Abstract
The synthesis of new 2,6-disubstituted purine 2′,3′-dideoxy-2′,3′-difluoro-D-arabino nucleosides is reported. Their ability to block HIV and HCV replication along with their cytotoxicity toward Huh-7 cells, human lymphocyte, CEM and Vero cells was also assessed. Among them, β-2,6-diaminopurine nucleoside 25 and guanosine derivative 27 demonstrate potent anti-HIV-1 activity (EC50 = 0.56 and 0.65 μm; EC90 = 4.2 and 3.1 μm) while displaying only moderate cytotoxicity in primary human lymphocytes.
Introduction
The chemical synthesis and biochemical properties of fluorine-containing nucleosides have been the focus of numerous studies over the years [1]. Recent advances in medicinal chemistry indicate conclusively that synthetic fluorinated nucleoside analogs represent a valuable class of drugs for the treatment of various diseases [2–5]. Introduction of a fluorine atom into nucleoside analogs can lead to a change in biological activity, lipophilicity, or bioavailability. The small size and strong electronegativity of the fluorine atom, which can mimic either a hydrogen or a hydroxyl group, may critically influence the pharmacokinetic properties and/or toxicity of a drug [1]. It has been established that fluorination of either the sugar moiety or the heterocyclic base of a nucleoside may alter its affinity with various metabolic enzymes and can radically affect the conformation of the pentofuranose ring of the nucleoside in solution [6, 7]. Nucleosides containing fluorine at C2′ exhibit potent biological activities [8, 9]. Among them, C2′-β-fluoro purine nucleosides are of special interest because the location of the fluorine atom in the β-orientation can affect their phoshorylation, the metabolic stability of the glycosidic bond and potentially their antiviral and anticancer activities. For instance, fluorinated nucleosides such as sofosbuvir and gemcitabine (Figure 1) have been approved for the treatment of hepatitis C virus (HCV) and various cancers, respectively. On the other hand, lodenosine (2′-β-fluoro-2′,3′-dideoxyadenosine, 1) displays in vitro activity against HIV-1 and appears chemically and metabolically stable (Figure 1) [10].
FDA approved 2′-fluoro nucleosides and biologically active fluorine containing purine nucleosides 1–4.
A number of other purine nucleosides with the 2′-fluoro-β-D-arabinofuranosyl moiety have been synthesized and tested for their antitumor activity, including 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)guanine (3), which displays activity against human leukemic T-cell lines [11, 12]. In addition, 3′-α-fluoro-2′,3′-dideoxyguanosine (4) was studied clinically for the treatment of HIV and HBV infected patients (Figure 1) [13, 14]. The HBV studies were subsequently abandoned due to a lack of advantage versus standard of care (http://www.medivir.se/v5/en/uptodate/pressrelease.cfm). In earlier investigations, we disclosed 9-(2′,3′-dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)adenine (2), a unique difluorinated nucleoside that was more potent in vitro against HIV-1 than lodenosine 1 [15]. Based on this work, we have decided to further evaluate this new class of compounds and wish to describe herein the synthesis of new D-arabino-2′,3′-dideoxy-2′,3′-difluoronucleosides along with their biological evaluation for in vitro anti-HIV-1 and anti-HCV activities.
Results and discussion
In order to prepare our library of purine nucleosides, the synthesis of key 2,6-dichloropurine nucleoside 9 was optimized (Scheme 1).
Reagents and conditions: (a) AcOH/Ac2O/cc H2SO4 0°C to 4°C (6, 77%; 7, 10–12%); or 0°C to rt, (6, 87%); (b) TMSBr/CH2Cl2/ZnBr2, 0°C to rt; 95%; (c) i) Na salt of 2,6-dichloropurine/CH3CN, rt (9, 55%, 10, 13%); ii) K salt of 2,6-dichloro purine/CH3CN, rt, (9, 73%, 10, 8%); (d) Ac2O/Py, rt, 86%; (e) AcOH/Ac2O/conc. H2SO4, rt, 97%; (f) TMSOTf, silylated 2,6-dichloropurine/CH3CN/DCE, rt, 89%; (g) NaHCO3, MeOH, rt, 63%; (h) DAST/CH2Cl2/Py, rt, 35%.
Thus, treatment of 5 in a mixture of acetic acid/acetic anhydride/H2SO4 at room temperature (instead of 4°C) [15], allowed for the formation of 1-O-acetyl derivative 6 in 87% yield (α/β ratio ca. 3:1) with no traces of acyclic compound 7 formed. Treatment of 6 with TMSBr in anhydrous CH2Cl2 in the presence of the inexpensive and easily available catalyst ZnBr2 lead to almost quantitative formation of 1′-α-bromide 8. The α-anomeric configuration of bromo sugar 8 was confirmed by the 3JH-1,F-2 and 3JH-1,H-2 values (12.6 Hz and < 1.0 Hz, respectively) observed by 1H NMR (Figure 2) and by comparison with known 1′-α-bromo anomer of 3,5-di-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranose [16]. Next, reaction of 5-O-benzoyl-2,3-dideoxy-2,3-difluoro-α-D-ara-binofuranosyl bromide (8) with the sodium salt of 2,6-dichloropurine [17] gave a 4:1 mixture of protected nucleosides 9 and 10, which were separated by column chromatography on silica gel (Scheme 1). In an effort to improve the β/α ratio during the glycosylation reaction [18–20], the use of the potassium salt of 2,6-dichloropurine was evaluated. Interestingly using this salt in anhydrous acetonitrile led to the formation of N9-β-D-nucleoside 9 and –α-D-protected nucleoside 10 in 73% and 8% yield, respectively (9/1 ratio). An alternative linear approach to the key intermediate 9 was also investigated from known fluorodeoxysugar 11 (Scheme 1) [21]. Thus, compound 11 was first acetylated in 86% and then allowed to react in a mixture of Ac2O/AcOH/H2SO4 to give acetyl derivatives 13 in 97% yield (α/β ratio ≈ 1:1). The glycosylation of 13 with a silylated 2,6-dichloropurine under Vorbrüggen conditions was investigated. Thus, the coupling reaction in the presence of TMSOTf under reflux in acetonitrile gave a complicated mixture of products from which β-2,6-dichloropurine nucleoside 14 (26%) was isolated. On the other hand, coupling of acetate derivative 13 with silylated 2,6-dichloropurine in a mixture of acetonitile-1,2-dichloroethane, and in the presence of TMSOTf gave desired 2,6-dichloropurine 14 in 89% yield. Selective deprotection of nucleoside 14 with NaHCO3 in methanol produced 15 in 63% yield and final fluorination with diethylaminosulfur trifluoride (DAST) in the presence of pyridine in dichloromethane at room temperature gave β-2′,3′-difluoro arabinonucleoside 9 in 35% yield.
1H NMR spectra of 1-α-bromide 8 and β-2′,3′-difluoroarabinonucleoside of 2,6-diaminopurine 26.
Treatment of protected nucleoside 9 with 1.1 equivalents of sodium methoxide in methanol at room temperature produced 2-chloro-6-methoxypurine arabinoside 16, in 84% yield. 2,6-Dimethoxypurine arabinoside 17 was prepared in 52% yield by reaction of 9 with 2.7 equivalents of sodium methoxide in methanol (Scheme 2) [22]. The treatment of nucleoside 9 with 5.0 equivalents of benzylamine in methanol at 55°C afforded 2-chloro-6-benzylaminopurine arabinoside 18 in 78% yield. Removal of the acyl group in 18 with saturated methanolic ammonia gave 2-chloro-6-benzylaminopurine analog 19 in 82% yield. Reaction of each β- and α-protected nucleosides of 2,6-dichloropurine 9 and 10 with LiN3 in EtOH under reflux afforded 2,6-diazido derivatives 20 and 21 in 97% yield. The reduction of both azido groups with SnCl2 in a mixture of dichloromethane-methanol [23] resulted in the formation of 5′-O-benzoyl derivatives of N9-β- and N9-α-arabinosides 23 (87%) and 24 (91%). It is noteworthy that 2-azido-6-amino derivative 22 was also isolated in 4% yield after reduction of β-protected nucleoside 20 with stannous chloride (Scheme 2). Subsequent debenzoylation of intermediates 23 and 24 with saturated methanolic ammonia, gave pure 2′,3′-dideoxy-2′,3′-difluoronucleosides 2,6-diaminopurine 25 and 26 in 72% and 79% yield, respectively. Guanine nucleoside 28 was prepared by enzymatic deamination of N9-β-nucleoside 25 in water with calf intestine adenosine deaminase in 85% yield (Scheme 2).
Reagents and conditions: (a) MeONa/MeOH, rt, 16, 84%; (b) MeONa/MeOH, rt and then reflux, 17, 52%; (c) BnNH2/MeOH, 55°C, 18, 78%; (d) LiN3/EtOH, reflux, (20, 97%, 21, 97%); (e) saturated NH3/MeOH, rt, (19, 82%, 25, 72%, 26, 79%); (f) SnCl2/CH2Cl2/MeOH, rt, (22, 4%, 23, 87%; 24, 91%); (g) Adenosine deaminase/H2O, rt, 27, 85%.
An alternative approach to prepare guanine analog 28 from bromide 8 was also investigated (Scheme 3). Treatment of 2-amino-6-chloropurine with potassium t-butoxide in 1,2-dimethoxyethane followed by coupling of the resulting salt with bromo sugar 8 in acetonitrile at room temperature gave a mixture of N9-β- and N9-α-nucleosides 28 and 29 which were separated by column chromatography in 50% and 4% yields, respectively. The benzoyl-protected 2-amino-6-chloropurine analog 28 was converted to guanine derivative 27 (71%) by treatment with 2-mercaptoethanol and sodium methoxide in refluxing methanol. Finally, in order to extend our series of novel purine nucleosides, debenzoylation of protected β-nucleoside 28 with saturated methanolic ammonia at room temperature afforded 2-amino-6-chloropurine analog 30 in 77% yield.
Reagents and conditions: (a) K salt of 2-amino-6-chloropurine/CH3CN, rt, (28, 50%, 29, 4%); (b) saturated NH3/MeOH, rt, 77%; (c) HSCH2CH2OH/MeONa/MeOH, reflux, 71%.
Structures of nucleosides 9, 10, 14, 16–30 were confirmed by 1H, 19F and 13C NMR, UV and mass spectroscopy. Assignment of the configurations of synthesized nucleosides at the anomeric centers are based upon 1H NMR analysis (long-range couplings between the H-8 proton of the purine and the 2′-β fluorine atom for the β-anomers) and 13C NMR data (characteristic JC-1′,F-2′ coupling constants for β- and α-anomers of purine 2′,3′-difluoro-D-arabinofuranosyl nucleosides) (Tables 1–4). The resonance signal of the purine H-8 proton for 2,6-diaminopurine nucleoside 25 is displayed as a doublet in its 1H NMR spectrum and the magnitude of the 5JH,F coupling is 2.4 Hz (Figure 2). It should be noted that two 4JH,F the long-range coupling constants of 2.0 Hz and 1.0 Hz between sugar H-1′ and H-5′ protons, and F-3′ substituent, respectively, are exhibited also in the spectrum of difluoride 25 due to the W-arrangements between these protons and fluorine atom at C-3′.
1H NMR Chemical shifts of 2′,3′-dideoxy-2′,3′-difluoro nucleosides 9, 10, 16, 17, 25–27 with D-arabino-configurations.
| Compound | H-1′ | H-2′ | H-3′ | H-4′ | H-5′ | H-5″ | Others |
|---|---|---|---|---|---|---|---|
| 9 | 6.60 dt | 5.46 br.dd | 5.35 ddd | 4.74 dm | 4.65–4.71 m | 8.32 (d, 1H, J=2.63, H-8), 7.46-8.07 (m, 5H, Bz) | |
| 10 | 6.55 d | 5.84 dd | 5.47 ddt | 5.09 ddt | 4.64 dd | 4.57 dd | 8.24 (s, 1H, H-8), 7.47–8.08 (m, 5H, Bz) |
| 16 | 6.55 dd | 5.52 dddd | 5.49 dddd | 4.29 dm | 3.87 dd | 3.84 dd | 8.46 (d, 1H, J=2.10, H-8), 4.17 (s, 3H, OCH3) |
| 17 | 6.49 ddd | 5.51 dddd | 5.45 dddd | 4.26 dm | 3.85 dd | 3.83 dd | 8.24 (d, 1H, J=2.13, H-8), 4.13 and 4.02 (2s, 6H, 2×OCH3) |
| 25 | 6.32 ddd | 5.44 dddd | 5.40 dddd | 4.22 dm | 3.84 ddd | 3.82 dd | 7.91 (d, 1H, J=2.42, H-8) |
| 26 | 6.22 dd | 5.99 ddt | 5.36 dddd | 4.69 ddt | 3.75 dd | 3.72 dd | 7.84 (s, 1H, H-8) |
| 27 | 6.15 dd | 5.60 dddd | 5.56 dddd | 4.10 dm | 3.65 br.m | 3.61 br.m | 10.66 (brs, 1H, NH), 7.74 (d, 1H, J=2.9, H-8), 6.51 (brs, 2H, NH2), 5.17 (t, 1H, J = 5.64, 5′-OH) |
Spectra were obtained in CDCl3 for nucleosides 9, 10; in CD3OD for 16, 17, 25, 26, and DMSO-d6 for nucleoside 27. δ in ppm, J in Hz.
Coupling constants (in Hz) for 1H NMR data of 2′,3′-dideoxy-2′,3′-difluoro nucleosides 9, 10, 16, 17, 25–27 with D-arabino- configurations.
| 3J(H,H) | 3J(H,F) | Others | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 1′,2′ | 2′,3′ | 3′,4′ | 4′,5′/4′,5″ | H1′,F2 | H3′,F2 | H2′,F3 | H4′,F3 | ||
| 9 | 2.56 | <1.0 | 2.38 | 4.37/n.d. | 21.8 | 9.2 | 11.94 | n.d | 5JF2′,H8 = 2.63 |
| 4J1′,F3′ = 2.56 | |||||||||
| gemJ2′,F2′ = 50.03 | |||||||||
| gemJ3′,F3′ = 49.51 | |||||||||
| JH5′,H5″ = 11.2 | |||||||||
| 10 | <1.0 | 1.8 | 1.7 | 5.48/5.5 | 15.02 | 12.6 | 11.22 | 23.34 | gemJ2′,F2′ = 48.14 |
| gemJ3′,F3′ = 50.12 | |||||||||
| JH5′, H5″ =12.14 | |||||||||
| 16 | 4.16 | 2.28 | 3.90 | 3.31/2.53 | 16.93 | 15.45 | 12.87 | 24.10 | 5JF2′,H8 = 2.1 |
| 4J1′,F3′ = 1.64 | |||||||||
| gemJ2′,F2′ = 50.65 | |||||||||
| gemJ3′,F3′ = 51.29 | |||||||||
| gemJH5′, H5″= 12.1 | |||||||||
| 17 | 3.89 | 2.19 | 3.80 | 3.85/5.12 | 17.29 | 15.70 | 12.50 | 24.36 | 5JF2′,H8 = 2.13 |
| 4J1′,F3′ = 1.8 | |||||||||
| gemJ2′,F2′ = 50.33 | |||||||||
| gemJ3′,F3′ = 49.51 | |||||||||
| JH5′, H5″ = 11.2 | |||||||||
| 25 | 3.87 | 2.0 | 3.85 | 4.62/5.33 | 18.37 | 12.18 | 14.69 | 25.0 | 5JF2′,H8 = 2.42 |
| 4J1′,F3′ = 2.0 | |||||||||
| gemJ2′,F2′ = 51.29 | |||||||||
| gemJ3′,F3′ = 50.65 | |||||||||
| gemJH5′, H5″= 12.8 | |||||||||
| 4J5′,F3′ = 1.0 | |||||||||
| 4J5′,F3′ = <1.0 | |||||||||
| 26 | 2.56 | 2.88 | 4.17 | 4.40/3.7 | 15.37 | 16.35 | 14.42 | 20.52 | gemJH5′, H5″= 11.5 |
| gemJ3′,F3′ = 52.25 | |||||||||
| gemJ2′,F2′ = 50.33 | |||||||||
| 27 | 4.16 | 3.2 | 3.21 | 5.48/5.42 | 16.34 | 16.03 | 14.42 | 22.91 | 5JF2′,H8 = 2.9 |
| gemJ2′,F2′ = 50.64 | |||||||||
| gemJ3′,F3′ = 51.61 | |||||||||
| gemJH5′, H5″= 12.8 | |||||||||
13C NMR Data of 2′,3′-difluoro nucleosides 9, 10, 16, 17, 25–27, 30.
| Compound | Chemical shifts, δTMS, ppm [J(C,F) in Hz] | Others | ||||
|---|---|---|---|---|---|---|
| C-1′ | C-2′ | C-3′ | C-4′ | C-5′ | ||
| 9 | 83.73 d | 93.64 dd | 91.57 dd | 81.31 d | 62.46 d | 166.17 (s, Ph-C=O), 128.81–133.85 (Ph-C=O and C-5), 153.57, 152.56, 152.35 (C-6, 2, 4), 144.88 (d, 4JC8,F2′ = 5.6, C-8) |
| 10 | 88.92 d | 96.09 dd | 94.64 dd | 84.23 d | 62.40 d | 166.08 (s, Ph-C=O), 128.13–133.78 (Ph-C=O and C-5), 153.71, 152.58, 152.29 (C-6, 2, 4), 143.35 (d, 4JC8,F2′ = 3.8, C-8) |
| 16 | 83.07 dd | 93.42 dd | 92.69 dd | 82.17 dd | 60.16 d | 161.35 (C-6) 153.34 (C-2) 152.63 (C-4) 142.79 (d, 4JC8,F2′ = 4.23, C-8) 119.48 (C-5) 54.44 (OCH3) |
| 17 | 82.79 dd | 93.51 dd | 92.76 dd | 81.96 dd | 60.19 d | 162.26 (C-6 or C-2) 162.02 (C-2 or C-6) 153.07 (C-4) 140.73 (d, 4JC8,F2′ = 4.14, C-8) 115.98 (C-5) 54.50 and 53.69 (2×OCH3) |
| 25 | 82.63 dd | 93.65 dd | 92.60 dd | 81.82 dd | 60.32 d | 160.74 (C-6) 156.33 (C-4) 151.32 (C-2) 137.19 (d, 5JC8,F2′ = 4.38, C-8) 112.36 (C-5) |
| 26 | 87.12 dd | 96.58 dd | 93.99 dd | 84.35 dd | 60.37 d | 160.77 (C-6) 156.36 (C-4) 151.35 (C-2) 136.23 (d, 5JC8,F2′ = <2.0, C-8) 113.23 (C-5) |
| 27 | 81.40 dd | 94.10 dd | 92.62 dd | 81.16 dd | 60.55 d | 157.24 (C-6) 154.48 (C-4) 151.46 (C-2) 136.60 (d, 5JC8,F2′ = 4.36, C-8) 116.59 (C-5) |
| 30 | 82.62 dd | 93.65 dd | 92.68 dd | 82.1 dd | 60.25 d | 160.51 (C-2) 153.46 (C-6) 150.52 (C-4) 141.79 (d, 5JC8,F2′ = 4.37, C-8) 123.16 (C-5) |
The 13C NMR Data of 2′,3′-difluoro nucleosides 9, 10, 16, 17, 25–27, 30 (Coupling constants given in Hz).
| Compound | F-2′ | F-3′ | ||||||
|---|---|---|---|---|---|---|---|---|
| 2J(C,F) | 3J(C,F) | 4J(C,F) | 3J(C,F) | 2J(C,F) | 3J(C,F) | |||
| C1′,F2′ | C3′,F2′ | C4′,F2′ | C5′,F2′ | C1′,F3′ | C2′,F3′ | C4′,F3′ | C5′,F3′ | |
| 9 | 16.71 | 30.20 | <1.0 | <1.0 | <1.0 | 30.70 | 27.27 | 8.71 |
| 10 | 36.25 | 29.92 | <1.0 | <1.0 | <1.0 | 30.92 | 26.00 | 7.67 |
| 16 | 17.21 | 28.81 | 3.08 | <1.0 | 3.76 | 28.28 | 25.45 | 6.26 |
| 17 | 17.88 | 28.15 | 2.12 | <1.0 | 3.35 | 28.65 | 25.43 | 6.10 |
| 25 | 17.08 | 28.67 | 1.48 | <1.0 | 2.35 | 28.74 | 24.93 | 6.51 |
| 26 | 35.54 | 28.12 | 1.50 | <1.0 | 4.99 | 28.16 | 24.92 | 5.14 |
| 27 | 16.85 | 25.08 | 3.99 | <1.0 | 4.99 | 26.85 | 24.83 | 5.22 |
| 30 | 16.90 | 28.80 | 2.99 | <1.0 | 2.90 | 28.90 | 24.93 | 5.31 |
To determine the spectrum of activity of the synthesized purine nucleosides, anti-HIV-1 activity was evaluated versus HIV-1LAI in primary human peripheral blood mononuclear (PBM) cells and 3′-azido-3′-deoxythymidine (AZT) was used as a positive control. Cytotoxicity was determined in human PBM, human T-lymphoblastoid (CEM), and African Green monkey (Vero) cells [24, 25]. All of the modified purine nucleosides were also evaluated for inhibition of HCV RNA replication at 10 μm in human hepatoma cells (Huh-7) using a subgenomic HCV replicon system and 2′-C-methyl-cytidine (2′-C-MeC) as a positive control [26]. Cytotoxicity in Huh-7 cells was determined simultaneously with anti-HCV activity by extraction and amplification of both HCV RNA and ribosomal RNA (rRNA) [27]. The antiviral and cytotoxicity results are summarized in Table 5. In general, all of the fluoro-containing nucleoside analogs inhibit HIV replication at micromolar concentrations, but show cytotoxicity in the same range in most of the cell systems tested. It is noteworthy though, that 2,6-diaminopurine analog 25 displays submicromolar activity against HIV (EC50 = 0.56 μm) while showing only modest cytotoxicity in PBM and CEM (CC50 = 58 and 91 μm, respectively) and no toxicity in Vero at concentration up to 100 μm.
In vitro antiviral activity and cytotoxicity of compounds 9, 10, 16, 17, 19, 20, 22, 25–28 and 30.
| Compound | Anti-HIV-1 activity (μm) | Anti-HCV activity (μm) | Cytotoxicity, CC50 (μm) | |||||
|---|---|---|---|---|---|---|---|---|
| EC50 | EC90 | EC50 | EC90 | PBM | CEM | Vero | Huh-7 | |
| 9 | 18 | 46 | <10a | <10a | 23 | 4.9 | 33 | <10a |
| 10 | 13 | 27 | <10a | <10a | 54 | 11 | 23 | <10a |
| 16 | 17 | 60 | >10 | >10 | >100 | >100 | >100 | >10 |
| 17 | >100 | >100 | >10 | >10 | >100 | >100 | >100 | >10 |
| 19 | 32 | >100 | 2.9 | 9.9 | 53 | 14 | 56 | 26 |
| 20 | 21 | >100 | >10 | >10 | 95 | 24 | >100 | >10 |
| 22 | 41 | >100 | >10 | >10 | 40 | 52 | >100 | >10 |
| 25 | 0.56 | 4.2 | >10 | >10 | 58 | 91 | >100 | >10 |
| 26 | 2.4 | 14 | >10 | >10 | 17 | 4.9 | >100 | >10 |
| 27 | 0.66 | 3.1 | >10 | >10 | 30 | 62 | 90 | >10 |
| 28 | 2.4 | 18 | 4.7 | 10 | 7.6 | ND | >100 | 9.1 |
| 30 | 0.65 | 2.4 | >10 | >10 | 14 | ND | >100 | >10 |
| AZT | 0.0037 | 0.047 | > 10 | > 10 | >100 | 14 | 56 | NDa |
| 2′<-C-MeCb | 48 | >100 | 1.8 | 5.8 | >100 | >100 | >100 | >10 |
aCytotoxicity in Huh-7 cells did not allow for anti-HCV activity determination.
bAZT and 2′-C-MeC (2′-C-methylcytidine) were used as positive controls for HIV and HCV assays, respectively.
Conclusions
A new and efficient procedure for the preparation of key 1′-α-bromosugar 8 by bromination of known acetate 6 under mild catalytic conditions was described. In addition, new and selective synthetic approaches to the key intermediates 9 and 28 were developed. With these compounds in hand, twelve 2′,3′-difluoro-D-arabinofuranosyl 2,6-disubstituted purine nucleosides were prepared which were evaluated as potential anti-HIV-1 and anti-HCV agents. The SAR results indicate that modifications at 2 and 6-positions have effects on antiviral activity and host cell toxicity. The β-2,6-diaminopurine nucleoside 25 demonstrates selective in vitro anti-HIV-1 activity (EC50 = 0.56 μm) while displaying only moderate cytotoxicity in human lymphocytes. Based on these encouraging results, further structural modifications of the base should allow us to improve the antiviral potency and selectivity of these compounds.
Experimental
Column chromatography was performed on silica gel 60 H (70–230 mesh; Merck, Darmstadt, Germany). All anhydrous solvents were distilled over CaH2, P2O5 or magnesium prior to the use. The UV spectra were recorded on Specord M-400 (Carl Zeiss, Germany). The 1H, 13C and 19F NMR spectra were recorded in CDCl3, CD3OD and DMSO-d6 with Bruker Avance-500-DRX spectrometer at 500.13, 126.76 and 470.59 MHz, respectively. Chemical shifts δ are reported in ppm downfield from internal SiMe4 (1H,13C) or external CFCl3 (19F). J values are reported in Hz. NMR assignments were confirmed by 2D (1H,1H and 1H,13C) correlation spectroscopy. Melting points were determined on a Boetius apparatus and were uncorrected. High resolution mass spectra were measured on a mass spectrometer Agilent Q-TOF 6550 (USA) using electrospray ionization.
1-O-Acetyl-5-O-benzoyl-2,3-dideoxy-2,3-difluoro-α/β- D-arabinofuranoside (6)
Method A
Acetolysis of 5 (0.337 g, 1.24 mmol) for 18 h in a mixture of acetic acid/acetic anhydride/H2SO4 was accomplished as described earlier [9]. The product was chromatographed on silica gel, eluting with EtOAc/hexane (ratio 1:5, 1:3 and 1:1) to give 6 (0.286 g, 77%) as a syrup and a diastereomeric mixture of 7 (0.046 g, 10%, oil). 1H NMR (CDCl3): (ratio of diastereomers α and β ca. 1.0:0.43), δ 7.45–8.05 (m, ArH), 6.06 (dd, 1H, H-1a, J1,F-2 = 5.2, J1,2 = 7.37), 5.99 (t, 0.43H, H-1b, J1,F-2 = 6.4, J1,2 = 6.4), 5.48 (m, H-4a and H-4b), 5.21 (dm, H-2b), 4.97 (ddd, H-3a), 4.80–4.86 (m, H-5a and H-5b), 4.65 (dm, H-3b), 4.50 (ddd, H-2a), 4.44–4.48 (m, H-5′a and H-5′b), 3.58 (s, OCH3 b), 3.54 (s, OCH3 a), 2.17 (s, OAc a), 2.15 (s, OAc b), 2.11 (s, OAc b), 2.10 (s, OAc a); 13C NMR (CDCl3): δ 170.7, 169.9, 169.4, 166.2 (4s, 2C=O, Ac and 2C=O, Bz), 133.4, 129.9, 129.8, 128.8, 129.7, 128.6 (s, 2C6H5CO-), 94.4 (dd, JC-1,F-2 = 30.1, JC-1,F-3 = 6.3, C-1b), 94.3 (dd, JC-1,F-2 = 29.2, JC-1,F-3 = 6.9, C-1a), 88.74 (dd, JC-2,F-2 = 185.5, JC-2,F-3 = 16.9, C-2b), 88.33 (dd, JC-2,F-2 = 183.5, JC-2,F-3 = 16.0, C-2a), 87.5 (dd, JC-3,F-3 = 181.5, JC-3,F-2 = 17.9, C-3a and C-3b), 68.1 (dd, C-4b), 67.9 (dd, C-4a), 62.0 (s, C-5a and C-5b), 58.63 (s, OCH3 b), 58.0 (s, OCH3 a), 21.06 (s, CH3CO), 21.02 (s, CH3CO), 20.9 (s, CH3CO); 19F NMR (CDCl3): δ -214.07 (F-2, dddd), -214.4 (F-3, m, JF-2,F-3 = 9.3) (compound α), -212.74 (F-2, dddd), -213.29 (F-3, m, JF-2,F-3 = 6.4) (compound β). HRMS (EI). Calcd for C17H20O7F2Na [M+Na]+: m/z 397.1100. Found: m/z 397.1106.
Method B
Concentrated H2SO4 (0.03 mL) was added to a solution of α-methyl glycoside 1 (0.1 g, 0.37 mmol) in acetic acid (0.78 mL) and acetic anhydride (0.19 mL) at 0°C. The reaction mixture was stirred at this temperature for 20 min and then 2 h at room temperature. The solution was poured into ice. After the ice melted, the aqueous phase was extracted with CH2Cl2 (3 × 20 mL). The combined organic phases were washed with aqueous NaHCO3, dried over anhydrous Na2SO4 and evaporated to dryness. The residue was chromatographed on a silica gel to afford 6 (0.096 g, 87%) as a syrup.
5-O-Benzoyl-2,3-dideoxy-2,3-difluoro-α- D-arabinofuranosyl bromide (8)
To a suspension of 6 (0.36 g, 1.2 mmol) and anhydrous ZnBr2 (0.065 g, 0.29 mmol) in anhydrous CH2Cl2 (10 mL) TMSBr (0.38 mL, 2.9 mmol) was added at 0°C. The resulting mixture was stirred at 0°C for 1 h, then 18 h at room temperature. The reaction mixture was poured into a cooled saturated solution of NaHCO3, extracted with CH2Cl2 (3 × 30 mL). The combined organic extracts were dried over anhydrous Na2SO4, evaporated to dryness, and co-evaporated with anhydrous toluene to give 8 (0.365 g, 95%) as a yellowish oil which was used in the next step without purification. 1H NMR (CDCl3): δ 7.46–8.04 (m, 5H, Bz), 6.55 (d, 1H, J1,2 < 1.0, J1,F-2 = 12.64, H-1), 5.57 (dd, 1H, J2,3 < 1.0, J2,F-2 = 49.65, J2,F-3 = 10.68, H-2), 5.21 (ddd, 1H, J3,4 = 3.7, J3,F-2 = 19.1, J3,F = 51.4, H-3), 4.85 (ddt, 1H, J4,F = 20.2, H-4), 4.67 (dd, 1H, J5,4 = 3.8, J5,5′ = 12.8, H-5), 4.62 (dd, 1H, J5,4 = 4.4, H-5′); 13C NMR (CDCl3): δ 166.1 (s, C=O, Bz), 133.6, 129.93, 129.28, 128.65 (4s, C6H5CO-), 100.1 (dd, JC-2,F-2 = 190.5, JC-2,F-3 = 26.37, C-2), 93.89 (dd, JC-3,F-2 = 32.01, JC-3,F-3 = 188.6, C-3), 86.57 (dd, JC-1,F-3 = 14.8, JC-1,F-2 = 31.95, C-1), 83.65 (d, JC-4, F-3 = 28.8, C-4), 61.9 (d, JC-5,F-3 = 5.65, C-5); 19F NMR (CDCl3): δ -170.98 (dm, F-2, JF-2,F-3 = 8.5), -188.31 (dt, F-3).
2,6-Dichloro-9-(5′-O-benzoyl-2′,3′-dideoxy-2′, 3′-difluoro-β-D-arabinofuranosyl)purine (9) and 2,6-dichloro-9-(5′-O-benzoyl-2′,3′-dideoxy-2′, 3′-difluoro-α-D-arabinofuranosyl)purine (10)
Method A
A solution of 1-α-bromide 8 (0.073 g, 0.227 mmol) in anhydrous MeCN (4 mL) was added to a suspension of the sodium salt of 2,6-dichloropurine, prepared from 2,6-dichloropurine (0.045 g, 0.238 mmol) and NaH (7.7 mg of 80% in oil, 0.024 mmol) in anhydrous MeCN (4 mL) under argon. The reaction mixture was stirred at room temperature overnight. Insoluble materials were removed by filtration and washed with MeCN (5 mL). The combined filtrate and washings were concentrated and the residue was chromatographed on silica gel (140 mL), eluting with EtOAc/toluene (ratio 1:8 and 1:4) to afford β- nucleoside 9 (0.054 g, 55%) as a colorless oil which crystallized during storage and α-nucleoside 10 (0.013 g, 13%) as a syrup.
Nucleoside 9
Mp 143–144°C; UV (EtOH), λmax, nm, (ε): 274 (5660), 231 (7300); 19F NMR (CDCl3): δ -188.77 (dm, F-2′or F-3′), -203.62 (m, F-3′ or F-2′). HRMS (EI). Calcd for C17H13N4O3F2Cl2 [M+H]+: m/z 429.0411. Found: m/z 429.0418.
Nucleoside 10
UV (EtOH), λmax, nm, (ε): 274 (5660), 231 (7300); 19F NMR (CDCl3): δ -190.48 (m, F-3′), -191.43 (m, F-2′). HRMS (EI). Calcd for C17H13N4O3F2Cl2 [M+H]+: m/z 429.0411. Found: m/z 429.0416.
Method B
To a solution of 2,6-dichloropurine (0.14 g, 0.74 mmol) in anhydrous 1,2-dimethoxyethane (11.5 mL) under argon at 0°C was added potassium t-butoxide (0.085 g, 0.75 mmol) and then the resulting solution was stirred for 12 min at room temperature before concentration. A solution of bromide 8 (0.2 g, 0.62 mmol) in anhydrous MeCN (17 mL) was added, under argon, to a suspension of the prepared potassium salt of purine in anhydrous MeCN (20 mL). The mixture was stirred under argon at room temperature for 18 h. Insoluble materials were removed by filtration and the solids were washed with MeCN (20 mL). The combined filtrate and washings were concentrated. The residue was dissolved in anhydrous CH2Cl2 (25 mL), and insoluble materials were filtered off and washed with CH2Cl2. The solvent was removed under reduced pressure and the residue was chromatographed on silica gel, eluting with EtOAc/toluene (ratio 1:8 and 1:4) to afford nucleoside 9 (0.195 g, 73%) and nucleoside 10 (0.02 g, 8%).
Methyl 5-O-benzoyl-2-O-acetyl-3-deoxy-3-fluoro-β- D-ribofuranoside (12)
Acetic anhydride (0.24 mL) was added to a solution of β-methyl riboside 11 (0.171 g, 0.63 mmol) in pyridine (3.5 mL) at room temperature. The mixture was stirred at this temperature for 48 h and then poured onto ice. After the ice melted, the aqueous phase was extracted with CH2Cl2 (3 × 20 mL). The combined organic phases were washed with aqueous NaHCO3, dried over anhydrous Na2SO4 and concentrated. The product was chromatographed on silica gel, eluting with EtOAc/hexane (ratio 1:6 and 1:4) to afford the acetate 12 (0.170 g, 86%) as a syrup. 1H NMR (CDCl3): δ 7.26–8.08 (m, 5H, Ar-H), 5.31 (dt, 1H, J3,F = 52.6, H-3), 5.19 (m, 1H, H-2), 4.99 (t, 1H, J1,2 = J1,F-3 =1.6, H-1), 4.52–4.91 (dm, 2H, 2H-5), 4.42 (dm, 1H, H-4), 3.36 (s, 3H, OCH3), 2.16 (s, 3H, COCH3); 13C NMR (CDCl3): δ 169.8 (s, C=O, Bz), 166.1 (s, C=O, OAc), 133.3, 129.75, 129.62, 129.40 (4s, C6H5CO-), 106.05 (s, C-1), 90.0 (d, JC-3,F-3 = 193.7, C-3), 79.25 (d, JC-2,F-2= 25.2, C-2), 75.0 (d, JC-4,F-3=13.97, C-4), 63.92 (d, JC-5,F-3 = 4.68, C-5), 55.75 (s, OCH3), 20.61 (s, COCH3); 19F NMR (CDCl3): δ -210.63 (ddd, F-3). HRMS (EI). Calcd for C15H17O6FNa [M+Na]+: m/z 335.0997. Found: m/z 335.1005.
1,2-Di-O-acetyl-5-O-benzoyl-3-deoxy-3-fluoro-α/β- D-ribofuranose (13)
β-Methyl riboside 12 (0.17 g, 0.54 mmol) was dissolved in a mixture of acetic acid (1.16 mL), acetic anhydride (0.28 mL) and concentrated H2SO4 (0.05 mL) at 0–5°C. The mixture was stirred at this temperature for 5 min and then 150 min at room temperature. The solution was poured onto ice. After the ice melted, the aqueous phase was extracted with CHCl3 (3 × 20 mL), and then after adding cold aqueous 5% NaHCO3 to the aqueous phase, it was again extracted with CHCl3 (2 × 20 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated to dryness to afford 13 (0.179 g, 97%) as a syrup. 1H NMR (CDCl3): (ratio of α and β anomers ca. 1.0:1.0), δ 8.09 (d, 2H, Bz), 8.04 (d, 2H, Bz), 7.60 (t, 2H, Bz), 7.49 (m, 4H, Bz), 6.53 (d,1H, J1,2 = 4.6, H-1a), 6.26 (t, 1H, J1,2 = J1,F-3 =1.8, H-1b), 5.37 (dt, 1H, H-3a), 5.26 (dm, 1H, H-3b), 4.81 (dm, 1H, H-4a), 4.63–4.71 (2m, 2H, 2H-5b), 4.55 (dd, 1H, H-5a), 4.49 (dd, 1H, H-5′a), 4.46 (dm, 1H, H-4b), 2.20, 2.19, 2.17 and 1.97 (4s, 12H, 4 COCH3); 13C NMR (CDCl3): δ 169.91, 169.78, 169.59, 169.10, 165.89, 165.85 (6s, C=O, 2Bz and 4Ac), 133.53, 133.45, 129.74, 129.45, 129.18, 128.65, 128.54 (8s, 2C6H5CO-), 98.2 and 93.7 (2s, C-1a, C-1b), 89.1 and 88.4 (2d, JC-3,F-3 = 194.4, JC-3,F-3 = 191.56, C-3a, C-3b), 82.25 and 80.7 (2d, JC-2,F-2 = 25.2, JC-2,F-2 = 25.0, C-2a, C-2b), 74.7 and 71.2 (2d, JC-4,F-3 = 14.4, JC-4,F-3 = 15.1, C-4a, C-4b), 63.36 and 63.14 (2d, JC-5a,F-3a = 9.16, JC-5b,F-3b = 5.21, C-5a, C-5b), 21.1, 20.1, 20.47, 20.37 (4s, COCH3); 19F NMR (CDCl3): δ 197.91 (dd, F-3a), -184.78 (dt, F-3b). HRMS (EI). Calcd for C16H17O7FNa [M+Na]+: m/z 363.0947. Found: m/z 363.0952.
2,6-Dichloro-9-(5′-O-benzoyl-2′-O-acetyl-3′-deoxy- 3′-fluoro-β-D-ribofuranosyl)purine (14)
A mixture of 2,6-dichloropurine (0.148 g, 0.33 mmol) and a catalytic amount of (NH4)2SO4, HMDS (3 mL) and anhydrous toluene (12 mL) was heated under reflux for 2 h under argon. After cooling the clear solution was concentrated under reduced pressure to give a residue. To a solution of this trimethylsilyl derivative and the diacetate 13 (0.217 g, 0.64 mmol) in a mixture of anhydrous MeCN (10.8 mL) and 1,2-dichloroethane (2.7 mL) was added TMSOTf (0.18 mL, 0.96 mmol) and the reaction mixture was stirred at room temperature for 90 min. After the standard work-up, the residue was purified by chromatography on silica gel, eluting with EtOAc/hexane (ratio 1:2 and 1:1) to yield nucleoside 14 (0.266 g, 89%) as a syrup. 1H NMR (CDCl3): δ 8.21 (s, 1H, H-8), 7.44-8.03 (m, 5H, Bz), 6.31 (d, 1H, J1′,2′ = 7.1, H-1′), 5.92 (ddd, 1H, J2′,3′ = 4.7, J2′,F-3′ = 18.65, H-2′), 5.65 (ddd, 1H, J3′,4′ = 1.82, J3′,F-3′ = 52.7, H-3′), 4.79–4.82 (m, 2H, H-4′ and H-5′), 4.61 (dm, 1H, H-5″), 2.19 (s, 3H, COCH3); 13C NMR (CDCl3): δ 169.7 (s, C=O, Bz), 165.8 (s, C=O, OAc), 153.4, 152.60, 152.40 (3s, C-2, C-4, C-6), 144.0 (s, C-8), 133.8, 131.4, 129.67, 129.58, 128.8 (5s, C6H5CO-, C-5), 89.3 (d, JC-3′,F-3′ = 190.98, C-3′), 85.9 (s, C-1′), 81.8 (d, JC-2′,F-3′ = 24.5, C-2′), 73.45 (dd, JC-4′,F-3′ = 15.58, C-4′), 62.95 (d, JC-5,F-3 = 8.93, C-5′), 20.6 (s, COCH3); 19F NMR (CDCl3): δ -198.59 (dt, F-3′); UV (EtOH), λmax, nm, (ε): 274 (6500), 230 (11300). HRMS (EI). Calcd for C14H14O5F [M-base]+: m/z 281.0925. Found: m/z 281.0928.
2,6-Dichloro-9-(5′-O-benzoyl-3′-deoxy-3′-fluoro-β- D-ribofuranosyl)purine (15)
A cooled solution of nucleoside 14 (0.175 g, 0.37 mmol) in anhydrous MeOH (14.0 mL) was treated with solid anhydrous NaHCO3 (0.117 g, 1.4 mmol). The reaction mixture was stirred at room temperature for 1 h, then neutralized with glacial acetic acid, concentrated, and co-evaporated with ethanol to dryness. The residue was chromatographed on silica gel, eluting with EtOAc/hexane (ratio 1:2 and 1:1) to afford nucleoside 15 (0.1 g, 63%) as an amorphous powder. 1H NMR (CDCl3 +CD3OD): δ 8.35 (s, 1H, H-8), 7.43–8.01 (m, 5H, Bz), 6.11 (d, 1H, J1′,2′ = 7.0, H-1′), 5.30 (ddd, 1H, J3′,2′ = 4.4, J3′,4′ = 1.85, J3′,F-3′ = 53.5, H-3′), 5.04 (ddd, 1H, J2′,F-3′ = 20.49, H-2′), 4.64–4.79 (m, 2H, H-4′ and H-5′), 4.58 (dd, 1H, H-5″); 13C NMR (CDCl3): δ 166.1 (s, C=O, Bz), 153.0, 152.6, 151.8 (3s, C-2, C-4, C-6), 145.1 (s, C-8), 133.5, 131.1, 128.80, 128.50 (4s, C6H5CO-), 129.4 (C-5), 91.1 (d, JC-3′,F-3′ = 190.98, C-3′), 88.0 (s, C-1′), 80.9 (d, JC-4′,F-3′ = 24.5, C-4′), 72.8 (dd, JC-2′,F-3′ = 15.58, C-2′), 63.0 (d, JC-5′,F-3′ = 8.93, C-5′); 19F NMR (CDCl3): -200.58 (dt, F-3′); UV (EtOH) λmax, nm, (ε): 274 (5580), 231 (9500). HRMS (EI). Calcd for C17H14N4O4FCl2 [M+H]+: m/z 427.0368. Found: m/z 427.0363.
2,6-Dichloro-9-(5′-O-benzoyl-2′,3′-dideoxy-2′, 3′-difluoro-β-D-arabinofuranosyl)purine (9) from nucleoside 15
To a suspension of nucleoside 15 (0.1 g, 0.23 mmol) in anhydrous dichloromethane (4.5 mL) was added pyridine (0.058 mL, 0.72 mmol) and diethylaminosulfur trifluoride (0.086 mL, 0.64 mmol) at 0°C. The reaction mixture was stirred at room temperature (25°C) for 14 h, and then poured onto cold aqueous 5% solution of NaHCO3. The aqueous phase was extracted with CHCl3 (3 × 30 mL), the combined organic phases were dried over anhydrous Na2SO4 and concentrated to dryness. The residue was chromatographed on silica gel, using a linear gradient AcOEt in hexane (0→33%) to afford nucleoside 9 (0.035 g, 35%) as a colorless oil.
2-Chloro-6-methoxy-9-(2′,3′-dideoxy-2′,3′-difluoro-β- D-arabinofuranosyl)purine (16)
To a solution of nucleoside 9 (0.008 g, 0.019 mmol) in anhydrous MeOH (1.7 mL), 0.1 mL of a 0.22 N solution of sodium methoxide in methanol was added. The reaction mixture was stirred at room temperature for 18 h, then neutralized with acetic acid, concentrated, and co-evaporated with a mixture of toluene/ethanol (1:1, 20 mL) to dryness. The residue was chromatographed on silica gel, eluting with CHCl3/MeOH (ratio 20:1 and 10:1) to afford nucleoside 16 (0.005 g, 84%); mp 174–176°C (EtOH); UV (EtOH) λmax, nm, (ε): 207 (14250), 258 (10100); 19F NMR (CD3OD): δ -196.61 (m, F-2′ or F-3′), -204.7 (m, F-3′ or F-2′). HRMS (EI). Calcd for C11H12N4O3F2Cl [M+H]+: m/z 321.0566. Found: m/z 321.0562.
2,6-Dimethoxy-9-(2′,3′-dideoxy-2′,3′-difluoro-β- D-arabinofuranosyl)purine (17)
To a solution of nucleoside 9 (0.031 g, 0.072 mmol) in anhydrous MeOH (2.5 mL), 0.22 mL of a 1 N solution of sodium methoxide in methanol was added. The reaction mixture was stirred at room temperature for 18 h, heated under reflux for 90 min, neutralized with acetic acid, and concentrated and co-evaporated with a mixture of toluene/ethanol (1:1, 50 mL) to dryness. The residue was chromatographed on silica gel, eluting with CHCl3, CHCl3/hexane/MeOH (10:5:1) to afford nucleoside 17 (0.012 g, 52%) as a syrup. UV (EtOH) λmax, nm, (ε): 212 (6500), 240 (9500), 262 (10300); 19F NMR (CD3OD): δ -196.28 (m, F-2′ or F-3′), -204.6 (m, F-3′ or F-2′). HRMS (EI). Calcd for C12H15N4O4F2 [M+H]+: m/z 317.1061. Found: m/z 317.1066. Calcd for C12H14N4O4F2Na [M+Na]+: m/z 339.0081. Found: m/z 339.0081.
2-Chloro-6-benzylamino-9-(5′-O-benzoyl-2′,3′-dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)purine (18)
To a solution of nucleoside 9 (0.011 g, 0.025 mmol) in anhydrous MeOH (2.3 mL) was added benzylamine (0.014 mL, 0.128 mmol). The reaction mixture was stirred at 55°C for 4 h, and then concentrated. The residue was chromatographed on silica gel, eluting with EtOAc/hexane (ratio 2:3 and 3:2) to afford nucleoside 18 (0.01 g, 78%); mp 164–166°C (MeOH); 1H NMR (CDCl3): δ 7.3–8.07 (5m, 10H, Bz and C6H5CH2-), 7.92 (br.s, 1H, H-8), 6.53 (dt, 1H, J1′,F-2′ = 22.6, H-1′), 6.29 (br.s, 1H, NH), 5.25-5.48 (m, 2H, H-2′ and H-3′), 4.82 (br.s, 2H, -CH2C6H5), 4.54-4.7 (m, 3H, H-5′, H-5″ and H-4′); 13C NMR (CDCl3): δ 166.2 (s, C=O, Bz), 156.1, 149.8, 137.9 (C-6, C-2, C-4), 139.45 (d, JC-8,F-2′ = 4.6, C-8), 137.9, 133.7, 129.94, 129.86, 129.25, 128.93, 128.75, 128.19, 127.85 (C6H5CO- and C6H5CH2-), 118.2 (C-5), 93.9 (dd, JC-2′,F-2′ = 183.5, JC-2′,F-3′ = 30.3, C-2′), 92.0 (dd, JC-3′,F-3′ = 192.49, JC-3′,F-2′ = 30.1, C-3′), 83.2 (d, JC-1′, F-2′ = 16.8, C-1′), 80.6 (d, JC-4′,F-3′ = 27.2, C-4′), 62.7 (d, JC-5′,F-3′ = 9.3, C-5′), 45.0 (s, C6H5CH2-); 19F NMR (CDCl3): δ -188.8 (m, F-2′ or F-3′), -203.78 (m, F-3′ or F-2); UV (MeOH) λmax, nm, (ε): 216 (17300) 232 sh, 272 (12100). HRMS (EI). Calcd for C24H20N5O3F2ClNa [M+Na]+: m/z 522.1120. Found: m/z 522.1126.
2-Chloro-6-benzylamino-9-(2′,3′-dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)purine (19)
A solution of nucleoside 18 (0.01 g, 0.02 mmol) in MeOH (6 mL) saturated at 0°C with ammonia was kept for 24 h at room temperature and then evaporated. The residue was chromatographed on silica gel, eluting with CH2Cl2, then CH2Cl2/MeOH (ratio 30:1 and 6:1) to afford nucleoside 19 (0.0065 g, 82%) as a syrup. 1H NMR (CD3OD): δ 8.19 (br.s, 1H, H-8), 7.21–7.39 (d and 2t, 5H, C6H5CH2-), 6.53 (ddd, 1H, H-1′), 5.36–5.45 (m, 2H, H-2′ and H-3′), 4.74 (br.s, 2H, -CH2C6H5), 4.26 (ddt, 1H, H-4′), 3.85 (dd, 1H, H-5′), 3.82 (dd, 1H, H-5″); 13C NMR (CD3OD): δ 154.6, 149.6, 131.6 (C-6, C-2, C-4), 140.0 (br.s, C-8), 129.4, 128.25, 127.5, 128.3, 127.0 (C6H5CH2-), 117.7 (C-5), 93.6 (dd, JC-2′,F-2′ = 182.1, JC-2′,F-3′ = 28.3, C-2′), 92.7 (dd, JC-3′,F-2′ = 28.6, JC-3′,F-3′ = 192.0, C-3′), 82.9 (d, JC-1′, F-2′ = 17.7, C-1′), 82.0 (d, JC-4′,F-3′ = 26.0, C-4′), 60.2 (d, JC-5′,F-3′ = 4.6, C-5′), 43.9 (s, C6H5CH2-); 19F NMR (CD3OD): δ -196.1 (m, F-2′ or F-3′), -204.58 (m, F-3′ or F-2′); UV (MeOH) λmax, nm, (ε): 211 (12600), 271 (8900). HRMS (EI). Calcd for C17H17N5O2F2Cl [M+H]+: m/z 396.1039. Found: m/z 396.1034.
2,6-Diazido-9-(5′-O-benzoyl-2′,3′-dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)purine (20)
Nucleoside 9 (0.075 g, 0.175 mmol) was treated with LiN3 (0.045 g, 0.92 mmol) in EtOH (10 mL) under reflux for 110 min. The reaction mixture was concentrated and the residue was dissolved in chloroform (5 mL). After filtration, the filtrate was concentrated and the residue was chromatographed on silica gel, eluting with EtOAc/petroleum ether (ratio 1:5, 1:4 and 1:2) to afford nucleoside 20 (0.075 g, 97%) as an amorphous powder. 1H NMR (CDCl3): δ 8.10 (d, 1H, JH-8, F-2′ = 1.93, H-8), 7.44–8.06 (3m, 5H, Bz), 6.53 (dt, 1H, J1′,2′ = 2.56, J1′,F-2′ = 22.1, J1′,F-3′ = 2.56, H-1′), 5.44 (dd, 1H, J3′,4′ < 1.0, J3′,F-2′ = 12.42, J3′,F′ = 50.44, H-3′), 5.31 (ddd, 1H, J2′,3′ < 1.0, J2′,F-2′ = 49.51, J2′,F-3′ = 9.46, H-2′), 4.62 (dm, 1H, H-4′), 4.69 (dd, 1H, H-5′), 4.65 (dd, 1H, H-5″); 13C NMR (CDCl3): δ 166.1 (s, C=O, Bz), 133.7, 129.85, 129.19, 128.8 (C6H5CO-), 156.6, 154.2, 153.5 (C-2, C-4, C-6), 142.5 (d, JC-8,F-2′ = 6.0, C-8), 121.0 (C-5), 93.04 (dd, JC-2′,F-2′ = 184.7, JC-2′,F-3′ = 30.3, C-2′), 93.89 (dd, JC-3′,F-2′ = 30.0, JC-3′,F-3′ = 192.1, C-3′), 83.4 (d, JC-1′, F-2′ = 16.8, C-1′), 80.8 (d, JC-4′,F-3′ = 27.1, C-1′), 62.55 (d, JC-5′,F-3′ = 8.9, C-5′); 19F NMR (CDCl3): δ -188.9 (m, F-2′), -203.74 (m, F-3′); UV (EtOH) λmax, nm, (ε): 232 (7300), 270 (2240), 297 (1120). HRMS (EI). Calcd for C17H13N10O3F2 [M+H]+: m/z 443.1140. Found: m/z 443.1140.
2,6-Diazido-9-(5′-O-benzoyl-2′,3′-dideoxy-2′,3′-difluoro-α-D-arabinofuranosyl)purine (21)
Starting from α-nucleoside 10 (0.014 g, 0.033 mmol) and using the procedure described above for the preparation of 20, nucleoside 21 (0.014 g, 97%) was obtained as a syrup. 1H NMR (CDCl3): δ 8.02 (s, 1H, H-8), 7.46–8.08 (m, 5H, Bz), 6.44 (dd, 1H, J1′,2′ = 1.0, J1′,F-2′ = 15.6, H-1′), 5.88 (ddt, 1H, J2′,F-2′ = 48.82, J2′,F-3′ = 12.3, H-2′), 5.44 (dddd, 1H, J3′,4′ = 2.5, J3′,F-2′ = 13.4, J3′,F-3′ = 50.0, H-3′), 5.05 (dm, 1H, H-4′), 4.62 (dd, 1H, H-5′), 4.57 (dd, 1H, H-5″); 13C NMR (CDCl3): δ 166.0 (C=O, Bz), 133.6, 129.83, 129.10, 128.6 (C6H5CO-), 156.6, 154.4, 153.1 (C-2, C-4, C-6), 141.2 (d, JC-8,F-2′ = 3.6, C-8), 121.7 (C-5), 96.3 (dd, JC-2′,F-2′ = 188.0, JC-2′,F-3′ = 28.9, C-2′), 94.2 (dd, JC-3′,F-2′ = 29.1, JC-3′,F-3′ = 184.87, C-3′), 88.5 (dd, JC-1′,F-2′ = 36.9, JC-1′,F-3′ = 2.18, C-1′), 83.45 (d, JC-4′,F-3′ = 25.88, C-4′), 62.4 (d, JC-5′,F-3′ = 7.3, C-5′); 19F NMR (CDCl3): δ -191.47 (dm, F-2′), -191.85 (m, F-3′); UV (EtOH) λmax, nm (ε): 228 (7300), 271 (2240), 298 (1120). HRMS (EI). Calcd for C17H13N10O3F2 [M+H]+: m/z 443.1140. Found: m/z 443.1142.
2,6-Diamino-9-(5′-O-benzoyl-2′,3′-dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)purine (23)
Anhydrous SnCl2 (0.081 g, 0.427 mmol) was added at room temperature, under argon, to a solution of nucleoside 20 (0.075 g, 0.169 mmol) in a mixture of anhydrous CH2Cl2 (10 mL) and MeOH (1.2 mL). The reaction mixture was stirred for 2 h, and then poured onto a cooled saturated aqueous solution of NaHCO3. After stirring, the prepared suspension was filtered and the precipitate was washed with CHCl3 (30 mL). After separation of the organic phase, the aqueous layer was extracted again with CHCl3 (3 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The residue was chromatographed on silica gel, eluting with EtOAc/hexane (2:1) and EtOAc/hexane/MeOH (ratio 20:10:2) to afford nucleoside 22 (0.003 g, 4%) as a syrup and nucleoside 23 (0.058 g, 87%).
Nucleoside 22
1H NMR (CDCl3): δ 7.92 (d, 1H, JH-8, F-2′ = 3.1, H-8), 7.46–8.08 (m, 5H, Bz), 6.48 (dt, 1H, J1′,2′ = J1′,F-3′ = 2.56, J1′,F-2′ = 22.7, H-1′), 5.73 (br.s, 2H, NH2), 5.44 (dd, 1H, J3′,4′ ~ 1.6, J3′,F-2′ = 12.6, J3′,F′ = 49.7, H-3′), 5.26 (ddd, 1H, J2′,3′ < 1.0, J2′,F-2′ = 49.4, J2′,F-3′ = 9.3, H-2′), 4.69 (dd, 1H, H-5′), 4.56–4.66 (m, 2H, H-5″ and H-4′); 13C NMR (CDCl3): δ 166.15 (s, C=O, Bz), 157.2, 156.0, 151.2 (C-6, C-4, C-2), 139.4 (d, JC-8,F-2′ = 6.0, C-8), 133.7, 129.86, 129.28, 128.7 (C6H5CO-), 116.6 (C-5), 93.9 (dd, JC-2′,F-2′ = 184.4, JC-2′,F-3′ = 30.16, C-2′), 91.7 (dd, JC-3′,F-2′ = 29.8, JC-3′,F-3′ = 191.29, C-3′), 83.2 (d, JC-1′,F-2′ = 16.9, C-1′), 80.5 (d, JC-4′,F-3′ = 27.9, C-4′), 62.7 (d, JC-5′,F-3′ = 8.9, C-5′); 19F NMR (CDCl3): δ -188.88 (m, F-2′ or F-3′), -203.85 (m, F-3′ or F-2′); IR (film): 2120 cm-1 (N3); UV (EtOH) λmax, nm, (ε): 232 (12400), 270 (8100). HRMS (EI). Calcd for C17H15N8O3F2 [M+H]+: m/z 417.1235. Found: m/z 417.1239.
Nucleoside 23
mp 90–92°C; 1H NMR (CDCl3): δ 7.73 (d, 1H, JH-8, F-2′ = 3.2, H-8), 7.46–8.05 (3m, 5H, Bz), 6.37 (dt, 1H, J1′,2′ = J1′,F-3′ = 3.2, J1′,F-2′ = 23.02, H-1′), 5.67 (br.s, 2H, NH2), 5.41 (ddd, 1H, J3′,4′ = 1.3, J3′,F-2′ = 12.9, J3′,F′ = 49.95, H-3′), 5.26 (ddd, 1H, J2′,3′ < 1.0, J2′,F-2′ = 49.46, J2′,F-3′ = 9.71, H-2′), 4.85 (br.s, 2H, NH2), 4.66 (dd, 1H, H-5′), 4.62 (dd, 1H, H-5″), 4.56 (ddt, 1H, H-4′); 13C NMR (CDCl3): δ 166.2 (s, C=O, Bz), 160.2, 156.1, 151.9 (C-6, C-4, C-2), 137.1 (d, JC-8,F-2′ = 6.4, C-8), 133.7, 129.88, 129.32, 128.73, 128.47 (C6H5CO-), 113.7 (C-5), 94.1 (dd, JC-2′,F-2′ = 183.7, JC-2′,F-3′ = 30.2, C-2′), 91.8 (dd, JC-3′,F-2′ = 29.8, JC-3′,F-3′ = 191.42, C-3′), 82.8 (d, JC-1′, F-2′ = 16.9, C-1′), 80.1 (d, JC-4′,F-3′ = 27.0, C-4′), 62.8 (d, JC-5′,F-3′ = 8.5,C-5′); 19F NMR (CDCl3): δ -188.85 (m, F-2′ or F-3′), -203.75 (m, F-3′ or F-2′); UV (EtOH) λmax, nm, (ε): 235 (7350), 256 (6690), 277 (6180). HRMS (EI). Calcd for C17H17N6O3F2 [M+H]+: m/z 391.1396. Found: m/z 391.1422.
2,6-Diamino-9-(5′-O-benzoyl-2′,3′-dideoxy-2′,3′-difluoro-α-D-arabinofuranosyl)purine (24)
Starting from α-nucleoside 21 (0.015 g, 0.034 mmol) and using the procedure described above for the preparation of 20, nucleoside 24 (0.012 g, 91%) was obtained as a syrup; 1H NMR (CDCl3): δ 7.63 (s, 1H, H-8), 7.42–8.07 (m, 5H, Bz), 6.26 (dd, 1H, J1′,2′<1.0, J1′,F-2′ = 16.5, J1′,F-3′ = 1.56, H-1′), 6.01 (ddt, 1H, J2′,3′ = 1.9, J2′,F-2′ = 49.7, J2′,F-3′ = 13.1, H-2′), 5.44 (br.s, 2H, NH2), 5.38 (ddd, 1H, J3′,4′ = 1.3, J3′,F-2′ = 16.0, J3′,F′ n.d., H-3′), 5.01 (dq, 1H, H-4′), 4.77 (br.s, 2H, NH2), 4.60 (dd, 1H, H-5′), 4.57 (dd, 1H, H-5″); 19F NMR (CDCl3): δ -191.72 (m, F-2′ or F-3′), -194.23 (m, F-3′ or F-2′); UV (EtOH) λmax, nm, (ε): 235 (7350), 256 (6600), 277 (6150). HRMS (EI). Calcd for C17H17N6O3F2 [M+H]+: m/z 391.1396. Found: m/z 391.1420.
2,6-Diamino-9-(2′,3′-dideoxy-2′,3′-difluoro-β- D-arabinofuranosyl)purine (25)
A solution of nucleoside 23 (0.058 g, 0.164 mmol) in MeOH (25 mL) saturated at 0°C with ammonia was kept for 18 h at room temperature and then concentrated. The residue was chromatographed on silica gel, eluting with CHCl3, CHCl3/MeOH (ratio 15:1 and 5:1) to afford nucleoside 25 (0.031 g, 72%); mp 236–238°C (EtOH); UV (EtOH) λmax, nm (ε): 215 (18450), 256 (7020), 277 (7280); 19F NMR (CD3OD): δ -195.19 (m, F-2′ or F-3′), -204.95 (m, F-3′ or F-2′). HRMS (EI). Calcd for C10H13N6O2F2 [M+H]+: m/z 287.1100. Found: m/z 287.1103.
2,6-Diamino-9-(2′,3′-dideoxy-2′,3′-difluoro-α- D-arabinofuranosyl)purine (26)
Starting from α-nucleoside 24 (0.012 g, 91%) and using the procedure described above for the preparation 23, nucleoside 26 (0.007 g, 79%) was prepared as an amorphous powder; UV (EtOH) λmax, nm (ε): 215 (18400), 256 (7000), 277 (7240); 19F NMR (CDCl3): δ -196.56 (m, F-2′ or F-3′), -197.764 (m, F-3′ or F-2′). HRMS (EI). Calcd for C10H13N6O2F2 [M+H]+: m/z 287.1100. Found: m/z 287.1104.
9-(2′,3′-Dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)guanine (27) from nucleoside 25
To a solution of nucleoside 25 (0.02 g, 0.07 mmol) in water (3 mL) was added adenosine deaminase (15 μL). The resulting solution was stirred at 23°C for 48 h, and concentrated after addition of methanol. The residue was chromatographed on silica gel, eluting with CHCl3, CHCl3/MeOH (ratio 10:1 and 5:1) to afford nucleoside 25 (0.017 g, 85%); mp > 260°C (MeOH); UV (H2O) λmax, nm (ε): 251 (14200), 270 (sh); 19F NMR (DMSO-d6): δ -191.72 (m, F-2′ or F-3′), -194.23 (m, F-3′ or F-2′). HRMS (EI). Calcd for C10H12N5O3F2 [M+H]+: m/z 288.0908. Found: m/z 288.0904. Calcd for C10H11N5O3F2Na [M+Na]+: m/z 310.0728. Found: m/z 310.0724.
2-Amino-6-chloro-9-(5′-O-benzoyl-2′,3′-dideoxy-2′, 3′-difluoro-β-D-arabinofuranosyl)purine (28) and 2-amino-6-chloro-9-(5′-O-benzoyl-2′,3′-dideoxy-2′, 3′-difluoro-α-D-arabinofuranosyl)purine (29)
2-Amino-6-chloropurine potassium salt was prepared by adding (0.024 g, 0.151 mmol) potassium t-butoxide to a solution of 2-amino-6-chloropurine (0.036 g, 0.21 mmol) in anhydrous 1,2-dimethoxyethane (11 mL) under argon at 0°C and the resulting solution was stirred for 40 min at room temperature and then concentrated. To a suspension of the prepared potassium salt of 2-amino-6-chloropurine in anhydrous MeCN (9 mL) was added, under argon, a solution of bromide 8 (0.060 g, 0.187 mmol) in anhydrous MeCN (5 mL). The reaction mixture was stirred under argon at room temperature for 18 h. Insoluble materials were removed by filtration and the solids were washed with MeCN (5 mL). The combined filtrate and washings were concentrated. The residue was dissolved in anhydrous CH2Cl2 (5 mL), filtered off and insoluble materials were washed with CH2Cl2. The solvent was removed under reduced pressure, and the residue was chromatographed on silica gel, eluting with EtOAc/hexane (1:1) to afford β-nucleoside 28 (0.038 g, 50%) as a white amorphous powder and α-nucleoside 29 (0.003 g, 8%) as a syrup.
β-Nucleoside 28
1H NMR (CDCl3): δ 7.97 (d, 1H, JH-8, F-2′ = 2.8, H-8), 7.45–8.05 (m, 5H, Bz), 6.41 (dt, 1H, J1′,2′ = 2.5, J1′,F-2′ = 19.8, H-1′), 5.46 (dd, 1H, J2′,3′ < 1.0, J2′,F-2′ = 49.9, J2′,F-3′ = 12.78, H-2′), 5.29 (ddd, 1H, J3′,4′ = 2.46, J3′,F-2′ = 25.0, J3′,F′ = 49.95, H-3′), 5.29 (br.s, 2H, NH2), 4.67 (d, 2H, H-5′ and H-5″), 4.58 (ddt, 1H, H-4″); 13C NMR (CDCl3): δ 166.2 (s, C=O, Bz), 133.7, 129.85, 129.19, 128.75 (C6H5CO-), 159.2, 153.4, 151.9 (C-2, C-6, C-4), 141.1 (d, JC-8,F-2′ = 6.1, C-8), 125.0 (C-5), 94.1 (dd, JC-2′,F-2′ = 183.9, JC-2′,F-3′ = 30.0, C-2′), 91.68 (dd, JC-3′,F-2′ = 30.0, JC-3′,F-3′ = 192.07, C-3′), 83.15 (d, JC-1′,F-2′ = 16.7, C-1′), 80.6 (d, JC-4′, F-3′ = 27.1, C-4′), 62.6 (d, JC-5′,F-3′ = 8.9, C-5′); 19F NMR (CDCl3): δ -188.82 (m, F-2′ or F-3′), -203.76 (m, F-3′ or F-2′); UV (MeOH) λmax, nm, (ε): 232 (16350), 308 (6600). HRMS (EI). Calcd for C17H15N5O3F2Cl [M+H]+: m/z 410.0831. Found: m/z 410.0834.
α-Nucleoside 29
1H NMR (CDCl3): δ 7.88 (s, 1H, H-8), 7.46–8.08 (3m, 5H, Bz), 6.34 (d, 1H, J1′,F-2′ = 15.8, H-1′), 5.90 (dd, 1H, J2′,3′ < 1.0, J2′,F-2′ = 48.6, J2′,F-3′ = 12.49, H-2′), 5.42 (ddd, 1H, J3′,4′ = 2.46, J3′,F-2′ = 25.0, J3′,F′ = 49.95, H-3′), 5.16 (br.s, 2H, NH2), 5.02 (dm, 1H, H-4′). 4.61 (dd, 1-H, H-5′), 4.58 (dd, 1H, H-5″); 13C NMR (CDCl3): δ 166.0 (C=O, Bz), 133.6, 129.83, 129.17, 128.6 (C6H5CO-), 159.1, 153.0, 152.0 (C-2, C-6, C-4), 139.8 (d, JC-8,F-2′ = 3.2, C-8), 125.7 (C-5), 96.3 (dd, JC-2′,F-2′ = 187.8, JC-2′,F-3′ = 29.1, C-2′), 94.3 (dd, JC-3′,F-2′ = 29.6, JC-3′,F-3′ = 185.7, C-3′), 88.2 (dd, JC-1′,F-2′ = 36.4, JC-1′,F-3′ = 2.9, C-1′), 83.1 (d, JC-4′,F-3′ = 25.6, C-4′), 62.5 (d, JC-5′,F-3′ = 6.6, C-5′); 19F NMR (CDCl3): δ -191.63 (m, F-2′ or F-3′), -192.6 (m, F-3′ or F-2′); UV (MeOH) λmax, nm, (ε): 232 (16450), 308 (6700). HRMS (EI). Calcd for C17H15N5O3F2Cl [M+H]+: m/z 410.0831. Found: m/z 410.0833.
2-Amino-6-chloro-9-(2′,3′-dideoxy-2′,3′-difluoro-β- D-arabinofuranosyl)purine (30)
A solution of nucleoside 28 (0.018 g, 0.043 mmol) in MeOH (5 mL) saturated at 0°C with ammonia was kept for 18 h at room temperature and then concentrated. The residue was chromatographed on silica gel, eluting with CHCl3, then CHCl3/MeOH (ratio 15:1 and 5:1) to afford nucleoside 30 (0.01 g, 77%) as a syrup; UV (EtOH) λmax, nm, (ε): 220 (18900), 247 (9000), 309 (7600); 1H NMR (CD3OD): δ 8.21 (d, 1H, JH-8, F-2′ = 2.4, H-8), 6.43 (ddd, 1H, J1′,2′ = 3.5, J1′,F-2′ = 17.3, J1′,F-3′ = 1.9, H-1′), 5.40–5.45 (dm, 2H, H-2′ and H-3′), 4.25 (dm, 1H, H-4′), 3.85 (dd, 1H, H-5′), 3.83 (dd, 1H, H-5″); 19F NMR (CD3OD): δ -195.72 (m, F-2′ or F-3′), -204.7 (m, F-3′ or F-2′). HRMS (EI). Calcd for C10H11N5O2F2Cl [M+H]+: m/z 306.0569. Found: m/z 306.0573.
9-(2′,3′-Dideoxy-2′,3′-difluoro-β-D-arabinofuranosyl)guanine (27) from nucleoside 28
To a solution of nucleoside 28 (0.02 g, 0.049 mmol) in MeOH (2 mL), 2-mercaptoethanol (0.14 mL, 0.2 mmol) and sodium methoxide 0.011 g (0.2 mmol) were added. The reaction mixture was heated under reflux for 3 h. After cooling to room temperature, the resulting solution was neutralized with acetic acid and solvents were removed under reduced pressure. The residue was purified on silica gel, eluting with CHCl3, then CHCl3/MeOH (ratio 10:1 and 5:1) to afford nucleoside 27 (0.01 g, 71%).
Dedication: This work is dedicated to our friend and colleague Dr. Kyoichi (Kyo) Watanabe who passed away on April 7, 2015. We will miss his wisdom, encyclopedic memory on nucleosides, and adorable smiling face.
Acknowledgments
This work was supported in part by NIH grant 5P30-AI-50409 (CFAR), by the Department of Veterans Affairs and by the Belarus State Program of FOI ‘Chempharmsynthesis’(Grants 2.19 and 4.20). Dr. Schinazi is the Chairman and a major shareholder of CoCrystal Pharma, Inc. Emory University received no funding from Cocrystal Pharma, Inc., to perform this work and vice versa.
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