Column chromatography was performed on silica gel 60 H (70-230 mesh; Merck, Darmstadt, Germany), and thin-layer chromatography (TLC) on Merck silica gel aluminum 60 F₂₅₄ precoated plates. The anhydrous solvents were distilled over CaH₂, P₂O₅ or magnesium prior to the use. All commercially available reagents were used without further purification. ¹H, ¹³C, and ¹⁹F NMR spectra were recorded in CDCl₃, CD₃OD and DMSO-d₆ with a Bruker Avance-500-DRX spectrometer at 500.13, 126.76 and 470.59 MHz, respectively. ¹H and¹³C NMR chemical shifts (δ, ppm) are relative to internal chloroform peak (7.26 ppm for ¹H and 77.0 for ¹³C NMR). Chemical shifts are also reported downfield from internal SiMe₄ (¹H) or external CFCl₃ (¹⁹F). Splitting patterns were reported as following: s: singlet, d: doublet, t: triplet, m: multiplet. J values are reported in Hz. Melting points were determined on a Boetius apparatus and were uncorrected. High resolution mass spectra (HRMS) were recorded on an Agilent Q-TOF 6550 Instrument (USA) using ESI (electrospray ionization).

Synthesis of clofarabine was studied using two synthetic routes from known carbohydrate derivatives. In the first approach, we have employed peracylated D-ribofuranose 12 prepared by acetolysis reaction of methyl 2,3,5-O-pivaloylated β-D-ribofuranoside (5) taking into account our previous investigations in this field of 2́-β-fluoro-substituted adenine nucleosides 21 (Scheme 1). However, we failed to reproduce our synthetic procedure 21 reported for preparation of the diacetate 12 through acetolysis of acylated methyl β-D-riboside in a mixture of Ac₂O/AcOH/H₂SO₄ (5.2% vol sulphuric acid) at room temperature. 1-O-Acetate 6 along with the target 1,2-diacetyl D-ribofuranose derivative 12 (21-45%), or only monoacetyl derivative, were prepared in a series of experiments (Scheme 1, conditions b). Further, we have undertaken study of acetolysis of 5 varying various reaction conditions in order to prepare 12 with high yield. Acetolysis (conditions c) in a mixture of reagents - AcOH, Ac₂O, H₂SO₄ (8.0:1.0:1.1) with increased content of sulphuric acid (9.6% vol) gave rise to the 1-O-acetyl derivative 6 as a mixture of β/α-anomers (68%) and acyclic product 9 (10%). Mechanism of the formation of by-product 9 via protonation of the monoacetate 6 and generation of open furanose derivative 8 is outlined in Scheme 1. 1-O-acetate 6 was prepared from methyl ribofuranoside derivative 5 without formation of the diacetate 12, using anhydrous conditions for acetolysis reaction explored earlier for preparation of 1,2-di-O-acetyl-3,5-di-O-pivaloyl-L-arabinofuranoses from pivaloylated methyl L-arabinofuranoside 22. As result of studying acetolysis reaction we have found optimal conditions to obtain 12 with high yield (72%, conditions d) after chromatography on silica gel.

Carrying out acetolysis of 5 in the presence of a small quantity of water resulted to the target diacetate with a high yield. Proposed mechanism of the formation of 12 is shown in Scheme 1. We propose that synthetic route to the target acylated D-ribofuranose derivative includes formation of the 1-O-acetate 6 on the first step of acetolysis (Ac₂O/AcOH/H₂SO₄) followed by generation, in the presence of water, intermediate D-ribofuranose derivatives 10 and 11, which lead to exchange of the 2-O-pivaloyl group in the starting riboside by the 2-O-acetyl group after acetylation at 2-hydroxyl group in selectively acylated ribofuranose 11 with acetic anhydride. It is important to note that the mechanism under above consideration for the acetolysis reaction of D-glycoside 5 with the formation of the 1,2-diacetate 12 differs from that suggested earlier in our previous work 22 for preparation of isomeric 1,2-di-O-acetyl-3,5-di-O-pivaloyl-L-arabinofuranoses via intermediate 1,2-pivaloyloxonium ion from protected L-arabinoside under anhydrous conditions.

Stereoselective glycosylation of silylated 2,6-dichloropurine with the 1,2-diacetate 12 in in acetonitrile in the presence of TMSOTf gave fully O-acylated β-D-riboside of 2,6-dichloropurine 13in 96% yield (Scheme 2).

Selective removal of 2′-O-acetyl group in protected nucleoside 13 was explored under different conditions which are summarized in Table 1 (entries 1-6). Mild removal of 2′-O-acetyl group in nucleoside 5 with the known method, with an excess of NaHCO₃ in methanol 10, gave rise to a mixture of 3′,5′-di-O-pivaloyl (14)and 2′,5′-di-O-pivaloyl (15) 2,6-dichloropurine β-D-ribosides, which were inseparable by column chromatography on silica gel, in an overall 51% yield. The deacetylation of 13 under the mild basic conditions was accompanied by migration the 3′-O-pivaloyl group of 14 from 3′-position to 2′-position with the formation of the 2′-O-pivaloylate 15 (a ratio of 14/15 – 1.7/2.0:1 according to ¹H NMR data of the crude reaction mixtures). We failed to isolate individual acylated nucleosides 14 and 15 from a mixture using chromatography on silica gel or crystallization. Second known approach with application of dibutylthin oxide (Bu₂SnO) in methanol 2324 was tested for selective removal of the 2́-O-acetyl group in nucleoside 13. Mixtures of 3′,5′-di-O-pivaloyl (14)and 2′,5′-di-O-pivaloyl (15) 2,6-dichloropurine ribosides were prepared in 55% and 69% yields, respectively, using 1.1 or 2.1 equiv of Bu₂SnO in anhydrous methanol (entries 2-3), the best ratio of isomeric ribosides being achieved under refluxing of 13 with 2.1 equiv of Bu₂SnO. Novel method for selective deacetylation of nucleoside 13 was explored using NaOCN under various conditions (solvent and excess of inorganic salt, entries 4-6). Best yield of a mixture of nucleosides 14/15 and regioselectivity of the deacylation reaction (a ratio of 14/15 –2.2:1) was prepared with 1.9 equiv of sodium cyanate in anhydrous methanol at room temperature (entry 5). The regioselective deacetylation of 13 with NaHCO₃, NaCNO or Bu₂SnO in methanol was accompanied by migration the 3′-O-pivaloyl group of 3′,5′-diprotected nucleoside 14 with the formation of the 2′-O-pivaloylate 15 (Table 1). Making use 4.3-fold excess of NaHCO₃ or 1.9 eqiuv of NaCNO in anhydrous methanol, mixtures of 14/15 were prepared in 51% and 56% yields, respectively. The use of a large excess of NaHCO₃ in methanol (entry 1) produced less yield of a mixture of isomeric nucleosides 14 and 15 than that isolated after selective deprotection of the 2′-O-acetate 13 with NaCNO (entry 5). These findings may be explained by different solubility of inorganic sodium salts at room temperature that is the important factor in development of efficient method for cleavage of 2′-O-acetyl group. The increase of the reaction time in control experiment with an excess of NaCNO in anhydrous methanol resulted in the formation of by-products (according to TLC data and ¹H NMR data of the reaction mixture) and reducing overall yield of isomeric nucleosides. Fast removing of 2´-O-acetyl protecting group in the intermediate nucleoside 13 (entry 5) likely proceeds by a transesterification mechanism via generation of methoxide anion from sodium cyanate in anhydrous methanol with a gradual release of HOCN.

Further, introduction of the C2′-β-fluorine atom via the nucleophilic replacement of an activated C2′-α-hydroxyl of 3′,5′-di-O-pivaloyl ribonucleoside 14 containing bulky directing groups with a fluoride anion was studied on the next step. The treatment of a mixture of selectively blocked ribonucleosides 14 and 15 (a ratio - 2.3:1 after column chromatography on silica gel)with an excess of DAST in CH₂Cl₂/pyridine at room temperature afforded protected 2′-β-fluoro nucleoside(16,59%).Isomeric 3′-β-fluoro(17, 2-7%)nucleoside was also isolated as by product under the fluorination step of mixtures of nucleosides 14 and 15 after column chromatography on silica gel. New 2,6-dichloropurine 3′,5′-di-O-pivaloyl β-D-2′-fluoroarabinonucleoside 16 was prepared as the key intermediate for synthesis of different purine modified 2′-β-F-nucleosides.Analysis of conformational peculiarities of selectively protected 2,6-dichloropurine ribonucleoside 14 based upon its ¹H NMR spectral data (J_1',2´ = 6.25, J_2´,3´ =5.6, J_3´,4´ =3.27 Hz) shows that the pentofuranose ring in this nucleoside takes a C-2′-endo conformation, S-type, as in the case of the adenine analogue 21 studied earlier in the DAST reaction which leads to adenine 2′-β-fluoro-nucleoside under reflux in moderate yield. However, despite the population of S-type conformation in N/S pseudorotational equilibrium is predominant in solution for the both 3′,5′-di-O-acyl purine ribonucleosides, there are some differences in the spatial organization of 3′,5′-di-O-pivaloyl-2,6-dichloropurine nucleoside and the corresponding adenosine derivative, their C2′-α-hydroxyl activated intermediates forming with DAST during the course of fluorination reactions. Selective amination of 2,6-dichloropurine derivative 16 with ammonia in 1,2-dimethoxyethanefollowed by the removal of acyl groups in 3′,5′-di-O-pivaloyl 2′-β-fluoro nucleoside of 2-chloroadenine 18 with sodium methoxide in methanol resulted in the target nucleoside 3 in 71% combined yield on two steps after column chromatography on silica gel. Thus, synthesis of clofarabine was carried out in seven steps from D-ribose in moderate overall yield exploiting the stereoselective glycosylation of 2,6-dichloropurine with available peracylated D-ribose, selective 2′-O-deacetylation of the intermediate tri-O-acylated nucleoside, and the DAST fluorination of 3′,5′-di-O-pivaloyl-2,6-dichloropurine ribonucleoside.

Next, a short approach to clofarabine (Scheme 3) was investigated from the glycosyl bromide 19 prepared by mild bromination of available perbenzoylated 2-deoxy-2-fluoro-α-D-arabinofuranose with TMSBr 17 taking into account the cost-effective synthesis reported earlier 11. Anion glycosylation of 2-chloroadenine salt, generated in the presence of potassium tert-butoxide and anhydrous potassium bromide as additive in 1,2-dimethoxyethane, with the 1-α-bromide 19 in acetonitrile resulted in a mixture of N9-β-anomer 20 (48%) and its α-anomer 21 (16%) which were separated by column chromatography on silica gel. Formation of protected β- and α-nucleosides (β/α ratio - 3:1) along with by-product (7-8%) was observed in this experiment according to ¹H NMR data of the crude reaction mixture in CDCl₃ (Scheme 2, conditions a₁).

The coupling of 19 with 2-chloroadenine salt, produced by treatment with t-BuOK in the presence of potassium bromide, in a mixture of CH₃CN/CH₂Cl₂ (2:1) (Scheme 2, a₂) at room temperaturegave rise to β-(55%) and α-(18%) protected nucleosides after column chromatography on silica gel with a similar β/α ratio and higher overall yield in comparison to the previous reaction conditions. No increase of anomeric stereoselectivity of the glycosylation reaction of 2-chloroadenine salt was observed in a mixture of CH₃CN/CH₂Cl₂ (1:1) in the presence of calcium hydride as additive and conversion of bromide was 86% for 36 h in this experiment. Synthesis of anomeric benzoylated nucleosides by anion glycosylation of 2-chloroadenine with 1-α-bromide involves competitive S_N2 and S_N1-reaction pathways, the latter occurs via generation of an oxonium ion intermediate. Formation of α-glycosylated product 21 in CH₃CN/CH₂Cl₂ with potassium bromide as additive likely to proceed via a 3,5-benzoxonium ion 25 deriving from the 1-α-bromide, and a subsequent nucleophilic α-attack of the nucleobase at C1.

Characteristic of the studied glycosylation reactions of the potassium salt of 2-chloroadenine, generated using t-BuOK in 1,2-dimethoxyethane, with 1-α-bromosugar are: i) the anion glycosylation reaction with a small excess of the heterobase proceeds in heterogeneous phase in acetonitrile or a mixture of solvents with different polarities at room temperature with a full conversion of bromosugar to nucleoside products; ii) KBr as additive results in a little increase of β/α-anomeric selectivity towards protected N9-β-nucleoside, and iii) good isolated yield of intermediate benzoylated β-2′F-arabinonucleoside is achieved after selective glycosylation in a binary solvent system and separation of N9-β- and α-glycosylated products by column chromatography on silica gel in detected conditions. Furthermore, based on our previous results on efficient synthesis of benzoylated 2-amino-6-chloropurine 2′-fluoro-β-arabinonucleoside from the bromide 1917 and the above findings tested for isomeric 2-chloroadenine nucleoside 20, we may conclude that a better solubility of the modified purine base in organic solvents provide a higher β/α-stereoselectivity of heterogeneous glycosylation reaction of the purine potassium salt in a binary solvent mixture.

Deprotection of benzoylated 2′-fluoro β-nucleoside 20 with ammonia in methanol yielded clofarabine 3 in 78% yield after crystallization. The target nucleoside was prepared in 35-43% overall yields using column chromatography. Synthesis of N6-substituted clofarabine derivative 14 was carried out by a direct alkylation of 3 with 1-methanesulfonyloxy-3-methylbutane (22) in anhydrous dimethylsulfoxide in the presence of t-BuOK at 89-90⁰C. The use of bulky alkylating agent prepared by mesylation of isoamyl alcohol with mesyl chloride resulted in a selective reaction on an exocyclic N6-amino group of the starting nucleoside under studied conditions to give new N6-isopentyl clofarabine analogue 23 in 73% yield after column chromatography on silica gel.