Abstract
New xylofuranosyl and glucopyranosyl nucleoside phosphoramidates were synthesized as potential mimetics of nucleoside 5′-monophosphates. Their access involved N-glycosylation of uracil and 2-acetamido-6-chloropurine with 5′/6′-azido-1,2-di-O-acetyl glycosyl donors and subsequent Staudinger-phosphite reaction of the resulting azido nucleosides. The coupling of the purine derivative with the pyranosyl donor furnished N9- and N7-linked nucleosides in 1:1 ratio, whereas with the furanosyl donor, the N9-nucleoside was the major regioisomer formed. When using uracil, only 5′/6′-azido N1-linked nucleosides were obtained. The purine 5′/6′-azido nucleosides were converted into corresponding phosphoramidates in good yields. The antiproliferative effects of the nucleoside phosphoramidates and those of the azido counterparts on cancer cells were evaluated. While the nucleoside phosphoramidates did not show significant activities, the purine 5′/6′-azido nucleosides displayed potent effects against K562, MCF-7 and BT474 cell lines. The 5′-azidofuranosyl N9 and N7-linked purine nucleosides exhibited highest activity towards the chronic myeloid leukemia cell line (K562) with GI50 values of 13.6 and 9.7 μM, respectively. Among pyranosyl nucleosides, the N7-linked nucleoside was the most active compound with efficacy towards all cell lines assayed and a highest effect on K562 cells (GI50=6.8 μM). Cell cycle analysis of K562 and MCF-7 cells showed that the most active compounds cause G2/M arrest.
Introduction
Nucleoside and nucleotide analogs represent privileged classes of molecules in medicinal chemistry due to their broad spectrum of biological effects, which include anticancer [1], [2], antiviral [2], [3], antibiotic [4], [5], [6] or cholinesterase inhibitory properties [7], [8], [9].
Their therapeutic potential is well demonstrated and validated by the several compounds of this type in clinical use towards cancer and viral infections. Their bioactivity arises from their ability to mimic natural nucleotides and to be recognized by nucleotide-dependent enzymes. Therefore, these molecules may interfere and inhibit crucial biological pathways, such as nucleic acid synthesis, cell signaling or cell cycle regulation, among other events, the alteration or over-activation of which is a hallmark and drive the progress of some diseases [2].
Various nucleoside or nucleotide analogs can interfere with nucleic acid biosynthesis by incorporation into DNA or RNA and inhibition of DNA or RNA polymerization, which induce inhibition of cellular division or of viral replication. In these cases, the active forms of these molecules are mostly the corresponding nucleoside triphosphate metabolites which are formed intracellularly through mediation of nucleoside/nucleotide kinases.
Other enzymes using nucleotides as substrates include DNA ligases [10], [11], DNA methyltransferases [12], [13], glycosyltransferases [14], [15] or kinases involved in metabolic and signaling pathways [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26].
Among the nucleoside derivatives that have shown potent biological effects associated with good pharmacokinetic properties for clinical application are the nucleoside phosphoramidates [27]. These nucleotide analogs, in which an amino group replaces a phosphate alkoxy or hydroxyl group, are neutral and moderately lipophilic molecules that show good cell permeability. This aspect makes their use advantageous relatively to that of cytotoxic or antiviral nucleoside and nucleoside monophosphates, which are not able to penetrate into cells due to their hydrophilic nature and often need nucleoside transporters to cross the cell membranes. Moreover, cancer cells and some virus-infected cells frequently decrease their level of nucleoside transporters as a mechanism of resistance [28], [29], [30] and therefore modified nucleosides that can enter cells by passive diffusion are desirable. The reported bioactive nucleoside phosphoramidates comprise the phosphoramidate moiety linked to the nucleoside at C-5′ by an oxygen atom and these compounds are converted to nucleoside monophosphates by intracellular enzymes, bypassing the resistance of some cells arising from deficiency of nucleoside kinases and leading ultimately to the active nucleoside triphosphates [27], [31]. Sofosbuvir is probably the best representative compound among antiviral nucleoside phosphoramidates, being the most effective agent towards hepatitis C virus (HCV) [31], [32], [33]. Other nucleotide analogs of this type also displayed strong anticancer activities, such as the cytarabine phosphoramidate, which showed higher cytotoxicity than cytarabine towards nucleoside transport-deficient and nucleoside kinase-deficient leukemia cell lines [34].
Owing to their biological potential, the access to new analogous and mimetic structures of nucleosides/nucleotides and the study of their bioactivities remains relevant.
We report herein on the synthesis of new furanosyl and pyranosyl nucleoside phosphoramidates, in which the phosphoramidate system is connected by the nitrogen atom to the sugar moiety at C-5′ or at C-6′, and evaluation of their antiproliferative activities towards cancer cells. Nucleoside phosphoramidates of this type are relatively unexploited with few synthetic reports published in the early nineties [35], [36], whereas concerning their biological interest, the only reported studies were on the incorporation of a guanosine derivative as initiating nucleotide in RNA polymerase-promoted transcriptions [37], [38].
The rational for the access of these molecules is based on their prospective intracellular conversion into nucleoside 5′/6′-phosphoramidic diphosphoric anhydrides, by successive kinase-mediated phosphorylations, and their further incorporation into nucleic acids, eventually stopping chain elongation and inducing cytotoxicity, in analogy with nucleoside triphosphates.
The ability of DNA polymerase I to incorporate nucleoside 5′-phosphoramidic diphosphoric anhydrides (designated as 5′-amino nucleoside-5′-N-triphosphates) into DNA by forming a internucleotide phosphoramidate linkage, has been reported [39], [40], [41], [42]. These mimetics of nucleotide triphosphates appeared to be sufficiently stable for incorporation in the polynucleotide chain. Recently, studies on their incorporation into RNA, using various DNA-dependent RNA polymerases [43], revealed the propensity of these compounds to inhibit transcription.
Moreover, as potential mimetics of nucleotides, these molecules may act as inhibitors of nucleotide-dependent enzymes.
Protected nucleoside phosphoramidates structurally appropriate for cell penetrability were envisaged. Hence, dialkyl phosphoramidate diesters were accessed taking into account that once inside the cell, the phosphoramidate moiety is susceptible to hydrolysis by phosphodiesterases, a step that normally occurs during intracellular activation of ester-type pronucleotides [27]. However, the resulting deprotected nucleoside phosphoramidates are also prompted to undergo cleavage by cellular phosphoramidases, a process that may take place in competition with kinase-mediated phosphorylation, leading to the corresponding amino nucleoside metabolites. Considering the biological effects reported for 5′-amino nucleosides, which include ability to inhibit thymidine kinases [44] and propensity for incorporation into DNA in HSV-infected cells via phosphorylation to the corresponding mimetics of nucleoside triphosphates [45], resulting in antiviral efficacy [45], [46], it is plausible to predict that such metabolites, if formed, also have potential to exhibit cytotoxicity.
Concerning the sugar moiety, since fully deprotected structures would be rather hydrophilic, hampering their penetration into cells, O-acetyl protection and a benzyloxy substituent were kept. While a benzyl group, besides introducing some hydrophobicity, may also allow additional interactions with aromatic amino acid residues of a biological target, the acetate groups are liable to intracellular cleavage by esterases. Ester protection is an approach that was shown to cause a significant improvement on the bioavailability, membrane permeability and sometimes on the bioactivity of nucleosides/nucleotides [47], [48], [49].
Motivated by the known anticancer activities of 3′- and 4′-azido nucleosides [50], [51], [52], in this work, the anticancer potential of the intermediate 5′- and 6′-azido furanosyl/pyranosyl nucleosides, which belong to a group of azido derivatives whose biological profile is less studied, was also evaluated.
Results and discussion
Azido sugars are useful synthetic intermediates in carbohydrate chemistry [53], [54], particularly for the preparation of various amino-functionalized derivatives, and were the suitable templates used herein for the further access to nucleoside phosphoramidates. Thus, the 5-azido-1,2-di-O-acetyl-3-O-benzyl xylofuranose derivative 3 was the glycosyl donor precursor for the synthesis of xylofuranosyl-based nucleosides and related phosphoramidates. Its preparation involved the benzylation of the 5-azido-5-deoxy-1,2-O-isopropylidene-α-d-xylofuranose (1) and subsequent acid-mediated hydrolysis of the acetonide functionality of 2, which was followed by acetylation (Scheme 1). The 1,2-di-O-acetyl derivative 3 was obtained as a anomeric mixture (3α/3β ratio, 1:1) in 80% yield and it was coupled with silylated 2-acetamido-6-chloropurine in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) under microwave irradiation (MW), affording the β-N9-linked azido nucleoside 4 and its N7 regioisomer 5 in 50% and 11% yields, respectively. The assignment of the regiochemistry of the N-glycosidic bonds of 4 and 5 was supported on HMBC experiments, which in case of the N9 nucleoside (4), showed a correlation between the anomeric proton (H-1′) and C-4 of the purine system. Moreover, the signals for H-1′, H-8, C-4 and C-8, which are deshielded in the N7 nucleoside 5, are also diagnostic 1H/13C NMR features for providing an unambiguous N9/N7 regiochemical elucidation.
Synthesis of 5′-azido xylofuranosyl purine nucleosides and a related nucleoside phosphoramidate.
The conversion of the azido N9-nucleoside 4 into the corresponding phosphoramidate by the Staudinger-phosphite reaction was subsequently performed. Preliminary trials were carried out with a primary sugar azide, namely the tri-O-benzylated 6-azido derivative of methyl glucopyranoside (8), which was prepared from the partially protected glycoside 7 through tosylation and further nucleophilic displacement with sodium azide (Scheme 2). The treatment of 8 with trimethyl phosphite in dichloromethane was effective when the reaction was performed under reflux, giving the sugar phosphoramidate 9 in 84% yield, while virtually no conversion was detected after one hour at room temperature. Hence, using the validated reaction conditions, the nucleoside phosphoramidate 6 was obtained in 72% yield from 4.
Synthesis of a 6-phosphoramidate derivative of methyl glucopyranoside and subsequent mono-dealkylation at the phosphoramidate moiety.
It is noteworthy to mention that in initial experiments in which the glucopyranoside 6-phosphoramidate 9 was subjected to the previously mentioned conditions for N-glycosylation, which are also reported to be effective for methyl glycosides, only demethylation at the phosphoramidate moiety leading to 10 occurred (Scheme 2). This transformation most probably follows a similar mechanism than that proposed for the dealkylation of phosphonate esters with trimethylsilyl chloride or bromide [55], [56]. It may involve the nucleophilic attack by a phosphoryl oxygen lone pair of 9 to the TMSOTf silicon center. The displaced triflate anion subsequently captures a methyl group reforming the P–O double bond. The resulting methyl trimethylsilyl phosphoramidate intermediate is then hydrolyzed into 10 during the work-up. This conversion, mediated by the Lewis acid used for N-glycosylation, indicated the need of synthesizing firstly the azido nucleosides and performing the Staundinger-phosphite reaction afterwards.
Coupling of the 1,2-di-O-acetyl glycosyl donor 3 with uracil under similar reaction conditions to those used for the purine derivative, afforded the xylofuranosyl uracil 11 in moderate yield (Scheme 3). The N1-C1′ linkage in 11 was assigned based on key HMBC correlations between H-1′ and both C-2/C-6. Treatment of 11 with trimethyl phosphite led to the corresponding nucleoside phosphoramidate 12 along with the N3-methyl derivative 13 in 76% total yield and a 12/13 ratio of 1:05. These products were inseparable by column chromatography and were clearly identified by NMR and HRMS analysis of the mixture. For the minor structure (13), a set of methyl protons, aside from those of the dimethylphosphoramidate moiety, were detected at δ=3.30 ppm exhibiting HMBC correlations with C-2 and C-4, in accordance with a N3-methyl uracil derivative, whereas the signal for the corresponding carbon atom appeared at δ=27.8 ppm.
Synthesis of a 5′-azido xylofuranosyl uracil nucleoside and related nucleoside phosphoramidates.
The formation of 13 likely arises from rearrangement of the intermediate phosphorimidate either by an inter- or intramolecular process. The thermal or Lewis acid-catalyzed rearrangement of phosphorimidates in which a alkyl group is transferred to the phosphoramidite nitrogen, leading to the N,N-disubstituted phosphoramidates, has been reported [57], [58]. In the present case, N-alkylation take places at the uracil moiety. As shown for the aza-ylide resonance form (A), an intramolecular rearrangement may occur through deprotonation of the uracil moiety by the negatively-charged nitrogen atom and further transferring of a methyl group to the uracil N3 atom with accompanying formation of the double P–O bond (B).
For the synthesis of azido nucleosides and related phosphoramidates embodying a glucopyranosyl unit, a tri-O-acetylated 6-azido-3-O-benzyl glycosyl donor (15) was prepared as a anomeric mixture (α/β ratio 1:0.7) through cleavage of the isopropylidene group of the 6-azido-3-O-benzyl glucofuranose precursor 14 [59], and subsequent acetylation (Scheme 4). MW-assisted TMSOTf-mediated N-glycosylation of silylated 2-acetamido-6-chloropurine with 15 furnished the purine N9-linked and N7-linked 6′-azido nucleosides 16 and 17 in ca. 1:1 ratio and a combined 74% yield, whose structures were assigned based on their characteristic NMR distinguishing data as for nucleosides 4/5. Their reactions with trimethyl phosphite furnished the corresponding phosphoramidates 18 and 19 in 83% and 86% yields, respectively.
Synthesis of 6′-azido glucopyranosyl nucleosides and related nucleoside phosphoramidates.
Similarly to 3, the 6-azido glucofuranosyl donor 15 was converted into its uracil N1-linked nucleoside 20 in good yield using the previously described N-glycosylation protocol.
The nucleoside phosphoramidates (6, 18, 19) as well as the azido nucleosides (4, 5, 11, 16, 17, 20) were evaluated for their antiproliferative effects in chronic myeloid leukemia cell line K562 and breast cancer cell lines MCF-7 and BT474 (Table 1). None of the phosphoramidate derivatives showed anticancer activity at concentrations below 100 μM. However, moderate activities were displayed by the azido nucleosides possessing a 2-acetamido-6-chloropurine moiety. The xylofuranosyl-based nucleosides (4, 5) displayed higher antiproliferative action in the leukemia cell line, with approximately similar effects from both N9 and N7-regioisomers (GI50=13.6 μM and 9.7 μM, respectively). The activity of the glucopyranosyl nucleosides proved to be influenced by the regiochemistry of the N-glycosidic bond. While the purine N9-linked nucleoside 16 showed weak effects towards the K562 and the BT474 cell lines, its N7-regioisomer 17 was the most active compound of this series, with significant activities in all cancer cells. K562 cells were the most sensitive to nucleoside 17, with a GI50 value of 6.8 μM. Although the activity of 17 in K562 is 10 fold lower than that of the antileukemic agent imatinib, the result obtained is satisfactory and motivates further structural optimization.
Antiproliferative activity of the new azido nucleosides and nucleoside phosphoramidates in cancer cell lines.
| Compound | GI50 (μM)a |
||
|---|---|---|---|
| K562 | MCF-7 | BT474 | |
| 4 | 13.6 | >100 | 93.1 |
| 5 | 9.7 | >100 | >100 |
| 6 | >100 | >100 | >100 |
| 11 | >100 | >100 | >100 |
| 16 | 48.5 | >100 | 50.9 |
| 17 | 6.8 | 29.6 | 13.6 |
| 18 | >100 | >100 | >100 |
| 19 | 98.1 | >100 | >100 |
| 20 | >100 | >100 | >100 |
| imatinib | 0.7 | >10 | >10 |
| 5-fluorouracil | >50 | 2.4 | NT |
-
aAll values were obtained from at least two determinations.
Finally, flow cytometric analysis of cells treated with the most active compounds was performed in order to determine their effect on the cell cycle (Fig. 1). The experiment revealed that 6′-azido nucleoside 17 (6.25 μM concentration) causes a significant G2/M arrest in K562 and MCF-7 cells after 24 h incubation. The 5′-azido furanosyl nucleoside 4 (25 μM concentration) showed the same effect in K562 cells (>20% G2/M cells than in control cells), while it did not produce any alteration in MCF-7 cells. In addition, increased subdiploid populations were observed in both cell lines treated with 17, while 4 affected only K562 cells.
Effect of 17 (6.25 μM) and 4 (25 μM) on the cell cycle of K562 and MCF-7 cancer cell lines following 24 h treatment. Flow cytometric analysis of the cell cycle (propidium iodide staining) was quantified by ModFit software, version 4.1.7.
Conclusions
Uracil and purine 5′-azido xylofuranosyl and 6′-azido glucopyranosyl-based nucleosides were synthesized from easily accessed azido-1,2-di-O-acetyl glycosyl donors and the purine derivatives were converted into the corresponding nucleoside 5′/6′-phosphoramidates. The latter compounds, planned as potential nucleotide mimetics, were devoid of any noticeable antiproliferative effect, which is probably due to the lability of the phosphoramidate function. In contrast, the purine 5′/6′-azido nucleosides showed significant anticancer activities, namely the furanosyl derivatives which displayed selectivity towards K562 cells and the N7-linked pyranosyl nucleoside with action on all cell lines, especially towards K562 and BT474 cells. The uracil nucleosides were nearly inactive. These findings indicate that the azido functionality as well as the 2-chloro-6-acetamido purine unit are important for the detected bioactivities. These results motivate compounds’ structural optimization aiming at improving their bioactivities and studying which structural features are essential for a selective action.
Moreover, with the preliminary studies highlighting a G2/M cell cycle arrest induced by the active azido nucleosides, further studies will be carried out to disclose their mechanism of action. That probably involves interference with DNA repair or replication machinery. Most likely, it differs from that of the known anticancer and antiviral azido nucleosides, since these new compounds lack a terminal hydroxyl group for further conversion into nucleoside triphosphates, not enabling their incorporation into DNA.
Experimental section
Chemistry
General methods
The reactions were monitored by TLC on Merck 60 F254 silica gel aluminium plates and the spots were detected under UV light (254 nm) and/or by spraying with a solution of 10% H2SO4 in EtOH. For column chromatography, silica gel 60 G (0.040–0.063 mm, E. Merck) was used. NMR spectra were acquired with a BRUKER Avance 400 spectrometer operating at 400.13 MHz for 1H, 100.62 MHz for 13C or at 161.91 MHz for 31P. Chemical shifts are expressed in parts per million and are reported relative to internal TMS, in the case of CDCl3, or relative to the respective solvent peak as reference. 31P Spectra were referenced according to the IUPAC recommendations for chemical shift referencing [60]. High-resolution mass measurements were performed on a High Resolution QqTOF Impact II mass spectrometer equipped with an ESI ion source (Bruker Daltonics). Spectra were recorded in positive ESI mode with external calibration. Melting points were determined with a Stuart Scientific SMP 3 apparatus and are uncorrected. Optical rotations were measured on a Perkin–Elmer 343 polarimeter.
5-Azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-α-d-xylofuranose (2)
To a solution of 5-azido-5-deoxy-1,2-O-isopropylidene-α-d-xylofuranose (222 mg, 1.03 mmol) in DMF (5 mL) at 0°C, NaH (60%, 82 mg, 2.06 mmol) was added. The suspension was stirred at 0°C for 10 min., whereupon benzyl bromide (0.25 mL, 2.06 mmol) was added. The mixture was stirred at room temperature overnight. After completion of the reaction, as shown by TLC (petroleum ether/EtOAc, 3:2), water was added at 0°C and the mixture was transferred to a separatory funnel. The aqueous phase was extracted with diethyl ether. The combined organic phases were dried with anhydrous magnesium sulfate, filtered and the solvent was evaporated under vacuum. The crude was subjected to flash column chromatography on silica-gel (petroleum ether/EtOAc, 7:1) to give the title compound as a colorless oil (286 mg, 91%).
1H RMN (CDCl3, 400 MHz)*: δ 7.39–7.26 (m, 5 H, Ph), 5.92 (d, 1 H, H-1, J1,2=3.8), 4.67 (d, part A of AB system, 1 H, H-a from CH2Ph, Ja,b=11.8), 4.62 (d, 1 H, H-2, J1,2=3.8), 4.50 (d, part B of AB system, 1 H, H-b from CH2Ph) 4.30 (ddd, 1 H, H-4), 3.94 (d, 1 H, H-3, J3,4=3), 3.58 (dd, part A of ABX system, 1 H, H-5a, J5a,5b=12.4, J4,5a=6.7), 3.47 (dd, part B of ABX system, 1 H, H-5b, J4,5b=6.4), 1.49 (s, 3 H, CH3), 1.32 (s, 3 H, CH3). 13C (RMN, 100 MHz)*: δ 137.2 (Cq, arom), 128.6, 128.1, 128.8 (CH, Ph), 111.9 (Cq, i-Pr), 105.2 (C-1), 82.1 (C-2), 81.5 (C-3), 78.8 (C-4), 71.9 (CH2, Bn), 49.3 (C-5), 26.8, 26.3 (2×CH3).
*NMR data were in agreement with the reported data [61].
1,2-Di-O-acetyl-5-azido-3-O-benzyl-5-deoxy-α,β-d-xylofuranose (3α, 3β)
A solution of 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-α-d-xylofuranose (109 mg, 0.36 mmol) in aqueous trifluoroacetic acid (65%, 3 mL) was stirred at room temp. for 30 min. The solvents were co-evaporated with toluene. The resulting residue was treated with pyridine (2 mL) and acetic anhydride (1.5 mL) and the mixture was stirred at room temp. for 1 h. The solvents were co-evaporated with toluene and the crude was purified by flash column chromatography (n-hexane/EtOAc, 6:1) to give the title compound (100 mg, 80%, two steps, anomeric mixture, α/β ratio, 1:1) as a colorless oil.
1H NMR (400 MHz, CDCl3): δ 7.44–7.29 (m, 10 H, CH, Ph, α, β), 6.44 (d, 1 H, H-1 α, J1,2 (α)=4.6), 6.17 (br. d, 1 H, H-1 β), 5.35–5.30 (m, 2 H, H-2 α, H-2 β), 4.81, 4.72 (2 d, 2×part A of AB system, 2 H, H-a, CH2, Bn, α, β, Ja,b (α)=12.0, Ja,b (β)=12.0), 4.62, 4.59 (2 d, 2×part B of AB system, 2 H, H-b, CH2, Bn, α, β), 4.48–4.39 (m, 4 H, H-4 α, H-4 β), 4.29 (t, 1 H, H-3α, J2,3~J3,4~5.4), 4.07 (br. dd, 1 H, H-3 β), 3.65–3.51 (m, 4 H, CH2-5, α, β), 2.14, 2.11, 2.10, 2.09 (4 s, 12 H, CH3, OAc, α, β). 13C NMR (100 MHz, CDCl3): δ 169.8, 169.7, 169.6, 169.4 (CO, Ac, α, β), 137.3, 134.2 (2×Cq, Ph, α, β), 128.7, 128.6, 128.6, 128.3, 128.2, 128.0 (CH, Ph, α, β), 99.7 (C-1 β), 94.0 (C-1 α), 81.9 (C-4 β), 80.2 (C-3 β), 79.6, 79.4 (C-2 β, C-3 α), 77.9 (C-4 α), 76.6 (C-2 α), 72.7, 72.1 (CH2Ph, α, β), 50.7, 50.4 (C-5, α, β), 21.3, 21.1, 21.0, 20.7 (CH3, OAc, α, β). HRMS: calcd for C16H19N3O6 [M+Na]+372.1166, found 372.1181.
General procedure for N-glycosylation of 2-acetamido-6-chloropurine or uracil with 1-O-acetyl glycosyl donors
To a suspension of nucleobase (0.255 mmol) in anhydrous acetonitrile (2 mL), N,O-bis(trimethylsilyl)acetamide (BSA, 0.13 mL, 0.51 mmol) was added and the mixture was stirred under N2 at room temp. for 20 min. A solution of 1-O-acetyl glycosyl donor (0.17 mmol) in anhydrous acetonitrile (2 mL) was added to the previous solution, which was followed by dropwise addition of trimethylsilyl triflate (TMSOTf, 0.2 mL, 1.1 mmol). The mixtute was stirred under microwave irradion (150 W, P max=250 Psi) at 65°C for 40–60 min. It was then diluted with DCM and neutralized with sat. aq. NaHCO3 soln. The aqueous phase was extracted with dichloromethane (3×) and the combined organic phases were dried with anhydrous MgSO4. After filtration and concentration under vacuum, the residue was purified by flash column chromatography on silica gel.
2-Acetamido-9-(2-O-acetyl-5-azido-3-O-benzyl-5-deoxy-β-d-xylofuranosyl)-6-chloropurine (4) and 2-acetamido-7-(2-O-acetyl-5-azido-3-O-benzyl-5-deoxy-β-d-xylofuranosyl)-6-chloropurine (5)
Compounds 4 and 5 were obtained according to the general procedure, starting from 1,2-di-O-acetyl-5-azido-3-O-benzyl-5-deoxy-α,β-d-xylofuranose (3, 59 mg, 0.17 mmol) and 2-acetamido-6-chloropurine (54 mg, 0.255 mmol). N-Glycosylation of the corresponding silylated purine with 3 in the presence of TMSOTf (0.2 mL, 1.1 mmol) was complete within 45 min. Purification by flash column chromatography on silica gel (EtOAc/hexane, from 1:1 to 3:1) afforded the N9 nucleoside 4 (42.1 mg, 50%) and its N7 regioisomer 5 (11 mg, 13%) as colorless oils.
Data for 4:
Data for 5:
1-(2-O-Acetyl-5-azido-3-O-benzyl-5-deoxy-β-d-xylofuranosyl)uracil (11)
Compound 11 was obtained according to the general procedure, starting from 1,2-di-O-acetyl-5-azido-3-O-benzyl-5-deoxy-α,β-d-xylofuranose (3, 30 mg, 86 μmol) and uracil (15 mg, 129 μmol). N-Glycosylation of the corresponding silylated uracil with 3 in the presence of TMSOTf (0.1 mL, 0.56 mmol) was complete within 45 min. Purification by flash column chromatography on silica gel (EtOAc/hexane, 1:1) afforded the title compound (18 mg, 52%) as a colorless oil.
General procedure for the Staudinger-phosphite reaction of azido derivatives with trimethyl phosphite
To a solution of azido derivative (0.05 mmol) in dichloromethane (5 mL), trimethyl phosphite (0.06 mmol, 7 μL) was added. The solution was stirred under reflux for 2–4 h, until complete conversion, as indicated by TLC. The solution was concentrated under vacuum and the residue was purified by flash column chromatography on silica gel.
2-Acetamido-9-[2-O-acetyl-3-O-benzyl-5-deoxy-5-(dimethoxyphosphoryl)amino-β-d-xylofuranosyl]-6-chloropurine (6)
Compound 6 was obtained according to the general procedure, treating 2-acetamido-9-(2-O-acetyl-5-azido-3-O-benzyl-5-deoxy-β-d-xylofuranosyl)-6-chloropurine (4, 25 mg, 0.05 mmol) with trimethyl phosphite (7 μL, 0.06 mmol). The reaction was complete within 2 h. Purification by flash column chromatography on silica gel (from EtOAc to EtOAc /MeOH, 4:1) afforded the title compound (21 mg, 72%) as a colorless oil.
1-[2-O-acetyl-3-O-benzyl-5-deoxy-5-(dimethoxyphosphoryl)amino-β-d-xylofuranosyl]uracil (12) and 1-[2-O-acetyl-3-O-benzyl-5-deoxy-5-(dimethoxyphosphoryl)amino-β-d-xylofuranosyl]- 3-methyluracil (13)
Compounds 12 and 13 were obtained according to the general procedure, treating 1-(2-O-acetyl-5-azido-3-O-benzyl-5-deoxy-β-d-xylofuranosyl)uracil (11, 14 mg, 35 μmol) with trimethyl phosphite (10 μL, 85 μmol). The reaction was complete within 2 h. Purification by flash column chromatography on silica gel (from EtOAc/hexane, 2:1 to EtOAc /MeOH, 9:1) afforded a mixture of the title compounds (13 mg, ratio 12/13, 1:0.5, 76%) which could not be separated despite repeated attempts.
NMR data for 12: 1H NMR (CDCl3, 400 MHz)*: δ 9.01 (s, NH), 7.58 (d, 1 H, H-6, J5,6=8.0), 7.46–7.22 (m, 5 H, Ph), 6.08 (br.s, 1 H, H-1′), 5.62 (d, 1 H, H-5), 5.15 (br.s, 1 H, H-2′), 4.72 (d, 1 H, part A of AB system, H-a, Bn, Ja,b=11.5), 4.56 (d, 1 H, part B of AB system, H-b, Bn), 4.17 (ddd, 1 H, H-4′), 3.90 (d, 1 H, H-3′, J3′,4′=2.8), 3.71, 3.68 (2 d, 6 H, 2×OCH3, JCH3,P=11.0), 3.41–3.21 (m, 2 H, CH2-5′), 2.99 (t, 1 H, NH), 2.14 (s, 3 H, CH3, OAc). 13C NMR (CDCl3, 100 MHz)*: δ 169.8 (CO, Ac), 163.0 (C-4), 150.2 (C-2), 140.3 (C-6), 136.4 (Cq, Ph), 128.9, 128.8, 128.5 (CH, Ph), 102.7 (C-5), 88.7 (C-1′), 82.4 (C-4′), 79.9, 79.8 (C-3′ C-2′), 72.9 (CH2, Bn), 53.5, 53.4 (2 d, OCH3, JCH3,P=5.6), 39.9 (C-5′), 21.0 (CH3, OAc).
NMR data for 13: 1H NMR (CDCl3, 400 MHz)*: δ 7.55 (d, 1 H, H-6, J5,6=8.0), 7.46–7.22 (m, 5 H, Ph), 6.08 (br.s, 1 H, H-1′), 5.70 (d, 1 H, H-5), 5.15 (br.s, 1 H, H-2′), 4.70 (d, 1 H, part A of AB system, H-a, Bn, Ja,b=11.5), 4.56 (d, 1 H, part B of AB system, H-b, Bn), 4.17 (ddd, 1 H, H-4′), 3.90 (d, 1 H, H-3′, J3′,4′=2.8), 3.71, 3.68 (2 d, 6 H, 2×OCH3, JCH3,P=11.0), 3.41–3.21 (m, 4 H, CH2-5′, N-CH3), 2.94 (t, 1 H, NH), 2.15 (s, 3 H, CH3, OAc). 13C NMR (CDCl3, 100 MHz)*: δ 169.8 (CO, Ac), 162.8 (C-4), 151.1 (C-2), 137.9 (C-6), 136.5 (Cq, Ph), 128.9, 128.7, 128.5 (CH, Ph), 101.9 (C-5), 89.7 (C-1′), 82.5 (C-4′), 79.9, 79.8 (C-3′, C-2′), 72.9 (CH2, Bn), 53.5, 53.4 (2 d, OCH3, JCH3,P=5.6), 39.9 (C-5′), 27.8 (NCH3), 21.0 (CH3, OAc).
*Data extracted from the spectrum of the mixture containing 12/13.
31P NMR (MeOD, 162 MHz) δ: 11.22, 11.23. HRMS: calcd for C20H26N3O9P [M+H]+484.1479, found 484.1488; calcd for [M+Na]+506.1299, found 506.1298. HRMS: calcd for C21H28N3O9P [M+H]+498.1636, found 498.1641; calcd for [M+Na]+520.1455, found 520.1454.
Methyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-α-d-glucopyranoside (8)
To a solution of methyl 2,3,4-tri-O-benzyl-α-d-glucopyranoside (7, 52 mg, 0.11 mmol) in dichloromethane and pyridine (3 mL, 1:1) under nitrogen, tosyl chloride (40 mg, 0.21 mmol) was added and the solution was stirred at room temp. for 24 h. Then, water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with water and dried with anhydrous MgSO4. After filtration and evaporation of the solvent under vacuum, the residue was dissolved in N,N-dimethylformamide (DMF, 3 mL) and sodium azide (40 mg, 0.62 mmol) was added. The mixture was stirred at 110 °C for 1.5 h. Then water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with water and dried with anhydrous MgSO4. After filtration and evaporation of the solvent, the residue was subjected to column chromatography (EtOAc/hexane, 1:4) to afford the title compound (52 mg, 95%) as a colorless oil.
NMR data: 1H NMR (400 MHz, CDCl3)*: δ 7.47–7.20 (m, 15 H, CH, Ph), 5.00 (d, part A of AB system, H-a, CH2Ph, Ja,b=10.8), 4.90 (d, part A of AB system, H-a, CH2Ph, Ja,b=10.9), 4.85–4.77 (m, 2 H, H-a, H-b, CH2Ph), 4.67 (d, part B of AB system, H-b, CH2Ph), 4.61 (d, 1 H, H-1, J1,2=3.5), 4.57 (d, part B of AB system, H-b, CH2Ph), 3.98 (t, 1 H, H-3, J2,3=J3,4=9.3), 3.78 (ddd, 1 H, H-5), 3.54 (dd, 1 H, H-2), 3.48–3.38 (m, 5 H, H-4, H-6a, OCH3), 3.33 (dd, part B of ABX system, 1 H, H-6 b, J5,6b=5.7, J6a,6b=13.1). 13C NMR (100 MHz, CDCl3): δ 138.7, 138.1, 138.0 (3×Cq, Ph), 128.6, 128.6, 128.2, 128.1, 128.1, 128.1, 128.1, 127.8 (CH, Ph), 98.2 (C-1), 82.0 (C-3), 80.1 (C-2), 78.4 (C-4), 75.9, 75.3, 73.6 (CH2, Bn), 70.0 (C-5), 55.5 (CH3, OMe), 51.5 (C-6).
*NMR data were in agreement with the reported data [62].
Methyl 2,3,4-tri-O-benzyl-6-deoxy-6-(dimethoxyphosphoryl)amino-α-d-glucopyranoside (9)
Compound 9 was obtained according to the general procedure, treating methyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-α-d-glucopyranoside (8, 50 mg, 0.1 mmol) with trimethyl phosphite (25 μL, 0.21 mmol). The reaction was complete within 2 h. Purification by flash column chromatography on silica gel (from EtOAc to EtOAc/MeOH, 9:1) afforded the title compound (49 mg, 84%) as a colorless oil.
Methyl 2,3,4-tri-O-benzyl-6-deoxy-6-(methoxyphosphoryl)amino-α-d-glucopyranoside (10)
Compound 10 was obtained according to the general procedure, starting from methyl 2,3,4-tri-O-benzyl-6-deoxy-6-(dimethoxyphosphoryl)amino-α-d-glucopyranoside (9, 42 mg, 0.074 mmol) and uracil (12.4 mg, 0.11 mmol). The reaction in the presence of TMSOTf (0.09 mL, 0.48 mmol) was complete within 30 min. Purification by flash column chromatography on silica gel (EtOAc/methanol, from 4:1 to 2:1) afforded the title compound (31.4 mg, 77%) as a colorless oil.
The conversion also occurred without silylated nucleobase, being the title compound obtained in similar yield (81%).
1,2,4-Tri-O-acetyl-6-azido-3-O-benzyl-6-deoxy-α,β-d-glucopyranose (15α, 15β)
A solution of 6-azido-3-O-benzyl-6-deoxy-1,2-O-isopropylidene-α-d-glucofuranose (14, 306 mg, 0.91 mmol) in aqueous trifluoroacetic acid (60%, 5 mL) was stirred at room temp. for 1 h. The solvents were co-evaporated with toluene. The resulting residue was treated with pyridine (4 mL) and acetic anhydride (3 mL) and the mixture was stirred at room temp. for 90 min. The solvents were co-evaporated with toluene and the crude was purified by flash column chromatography (n-hexane/EtOAc, 1:1) to give the title compound (377 mg, 98%, two steps, anomeric mixture, α/β ratio, 1:0.7) as a colorless oil.
1H NMR (CDCl3, 400 MHz): δ 7.41–7.17 (m, 8.5 H, Ph), 6.33 (d, 1 H, H-1α, J1,2 (α)=3.5), 5.65 (d, 0,7 H, H-1β, J1,2 (β)=8.4), 5.15 (t, 0.7 H, H-2 β), 5.11–5.01 (m, 2.7 H, H-2α, H-4α, H-4β), 4.71 (d, 1 H, H-a, Bn, α, Ja,b=11.9), 4.65–4.58 (m, 2.4 H, H-b, Bn, α, CH2Ph β), 4.02–3.90 (m, 2 H, H-5α, H-3α), 3.75 (t, 0.7 H, H-3 β, J2,3 ~ J3,4~ 9.3), 3.69 (ddd, 0.7 H, H-5 β, J5,6a=6.6, J5,6b=2.8, J4,5=9.9), 3.39–3.22 (m, CH2-6 α, CH2-6 β, J6a,6b(α)=13.2), 2.18, 2.11, 1.99, 1.99, 1.98 (5×s, 15.3 H, CH3, OAc, α, β). 13C NMR (CDCl3, 100 MHz): δ 169.7, 169.5, 169.5, 169.3, 169.2, 168.8 (6×CO, OAc,α, β), 138.0, (Cq, Bn, α), 137.6 (Cq, Bn, β), 128.6, 128.6, 128.1, 127.9, 127.9, 127.6 (CH, Ph, α, β), 91.9 (C1-β), 89.3 (C1-α), 79.9 (C-3 β), 76.9 (C-5 α), 74.9 (CH2, Bn, α) 74.3 (C-5 β, CH2, Bn, β), 71.5, 71.5, 71.4 (C-3α, C-2 α, C-2 β), 70.5, 70.4 (C-4 α, C-4 β), 51.1 (C-6 α), 51.0 (C-6 β), 21.0, 20.9, 20.9, 20.8, 20.8, 20.7 (6×CH3, OAc, α, β). HRMS: calcd forC19H23N3O8 [M+Na]+444.1377, found 444.1380.
2-Acetamido-9-(2,4-di-O-acetyl-6-azido-3-O-benzyl-6-deoxy-β-d-glucopyranosyl)-6-chloropurine (16) and 2-acetamido-7-(2,4-di-O-acetyl-6-azido-3-O-benzyl-6-deoxy-β-d-glucopyranosyl)-6-chloropurine (17)
Compound 16 and 17 were obtained according to the general procedure, starting from 1,2,4-tri-O-acetyl-6-azido-3-O-benzyl-6-deoxy-α,β-d-glucopyranose (15, 200 mg, 0.48 mmol) and 2-acetamido-6-chloropurine (172 mg, 0.81 mmol). N-Glycosylation of the corresponding silylated purine with 15 in the presence of TMSOTf (0.64 mL, 3.74 mmol) was complete within 40 min. Purification by flash column chromatography on silica gel (EtOAc/hexane, from 1:4 to 3:1) afforded the N9 nucleoside 16 (98.5 mg, 36%) as a colorless oil and its N7 regioisomer 17 (104.3 mg, 38%) as a white solid.
Data for 16:
Data for 17: m. p.=108.3–110 °C.
2-Acetamido-9-[2,4-di-O-acetyl-3-O-benzyl-6-deoxy-6-(dimethoxyphosphoryl)amino-β-d-glucopyranosyl]-6-chloropurine (18)
Compound 18 was obtained according to the general procedure, treating 2-acetamido-9-(2,4-di-O-acetyl-6-azido-3-O-benzyl-6-deoxy-β-d-glucopyranosyl)-6-chloropurine (16, 70 mg, 0.122 mmol) with trimethyl phosphite (350 μL, 2.97 mmol). The reaction was complete within 3 h. Purification by flash column chromatography on silica gel (from EtOAc to EtOAc /MeOH, 5:1) afforded the title compound (66 mg, 83%) as a colorless oil.
2-Acetamido-7-[2,4-di-O-acetyl-3-O-benzyl-6-deoxy-6-(dimethoxyphosphoryl)amino-β-d-glucopyranosyl]-6-chloropurine (19)
Compound 19 was obtained according to the general procedure, treating 2-acetamido-7-(2,4-di-O-acetyl-6-azido-3-O-benzyl-6-deoxy-β-d-glucopyranosyl)-6-chloropurine (17, 72 mg, 0.126 mmol) with trimethyl phosphite (360 μL, 3.05 mmol). The reaction was complete within 4 h. Purification by flash column chromatography on silica gel (from EtOAc to EtOAc /MeOH, 3:1) afforded the title compound (71 mg, 86%) as a colorless oil.
1-(2,4-di-O-Acetyl-6-azido-3-O-benzyl-6-deoxy-β-d-glucopyranosyl)uracil (20)
Compound 20 was obtained according to the general procedure, starting from 1,2,4-tri-O-acetyl-6-azido-3-O-benzyl-6-deoxy-α,β-d-glucopyranose (15, 50 mg, 0.12 mmol) and uracil (20 mg, 0.18 mmol). N-Glycosylation of the corresponding silylated uracil with 15 in the presence of TMSOTf (0.16 mL, 0.88 mmol) was complete within 1 h. Purification by flash column chromatography on silica gel (EtOAc/hexane, from 1:2 to 1:1) afforded the title compound (36 mg, 65%) as a white solid. m.p.=128.7–130.4 °C.
Biological assays
Proliferation assay
Cancer cell lines of different histological origin were cultivated according to the recommendations of the supplier. The cells were assayed with compounds using three-fold dilutions in triplicate. Treatment lasted for 72 h, followed by addition of Calcein AM solution, and measurement of the fluorescence of live cells at 485 nm/538 nm (ex/em) with a Fluoroskan Ascent microplate reader (Labsystems). The GI50 value, the drug concentration lethal to 50% of the cancer cells, was calculated from the obtained dose response curves.
Cell cycle analysis
Asynchronous cells were seeded into 96 well plate and then, after preincubation period, treated with tested compound for 24 h. Adherent MCF-7 cells were first washed with PBS, then trypsinized and finally the solution of trypsin inhibitor was added. After incubation, 5× staining solution (17 mM trisodium citrate dihydrate, 0.5% IGEPAL® CA-630, 7.5 mM spermine tetrahydrochloride, 2.5 mM Tris; pH 7.6 containing 50 μg/mL propidium iodide) was added. K562 cells were stained by 5× staining solution directly (i.e. without a trypsinization). Cell cycle was analyzed by flow cytometry using a 488 nm laser (BD FACS Verse with software BD FACSuite™, version 1.0.6.).
Article note
A collection of invited papers based on presentations at the XXVIII International Carbohydrate Symposium (ICS-28),New Orleans, July 17–21 2016.
Acknowledgments
‘Fundação para a Ciência e Tecnologia’ (FCT) is acknowledged for funding through the FCT Investigator Program (IF/01488/2013), the exploratory project IF/01488/2013/CP1159/CT0006 and the strategic project UID/MULTI/00612/2013. RJ, EŘ and VK gratefully acknowledge support from the Ministry of Education, Youth and Sports of the Czech Republic (the National Program of Sustainability I – Grant LO1204).
References
[1] C. M. Galmarini, F. Popowycz, B. Joseph. Curr. Med. Chem.15, 1072 (2008).10.2174/092986708784221449Search in Google Scholar PubMed
[2] L. P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet. Nat. Rev. Drug. Discov.12, 447 (2013).10.1038/nrd4010Search in Google Scholar PubMed
[3] C. Simons, Q. Wu, T. T. Htar. Curr. Top. Med. Chem.5, 1191 (2005).10.2174/156802605774463051Search in Google Scholar PubMed
[4] S. Rachakonda, L. Cartee. Curr. Med. Chem.11, 775 (2004)10.2174/0929867043455774Search in Google Scholar PubMed
[5] M. Winn, R. J. M. Goss, K.-i. Kimura, T. D. H. Bugg. Nat. Prod. Rep.27, 279 (2010)10.1039/B816215HSearch in Google Scholar PubMed
[6] M. Serpi, V. Ferrari, F. Pertusati. J. Med. Chem.59, 10343 (2016).10.1021/acs.jmedchem.6b00325Search in Google Scholar PubMed
[7] N. M. Xavier, S. Schwarz, P. D. Vaz, R. Csuk, A. P. Rauter. Eur. J. Org. Chem.2014, 2770 (2014).10.1002/ejoc.201301913Search in Google Scholar
[8] S. Schwarz, R. Csuk, A. P. Rauter. Org. Biomol. Chem.12, 2446 (2014).10.1039/C4OB00142GSearch in Google Scholar PubMed
[9] D. Batista, S. Schwarz, A. Loesche, R.Csuk, P. J. Costa, M. C. Oliveira, N. M. Xavier. Pure Appl. Chem.88, 363 (2016).10.1515/pac-2016-0102Search in Google Scholar
[10] A. E. Tomkinson, T. R. L. Howes, N. E. Wiest. Transl. Cancer Res.2, 1219 (2013)Search in Google Scholar
[11] T. Helleday, E. Petermann, C. Lundin, B. Hodgson, R. A. Sharma. Nat. Rev. Cancer8, 193 (2008).10.1038/nrc2342Search in Google Scholar PubMed
[12] K. Ghoshal, S. Bai. Drugs Today43, 395 (2007).10.1358/dot.2007.43.6.1062666Search in Google Scholar PubMed
[13] D. Subramaniam, R. Thombre, A. Dhar, S. Anant. Front. Oncol.4, 80 (2014).Search in Google Scholar
[14] L. Tedaldia, G. K. Wagner. Med. Chem. Commun.5, 1106 (2014).10.1039/C4MD00086BSearch in Google Scholar
[15] S. S. Pinho, C. A. Reis. Nat. Rev. Cancer15, 540 (2015).10.1038/nrc3982Search in Google Scholar PubMed
[16] A. M. Fajardo, G. A. Piazza, H. N. Tinsley. Cancers6, 436 (2014).10.3390/cancers6010436Search in Google Scholar PubMed PubMed Central
[17] K. Yan, L. N. Gao, Y. L. Cui, Y. Zhang, X. Zhou. Mol. Med. Rep.13, 3715 (2016).10.3892/mmr.2016.5005Search in Google Scholar PubMed PubMed Central
[18] A. Caretta, C. Mucignat-Caretta. Cancers3, 913 (2011).10.3390/cancers3010913Search in Google Scholar PubMed PubMed Central
[19] K. Bian, F. Murad. Adv Exp Med Biol.814, 5 (2014).10.1007/978-1-4939-1031-1_2Search in Google Scholar PubMed
[20] P. Liu, H. Cheng, T. M. Roberts, J. J. Zhao. Nat. Rev. Drug Discov.8, 627 (2009).10.1038/nrd2926Search in Google Scholar PubMed PubMed Central
[21] P. Wu, Y. Z. Hu. Curr. Med. Chem.17, 4326 (2010).10.2174/092986710793361234Search in Google Scholar PubMed
[22] J. Polivka Jr., F. Janku. Pharmacol. Ther.142, 164 (2014).10.1016/j.pharmthera.2013.12.004Search in Google Scholar PubMed
[23] S. Lapenna, A. Giordano. Nat. Rev. Drug Discov.8, 547 (2009).10.1038/nrd2907Search in Google Scholar PubMed
[24] M. Aarts, S. Linardopoulos, N. C. Turner. Curr. Opin. Pharmacol.13, 529 (2013).10.1016/j.coph.2013.03.012Search in Google Scholar PubMed
[25] T. A. Chohan, H. Qian, Y. Pan, J.-Z. Chen. Curr. Med. Chem.22, 237 (2015).10.2174/0929867321666141106113633Search in Google Scholar PubMed
[26] U. Asghar, A. K. Witkiewicz, N. C. Turner, E. S. Knudsen. Nat. Rev. Drug Discov.14, 130 (2015).10.1038/nrd4504Search in Google Scholar PubMed PubMed Central
[27] U. Pradere, E. C. Garnier-Amblard, S. J. Coats, F. Amblard, R. F. Schinazi. Chem. Rev.114, 9154 (2014).10.1021/cr5002035Search in Google Scholar PubMed PubMed Central
[28] C. M. Galmarini, J. R. Mackey, C. Dumontet. Leukemia15, 875 (2001).10.1038/sj.leu.2402114Search in Google Scholar PubMed
[29] V. L. Damaraju, S. Damaraju, J. D. Young, S. A. Baldwin, J. Mackey, M. B. Sawyer, C. E. Cass. Oncogene22, 7524 (2003).10.1038/sj.onc.1206952Search in Google Scholar PubMed
[30] J. Zhang, F. Visser, K. M. King, S. A. Baldwin, J. D. Young, C. E. Cass. Cancer Metastasis Rev.26, 85 (2007).10.1007/s10555-007-9044-4Search in Google Scholar PubMed
[31] H. K. Bhatia, H. Singh, N. Grewal, N. K. Natt. J. Pharmacol. Pharmacother.5, 278 (2014).10.4103/0976-500X.142464Search in Google Scholar PubMed PubMed Central
[32] L. Tang, H. Ward, S. Kattakuzhy, E. Wilson, S. Kottilil. Expert Rev. Gastroenterol. Hepatol.10, 21, (2016).10.1586/17474124.2016.1119042Search in Google Scholar PubMed
[33] G.M. Keating, Drugs74, 1127 (2014).10.1007/s40265-014-0247-zSearch in Google Scholar
[34] S. C. Tobias, R. F. Borch. Mol. Pharm.1, 112 (2004).10.1021/mp034019vSearch in Google Scholar
[35] D. Katalenić, V. Škarić. J. Chem. Soc., Perkin Trans. 1 1065 (1992).10.1039/P19920001065Search in Google Scholar
[36] D. Katalenić, V. Škarić, B. Klaić. Tetrahedron Lett.35, 2743 (1994).10.1016/S0040-4039(00)77021-3Search in Google Scholar
[37] D. Williamson, M. J. Cann, D. R. W. Hodgson. Chem. Commun. 5096 (2007).10.1039/b712066dSearch in Google Scholar PubMed
[38] D. Williamson, D. R. W. Hodgson. Org. Biomol. Chem.6, 1056 (2008).10.1039/b717896dSearch in Google Scholar PubMed
[39] R. L. Letsinger, J. S. Wilkes, L. B. Dumas. J. Am. Chem. Soc.94, 292 (1972).10.1021/ja00756a060Search in Google Scholar PubMed
[40] R. L. Letsinger, B. Hapke, G. R. Petersen, L. B. Dumas. Nucleic Acids Res.3, 1053 (1976).10.1093/nar/3.4.1053Search in Google Scholar PubMed PubMed Central
[41] R. L. Letsinger, J. S. Wilkes. Biochemistry15, 2810 (1976).10.1021/bi00658a017Search in Google Scholar PubMed
[42] J. L. Wolfe, T. Kawate, A. Belenky, V. Stanton Jr. Nucl. Acids Res.30, 3739 (2002).10.1093/nar/gkf502Search in Google Scholar PubMed PubMed Central
[43] W. Kotkowiak, A. Pasternak, R. Kierzek. PLoS One11, e0148282 (2016).10.1371/journal.pone.0148282Search in Google Scholar
[44] J. P. Neenan, W. Rohde. J. Med. Chem.16, 580 (1973).10.1021/jm00263a046Search in Google Scholar
[45] M. S. Chen, D. C. Ward, W. H. Prusoff. J. Biol Chem.251, 4833 (1976).10.1016/S0021-9258(17)33191-5Search in Google Scholar
[46] T.-S. Lin, J. P. Neenan, Y.-C. Cheng, W. H. Prusoff. J. Med. Chem. 19, 495 (1976).10.1021/jm00226a009Search in Google Scholar PubMed
[47] S. K. Roberts, G. Cooksley, G. J. Dore, R. Robson, D. Shaw, H. Berns, G. Hill, K. Klumpp, I. Najera, C. Washington. Hepatology48, 398 (2008).10.1002/hep.22321Search in Google Scholar PubMed
[48] M. R. Mish, A. Cho, T. Kirschberg, J. Xu, C. S. Zonte, M. Fenaux, Y. Park, D. Babusis, J. Y. Feng, A. S. Ray, C. U. Kim. Bioorg. Med. Chem. Lett.24, 3092 (2014).10.1016/j.bmcl.2014.05.015Search in Google Scholar PubMed
[49] M. Brandl, X. Wu, M. Holper, L.Hong, Z. Jia, R. Birudaraj, M. Reddy, T. Alfredson, T. Tran, S. Larrabee, X. Hadig, K. Sarma, C. Washington, G. Hill, D. B. Smith. Drug Devel. Ind. Pharm., 34, 683 (2008).10.1080/03639040701836636Search in Google Scholar PubMed
[50] T. Pathak. Chem. Rev.102, 1623 (2002).10.1021/cr0104532Search in Google Scholar PubMed
[51] J. Pereira, D. Levy, J. L. Ruiz, G. A. Brocardo, K. A. Ferreira, R. O. Costa, R. G. Queiroz, D. A. Maria, A. E. Neto, D. A. Chamone, S. P. Bydlowski. Anti-cancer Agents Med. Chem.13, 186 (2013).10.2174/187152013804487416Search in Google Scholar
[52] Q. Wang, X. Liu, Q. Wang, Y. Zhang, J. Jiang, X. Guo, Q. Fan, L. Zheng, X. Yu, N. Wang, Z. Pan, C. Song, W. Qi, J. Chang. Biochem. Pharmacol.81, 848 (2011).10.1016/j.bcp.2011.01.001Search in Google Scholar PubMed
[53] Z. Györgideák, J. Thiem. Adv. Carbohydr. Chem. Biochem.60, 103 (2006).10.1016/S0065-2318(06)60004-8Search in Google Scholar
[54] Z. J. Witczak. Recent advances in the synthesis of functionalized carbohydrate azides. in Specialist Periodical Reports: Carbohydrate Chemistry, A. P. Rauter, T. Lindhorst, (Eds.), Vol. 36, pp. 176–193, Royal Society of Chemistry, London (2010).10.1039/9781849730891-00176Search in Google Scholar
[55] C. E. McKenna, M. T. Higa, N. H. Cheung, M.-C. McKenna. Tetrahedron Lett.18, 155 (1977).10.1016/S0040-4039(01)92575-4Search in Google Scholar
[56] A. J. Gutierrez, E. J. Prisbe, J. C. Rohloff. Nucleos. Nucleot. Nucleic Acids20, 1299 (2001).10.1081/NCN-100002541Search in Google Scholar PubMed
[57] B. C. Challis, J. A. Challis, J. N. Iley. J. Chem. Soc., Perkin Trans. 2 813 (1978).10.1039/p29780000813Search in Google Scholar
[58] I. Wilkening, G. del Signore, C. P. R. Hackenberger. Chem. Commun. 2932 (2008).10.1039/b802030bSearch in Google Scholar PubMed
[59] J. Wang, J. Li, D. Tuttle, J. Y. Takemoto, C.-W. T. Chang. Org. Lett.4, 3997 (2002).10.1021/ol026588rSearch in Google Scholar PubMed
[60] R. K. Harris, E. D. Becker, S. M. Cabral de Menezes, R. Goodfellow, P. Granger. Pure Appl. Chem.73, 1795 (2001).10.1351/pac200173111795Search in Google Scholar
[61] B. K. Singh, A. K. Yadav, B. Kumar, A. Gaikwad, S. K. Sinha, V. Chaturvedic, R. P. Tripathi. Carbohydr. Res.343, 1153 (2008).10.1016/j.carres.2008.02.013Search in Google Scholar PubMed
[62] S. G. Hansen, H. H. Jensen. Synlett20, 3275 (2009).10.1055/s-0029-1218366Search in Google Scholar
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