spectra were performed on a Bruker maXis mass spectrometer by the “Federation de Recherche” ICOA/CBM (FR2708) platform. The following products are known products or previously reported by our group: N³-benzoyluracil (7a), CAS registration 2775-87-3; N³-benzoylthymine (7b), CAS registration 4330-20-5; N⁴,N⁴-bis(Boc)-cytosine (11a) CAS registration 1108637-28-0; 5-fluoro-N⁴,N⁴-bis(Boc)-cytosine (11b) CAS registration: 1450880-36-0.

(E)-1-Bromo-5-diethoxyphosphoryl-5,5-difluoro-pent-2-ene (5)

A suspension of zinc powder (441.3 mg, 6.75 mmol, 99.99% purity) in THF (0.4 mL) was added to a solution of 1,2-dibromoethane (0.96 mL, 1.13 mmol) in THF (5 mL) at room temperature, and was then warmed up to 65 °C. After 1 min, chlorotrimethylsilane (0.12 mL, 0.8 mmol) was added and the resulting mixture was stirred at 25 °C. After 15 min, the suspension was added dropwise to a solution of diethyl(bromodifluoro)methylphosphonate (0.8 mL, 4.5 mmol) in THF (2 mL), then the reaction mixture was stirred 12 h at 45 °C. CuBr (1.16 g, 8.1 mmol), activated LiCl (344.0 mg 8.1 mmol) and THF (4 mL) was added to the yellow solution at 0 °C under nitrogen, and then the resulting blue solution was stirred at 0 °C. After 10 min, trans-1,4-dibromo-2-butene (1.44 g, 6.75 mmol) was added and the reaction was stirred during 4 h at 0 °C. The mixture was filtered through celite and the filtrate was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO₄, and concentrated under vacuum. The residue was purified by silica gel column chromatography with petroleum ether–EtOAc (4 : 1) to give 2 (1.15 g, 80%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.88–5.76 (m, 1H), 5.29–5.22 (m, 1H), 4.27 (q, J = 7.3 Hz, 2H), 4.25 (q, J = 7.3 Hz, 2H), 2.81 (m, 2H), 1.36 (t, J = 7.3 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 125.2, 127.1, 127.0, 126.9, 121.4, 64.6, 64.5, 39.1, 38.9, 38.7, 38.5, 16.5, 16.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −111.1, −111.4. ³¹P NMR (162 MHz, CDCl₃) δ 6.9; HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₁₇BrF₂O₃P: 321.006487, found: 321.006126.

General procedure A: alkylation with nucleobases

A solution of nucleobase (1.3 equiv.) in dry DMF (3 mL), Cs₂CO₃ (1.3 equiv.) and (E)-diethyl-(5-bromo-1,1-difluoro)pent-3-enylphosphonate (1 equiv.) was stirred at room temperature under argon for 16 h. After removal of DMF under vacuum, the residue was purified by silica gel column chromatography with CH₂Cl₂–MeOH (99 : 1 to 96 : 4) to the desired product.

N³-Benzoyl-1-[(E)-5-diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-uracil (8a). The title compound was prepared from N³-benzoyluracil 7a following procedure A to give 8a (86%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (dd, J = 8.0, 1.2 Hz, 2H), 7.62 (dd, J = 8.0, 1.2 Hz, 1H), 7.47 (t, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 5.78 (m, 2H), 5.77 (d, J = 8.0 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 4.26 (q, J = 7.2 Hz, 2H), 4.24 (q, J = 7.2 Hz, 2H), 2.84 (m, 2H), 1.35 (t, J = 7.2 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 168.9, 162.5, 149.8, 143.7, 135.2, 131.5, 130.5, 129.6, 129.3, 125.7, 102.4, 64.8, 49.5, 37.7, 37.5, 37.4, 37.1, 16.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −111.1, −111.4; ³¹P NMR (162 MHz, CDCl₃) δ 6.3; HRMS (ESI): m/z [M + H]⁺ calcd for C₂₀H₂₄F₂N₂O₆P: 457.134078, found: 457.133456.

N³-Benzoyl-1-[(E)-5-diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-thymine (8b). The title compound was prepared from N³-benzoylthymine with typical procedure A to give 8b (81%) as a colourless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (dd, J = 8.0, 1.2 Hz, 2H), 7.63 (dd, J = 8.0, 1.2 Hz, 1H), 7.48 (t, J = 8.0 Hz, 2H), 7.17 (s, 1H), 5.79 (m, 2H), 4.35 (d, J = 6.0 Hz, 2H), 4.27 (q, J = 7.2 Hz, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.87 (m, 2H), 1.95 (s, 3H), 1.37 (t, J = 7.2 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 169.2, 163.2, 149.9, 139.5, 135.1, 131.7, 130.5, 130.0, 129.2, 125.7, 125.6, 125.5, 125.5, 111.2, 64.7, 49.2, 37.8, 37.6, 37.4, 37.2, 29.8, 16.5, 12.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −110.7 (t, J = 18.8 Hz), −111.0 (t, J = 18.8 Hz); ³¹P NMR (162 MHz, CDCl₃) δ 6.4; HRMS (ESI): m/z [M + H]⁺ calcd for C₂₁H₂₆F₂N₂O₆P: 471.148990, found: 471.149106.

General procedure B: deprotection of diethylphosphonate nucleosides

A septum-sealed microwave tube charged with diethyl phosphonate derivative and trimethylsilylbromide (10.0 equiv.) in CH₃CN (0.1 M) was irradiated at 70 °C under microwave irradiation during 30 min. The progress of the reaction was monitored by TLC analysis. The reaction mixture was quenched with CH₃OH and concentrated under vacuum, then deionized H₂O (ELGA® water, 10 mL) was added and the aqueous layer was washed with CH₂Cl₂ (3 × 5 mL) and lyophilised to yield the expected phosphonic acid derivative.

N¹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-uracil (9a). The title compound was prepared from phosphonate 8a following procedure B to give (9a) (75%) as a colourless oil. ¹H NMR (400 MHz, MeOD) δ 7.57 (d, J = 8.0 Hz, 1H), 5.79 (m, 2H), 5.67 (d, J = 8.0 Hz, 1H), 2.85 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 166.9, 152.7, 147.0, 131.1, 126.2, 126.1, 102.55, 38.6, 38.34, 38.2, 38.0, 20.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −113.7, −114.0. ³¹P NMR (162 MHz, CDCl₃) δ 5.3; HRMS (ESI): m/z [M + H]⁺ calcd for C₉H₁₂F₂N₂O₅P: 297.044718, found: 297.044641.

N¹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-thymine (9b). The title compound was prepared from phosphonate 8b following procedure B to give (9b) (53%) as a colourless oil. ¹H NMR (400 MHz MeOD) δ 7.40 (s, 1H), 5.79 (m, 2H), 4.34 (d, J = 3.9 Hz, 4H), 2.85 (m, 2H), 1.87 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 167.0, 152.9, 142.7, 131.3, 126.0, 111.6, 38.5, 38.4, 38.3, 38.2, 21.1, 12.3; ¹⁹F NMR (376 MHz, CDCl₃) δ −113.7, −114.0; ³¹P NMR (162 MHz, CDCl₃) δ 5.3; HRMS (ESI): m/z [M + H]⁺ calcd for C₁₀H₁₄F₂N₂O₅P: 311.060279, found: 311.060291.

N⁴,N⁴-Bis(Boc)-1-[(E)-5-diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-cytosine (12a). To a solution of N⁴,N⁴-bis(Boc)cytosine 11a (186 mg, 0.59 mmol, 1.0 equiv.) in dry DMF (2 mL) was added Cs₂CO₃ (195 mg, 0.65 mmol, 1.1 equiv.) and the gem difluorinated phosphonate 5 (211 mg, 0.65 mmol, 1.1 equiv.) at room temperature and stirred under an argon atmosphere for 3 h. The resulting mixture was then diluted with EtOAc (2 × 20 mL), quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO₄, and concentrated under vacuum. The residue was purified by silica gel column chromatography with petroleum ether/EtOAc (98 : 2 to 1 : 2) to give product 12a (279 mg, 85%) as a colourless oil; ¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J = 7.4 Hz, 1H), 6.99 (d, J = 7.4 Hz, 1H), 5.79 (m, 2H), 4.48 (d, J = 4.8 Hz, 2H), 4.27 (q, J = 7.3 Hz, 2H), 4.25 (q, J = 7.3 Hz, 2H), 2.83 (m, 2H), 1.37 (t, J = 7.1 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 162.3, 154.9, 149.6, 147.1, 130.2, 124.9, 96.5, 84.8, 64.5, 51.1, 37.4 (td, J = 21.5, 15.4 Hz), 27.7, 16.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −110.8 (t, J = 19.0 Hz), −111.1 (t, J = 19.0 Hz); ³¹P NMR (162 MHz, CDCl₃) δ 7.09 (s), 6.44 (s), 5.78 (s); HRMS (ESI) m/z [M + H]⁺ calcd for C₂₃H₃₇F₂N₃O₈P 552.2281, found 552.2284.

N⁴,N⁴-Bis(Boc)-1-[(E)-5-diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-5-fluorocytosine (12b). To a solution of N⁴,N⁴-bis(Boc)-5-fluorocytosine 11b (113.5 mg, 0.34 mmol, 1.0 equiv.) in dry DMF (1.5 mL) was added Cs₂CO₃ (123 mg, 0.38 mmol, 1.1 equiv.) and the gem difluorinated phosphonate 5 (121 mg, 0.38 mmol, 1.1 equiv.) at room temperature and stirred under an argon atmosphere for 3 h. The resulting mixture was then diluted with EtOAc (2 × 15 mL), quenched with water and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO₄, and concentrated under vacuum. The residue was purified by silica gel column chromatography with petroleum ether/EtOAc (98 : 2 to 1 : 2) to give product 12a (149 mg, 77%) as a yellow oil; ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J = 4.5 Hz, 1H), 5.90–5.76 (m, 2H), 4.53 (d, J = 5.4 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 4.25 (q, J = 7.1 Hz, 2H), 2.87 (m, 2H), 1.47 (s, 18H), 1.37 (t, J = 7.1 Hz, 6H).;¹³C NMR (101 MHz, CDCl₃) δ 155.95 (d, J = 14.3 Hz), 153.84, 149.02, 142.02, 139.56, 133.07 (d, J = 34.73 Hz), 129.21, 126.94 (m), 84.89, 64.80 (d, J = 8.5 Hz), 51.57, 37.60 (m), 29.82, 27.86, 16.53 (d, J = 5.5 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ −110.8, −111.1, −156.23; ³¹P NMR (162 MHz, CDCl₃) δ 6.23 (t, J = 105.8 Hz) HRMS (ESI) m/z [M + H]⁺ calcd for C₂₃H₃₅F₃N₃O₈P 570.2189, found 570.2187.

N¹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-cytosine (13a). The title compound was prepared from phosphonate 12a following procedure B to give 13a (>98%) as a colorless oil; ¹H NMR (250 MHz, MeOD) δ 7.95 (d, J = 5.8 Hz, 1H), 6.14 (d, J = 5.8 Hz, 1H), 5.87 (m, 2H), 4.44 (d, J = 8.50 Hz, 2H), 2.87 (m, 2H); ¹³C NMR (101 MHz, MeOD) δ 153.7, 153.4, 146.6, 136.0, 134.0, 133.7, 120.6, 36.9 (td, J = 22.1, 15.6 Hz); ¹⁹F NMR (376 MHz, MeOD) δ −113.19, −113.46, −170.84; ³¹P NMR (162 MHz, MeOD) δ 4.79 (t, J = 106.19 Hz); HRMS (ESI) m/z [M + H]⁺ calcd for C₉H₁₁F₃N₃O₄P 312.03665 found 312.03645.

N¹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-5-fluorocytosine (13b). The title compound was prepared from phosphonate following procedure B to give 13b (>98%) as a colorless oil; ¹H NMR (250 MHz, MeOD) δ 7.95 (d, J = 7.7 Hz, 1H), 6.17–6.08 (m, 1H), 5.88 (d, J = 4.3 Hz, 1H), 4.50 (d, J = 5.0 Hz, 2H), 3.02–2.77 (m, 2H); ¹³C NMR (101 MHz, MeOD) δ 153.7, 153.4, 146.6, 136.0, 134.0, 133.7, 120.6, 36.9 (td, J = 22.1, 15.6 Hz); ¹⁹F NMR (376 MHz, MeOD) δ −113.3 (t, J = 19.0 Hz), −113.6 (t, J = 18.9 Hz); ³¹P NMR (162 MHz, MeOD) δ 5.68 (s), 5.04 (s), 4.40 (s); HRMS (ESI) m/z [M + H]⁺ calcd for C₉H₁₃F₂N₃O₄P 295.0606 found 295.0607.

N⁹-[(E)-5-Diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-(6-chloro)purine (15a). The title compound was prepared from 6-chloropurine 14a following procedure A to give 15a (50%) as a colourless oil. ¹H NMR (400 MHz CDCl₃) δ 8.55 (s, 1H), 7.96 (s, 1H), 5.80 (m, 2H), 5.80 (dt, J = 15.6, 6.8 Hz, 1H), 4.87 (d, J = 6.4 Hz, 2H), 4.27 (q, J = 7.2 Hz, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.86 (m, 2H), 1.36 (t, J = 7.2 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 152.2, 152.1, 151.7, 151.1, 145.1, 131.6, 130.1, 129.2, 125.8, 64.7, 45.5, 37.6, 37.4, 37.2, 37.0, 16.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −114.1, −113.7; ³¹P NMR (162 MHz, CDCl₃) δ 6.3; HRMS (ESI): m/z [M + H]⁺ calcd for C₁₄H₁₉ClF₂N₄O₃P: 395.084844, found: 395.084588.

N⁹-[(E)-5-Diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-(2-amino-6-chloro)purine (15b). The title compound was prepared from 6-chloro-2-aminopurine (14b) following procedure A to give 15b (59%) as a colorless oil; ¹H NMR (400 MHz, CDCl₃) δ 7.80 (s, 1H), 5.90–5.75 (m, 2H), 5.21 (s, 2H), 4.68 (d, J = 5.7 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 4.24 (q, J = 7.1 Hz, 2H), 2.85 (m, 2H), 1.35 (t, J = 7.1 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 159.1, 153.6, 151.3, 142.0, 129.7, 125.2, 124.8 (dd, J = 11.0, 5.6 Hz), 64.6 (d, J = 6.9 Hz), 44.9, 37.2 (td, J = 21.6, 15.6 Hz), 16.4 (d, J = 5.4 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ −110.9 (t, J = 18.9 Hz), −111.2 (t, J = 18.9 Hz); ³¹P NMR (162 MHz, CDCl₃) δ 7.10 (dt, J = 14.8, 7.3 Hz), 6.44 (dt, J = 14.6, 7.1 Hz), 5.78 (dt, J = 15.0, 7.3 Hz); HRMS (ESI) m/z [M + H]⁺ calcd for C₁₄H₂₀ClF₂N₅O₃P 410.0955 found 410.0958.

N⁹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-hypoxanthine (16a). The title compound was prepared from the 6-chlorouracil phosphonate analogue 15a by treatment reflux acid (HCl, 1 M) to give 16a (54%) as a colorless oil; ¹H NMR (400 MHz, DMSO) δ 12.31 (s, 1H), 8.08 (t, J = 10.6 Hz, 2H), 7.29–6.90 (m, 1H), 5.95–5.88 (m, 1H), 5.73–5.65 (m, 1H), 4.78 (d, J = 5.9 Hz, 2H), 2.76 (t, J = 19.7 Hz, 2H); ¹³C NMR (101 MHz, DMSO) δ 157.0, 148.6, 146.1, 140.4, 130.5, 124.7 (d, J = 5.0 Hz), 124.1, 45.1, 37.2, 37.0–36.5 (m); ¹⁹F NMR (376 MHz, DMSO) δ −111.8 (t, J = 19.8 Hz), −112.1 (t, J = 19.8 Hz); ³¹P NMR (162 MHz, DMSO) δ 4.12 (t, J = 4.0 Hz), 3.51 (t, J = 4.6 Hz), 2.91 (t, J = 4.5 Hz); HRMS (ESI) m/z [M − H]⁻ calcd for C₁₀H₁₀F₂N₄O₄P 319.0413, found 319.0417.

N⁹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-guanine (16b). The title compound was prepared from phosphonate 15b by treatment reflux acid (HCl, 1 M) to give 16b (60%) as a colorless oil; ¹H NMR (400 MHz, MeOD) δ 9.08 (s, 2H), 8.11 (s, 1H), 6.02 (s, 4H), 4.88 (d, J = 3.9 Hz, 4H), 2.92 (t, J = 18.7 Hz, 4H); ¹³C NMR (101 MHz, MeOD) δ 162.0, 155.9, 153.7, 150.1, 136.6, 127.9–127.4 (m), 107.4, 46.1, 36.9 (d, J = 15.5 Hz); ¹⁹F NMR (376 MHz, MeOD) δ −113.1 (t, J = 18.8 Hz), −113.4 (t, J = 18.8 Hz); ³¹P NMR (162 MHz, MeOD) δ 5.68 (s), 5.30 (d, J = 103.5 Hz), 4.35 (s); HRMS (ESI) m/z [M − H]⁻ calcd for C₁₀H₁₁F₂N₅O₄P 334.0522, found 334.0529.

N¹-[(E)-5-Diethoxyphosphoryl-5,5-difluoro-pent-2-en-1-yl]-(3-carbamoyl)-1,2,4-triazol (18). The title compound was prepared from 1H-1,2,4-triazole-3-carboxamide (17) following procedure A to give 18 (42%) as a colorless oil; ¹H NMR (400 MHz, MeOD) δ 8.49 (s, 1H), 6.04–5.96 (m, 1H), 5.85–5.71 (m, 1H), 4.93 (d, J = 6.1 Hz, 2H), 4.34–4.22 (m, 4H), 2.99–2.80 (m, 2H), 1.36 (t, J = 7.1 Hz, 6H); ¹³C NMR (101 MHz, MeOD) δ 159.4, 149.6, 149.5–149.1 (m), 145.9, 130.7, 123.2 (dd, J = 10.9, 6.0 Hz), 120.4, 118.2, 64.8 (d, J = 7.0 Hz), 51.6, 45.7, 36.9 (dd, J = 21.6, 6.3 Hz), 15.4; ¹⁹F NMR (376 MHz, MeOD) δ −111.8 (t, J = 19.2 Hz), −112.1 (t, J = 19.2 Hz); ³¹P NMR (162 MHz, MeOD) δ 7.21 (dt, J = 15.7, 7.9 Hz), 6.54 (dt, J = 15.8, 7.9 Hz), 5.87 (dt, J = 15.9, 7.9 Hz); HRMS (ESI) m/z [M + H]⁺ calcd for C₁₂H₂₀F₂N₄O₄P 354.1185, found 354.1187.

N¹-[(E)-5,5-Difluoro-5-phosphono-pent-2-en-1-yl]-(3-carbamoyl)-1,2,4-triazole (19). The title compound was prepared from phosphonate following procedure B from 18 to give 19 (>98%) as an oil; ¹H NMR (400 MHz, MeOD) δ 8.07 (s, 1H), 6.02–5.73 (m, 2H), 5.29 (d, J = 5.8 Hz, 2H), 2.97–2.72 (m, 2H); ¹³C NMR (101 MHz, MeOD) δ 159.5, 149.5, 145.9, 129.8, 127.3, 124.4 (d, J = 5.8 Hz), 51.8, 36.9–36.4 (m); ¹⁹F NMR (376 MHz, MeOD) δ −114.3 (t, J = 19.3 Hz), −114.6 (t, J = 19.3 Hz); ³¹P NMR (162 MHz, MeOD) δ 5.68 (s), 5.30 (d, J = 103.5 Hz), 4.75 (s); HRMS (ESI) m/z [M + H]⁺ calcd for C₈H₁₀F₂N₄O₄P 295.0413 found 295.0414.

Antiviral activity assays

The antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation in HEL 299 (ATCC® CCL-137™) cell culture against herpes simplex virus 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus, vesicular stomatitis virus, cytomegalovirus (HCMV), and varicella-zoster virus (VZV). Moreover, the Vero (ATCC® CCL-81™) cell culture was utilized to test such compounds against parainfluenza-3, reovirus-1, Sindbis virus and Coxsackie B4. Furthermore, the novel compounds were evaluated in HeLa cell culture against vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus or MDCK (ATCC® CCL-34™) [influenza A (H1N1; H3N2) and influenza B]. Confluent cell cultures (or nearly confluent for MDCK cells) in microtiter 96-well plates were inoculated with 100 CCID₅₀ of virus (1 CCID₅₀ being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU). After 1–2 h virus adsorption period, residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations (200, 40, 8, 1.6, 0.32 μM) of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC₅₀ or concentration required reducing virus-induced cytopathogenicity or viral plaque (VZV) plaque formation by 50%. The minimal cytotoxic concentration (MCC) of the compounds was defined as the compound concentration that caused a microscopically visible alteration of cell morphology. Alternatively, cytotoxicity of the test compounds was measured based on inhibition of cell growth. HEL cells were seeded at a rate of 5 × 10³ cells per well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37 °C, the cell number was determined with a Coulter counter.

The cytostatic concentration was calculated as the CC₅₀, or the compound concentration required reducing cell proliferation by 50% relative to the number of cells in the untreated controls.

Acknowledgements

This work was supported in part by the grant from LabEx SYNORG and FEDER (program COSMI). The authors's thanks are due to Mrs Leentje Persoons, Mrs Lies Van Den Heurck, Mrs Ellen De Waegenaere for the biological assays.

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Footnote

† Electronic supplementary information (ESI) available: ¹H, ¹³C, ³¹P and ¹⁹F NMR data of selected compounds. See DOI: 10.1039/c7ra05153k

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