Carbohydrate Research 346 (2011) 2011–2015

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Carbohydrate Research

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Keto-fluorothiopyranosyl nucleosides: a convenient synthesisof 2- and 4-keto-3-fluoro-5-thioxylopyranosyl thymine analogs

Evangelia Tsoukala, Stella Manta, Niki Tzioumaki, Christos Kiritsis, Dimitri Komiotis ⇑Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 26 Ploutonos Str., 41221 Larissa, Greece

a r t i c l e i n f o

Article history:Received 21 January 2011Received in revised form 9 May 2011Accepted 12 May 2011Available online 19 May 2011

Keywords:XylopyranonucleosidesKetonucleosidesFluoronucleosides5-Thionucleosides

0008-6215/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.carres.2011.05.013

⇑ Corresponding author. Tel.: +30 2410 565285; faxE-mail address: dkom@bio.uth.gr (D. Komiotis).

a b s t r a c t

A novel series of fluorinated keto-b-D-5-thioxylopyranonucleosides bearing thymine as the heterocyclicbase have been designed and synthesized. Deprotection of 3-deoxy-3-fluoro-5-S-acetyl-5-thio-D-xylof-uranose (1) and selective acetalation gave the desired isopropylidene 5-thioxylopyranose precursor 3.Acetylation and isopropylidene removal followed by benzoylation led to 3-deoxy-3-fluoro-1,2-di-J-ben-zoyl-4-O-acetyl-50-thio-D-xylopyranose (6). This was condensed with silylated thymine and selectivelydeacetylated to afford 1-(20-J-benzoyl-30-deoxy-30-fluoro-50-thio-b-D-xylopyranosyl)thymine (8). Oxida-tion of the free hydroxyl group in the 40-position of the sugar led to the formation of the target 40-ketocompound together with the concomitant displacement of the benzoyl group by an acetyl affording, 1-(20-O-acetyl-30-deoxy-30-fluoro-b-D-xylopyranosyl-40-ulose)thymine (9). Benzoylation of 3 and removalof the isopropylidene group followed by acetylation, furnished 3-deoxy-3-fluoro-1,2-di-J-acetyl-4-O-benzoyl-50-thio-D-xylopyranose (12). Condensation of thiosugar 12 with silylated thymine followed byselective deacetylation led to the 1-(40-J-benzoyl-30-fluoro-50-thio-b-D-xylopyranosyl)thymine (14). Oxi-dation of the free hydroxyl group in the 20-position and concomitant displacement of the benzoyl groupby an acetyl gave target 1-(40-O-acetyl-30-deoxy-30-fluoro-b-D-xylopyranosyl-20-ulose)thymine (15).

� 2011 Elsevier Ltd. All rights reserved.

Nucleosides and their analogs have far been proven to take animportant place in medicinal chemistry as the structural basis forthe development of therapeutic agents.1–3 In spite of the initial suc-cess obtained with modified nucleosides, both the undesirable sideeffects of certain nucleosides and the demand for new antiviral andantitumor agents have prompted the search for further novelnucleosides with improved biological and chemical properties.4–6

In seeking to investigate new biologically active agents, we havepreviously designed and synthesized fluorinated pyranonucleo-sides, evaluated their potential antiviral, antitumor,7,8 and antioxi-dant9 properties and their activity at molecular level.10,11 Morerecently, in our attempts to arrive at new modified analogs, we havedemonstrated that insertion of a keto group and removal of the pri-mary hydroxymethyl function in the sugar moiety led to variousuncommon nucleosides,7,8,12–15 which showed promising antitu-mor and antiviral activities. It also appeared that these nucleosidesrepresent novel types of prodrugs,13 while they may act as acceptorsin a Michael-addition mechanism.12

In the field of modified nucleosides, considerable interest hasbeen drawn in the synthesis of thiosugars16 and thionucleosideanalogs17–19 in which the ring oxygen atom is replaced by sulfur.The biological interest in thiosugars has expanded to studies on dia-betes, enzyme inhibition, and antiviral and antitumor activities,20

ll rights reserved.

: +30 2410 565290.

while thionucleosides have been recognized as a novel and impor-tant class of antiviral agents and promising antitumor candidates.21

It is noteworthy that the replacement of the oxygen atom by sulfurin the sugar ring of the fluorinated analog of cytosine (FAC) led to anincrease of its activity against various human tumor cell lines, whilea new more biologically important compound was obtained.22,23

In view of the above observations and considering the utility ofderivatives containing sulfur in the ring, as biologically importanttargets in medicinal chemistry, we found intriguing to further ex-plore structure–activity relationships of fluorinated ketopyranonu-cleosides, by replacing the ring oxygen atom by sulfur. We thuspresent the synthesis of a novel class of xylopyranonucleosides ofthymine, possessing a ring sulfur atom and a keto group in the20- or 40-position of the sugar portion.

Retrosynthetic analysis suggested that 20-keto- and 40-keto-thionucleosides could be obtained by utilizing the available 5-thio-xylopyranosyl nucleoside of thymine I and its corresponding fullyunprotected analog II, respectively (Scheme 1). However, neitherselective deacetylation24,25 of thio-analog I, nor specific benzoyla-tion,26 pivaloylation,27 or silyl protection28 of 5-thioxylopyranonu-cleoside II led to the desirable partially protected precursors, butonly to the corresponding deacetylated derivative II, along withthe fully protected analogs III, respectively.

Since all attempts to selectively deprotect or protect the 5-thio-xylopyranosyl analogs of thymine were unsuccessful, we alteredthe retrosynthetic routes for the synthesis of the desirable

SAcO

FOAc

NH

N

O

O

I

SHO

FOH

NH

N

O

O

II

i SRO

FOR

NH

N

O

O

III

ii

R=Bz or Pv or TBDMSi

Scheme 1. Reagents: (i) (a) NaOH/EtOH/pyridine or (b) HONH2�HCl/NaOAc/pyridine; (ii) (a) BzCl/pyridine or (b) PvCl/pyridine or (c) TBDMSCl/pyridine.

2012 E. Tsoukala et al. / Carbohydrate Research 346 (2011) 2011–2015

40-keto- and 20-keto-30-fluoro-5-thioxylopyranosyl nucleosides, 9and 15, respectively. In both routes, the suitable key precursor isthe newly synthesized 1,2-O-isopropylidene analog of 5-thioxylo-pyranose 3.

30-Deoxy-30-fluoro-40-keto-5-thio-b-D-xylopyranonucleoside(9) was prepared according to the synthetic route outlined inScheme 2. Deprotection of 3-deoxy-3-fluoro-5-S-acetyl-5-thio-D-xylofuranose (1) in methanolic ammonia29 gave the glycosyl do-nor, 5-thioxylopyranose 2. Specific acetalation of the free hydroxylgroups in the 1,2-position of the sugar moiety of 2 with 2,2-dime-thoxypropane [(CH3)2C(OCH3)2], in the presence of p-toluenesul-fonic acid (p-TsOH) in N,N-dimethylformamide (DMF),30 affordedthe desired isopropylidene 5-thioxylopyranose 3, in 70% yield.Acetylation of the free hydroxyl group in the 4-position of the thio-sugar 3 with Ac2O/pyridine furnished the acetylated derivative 4.The isopropylidene group of compound 4 was removed underacidic conditions and the resulting compound 5 was reacted withbenzoyl chloride (BzCl) and pyridine to give the 1,2-di-O-benzoylderivative 6. Compound 6 was readily converted into the 1-(20-J-benzoyl-40-O-acetyl-30-deoxy-30-fluoro-50-thio-b-D-xylopyrano-syl)thymine (7), in 68% yield, upon reaction with silyl-protectedthymine in the presence of tin chloride (IV) as catalyst.31 Theparticipation of 20-benzoyloxy group led to the exclusive formationof the b-anomer 7. As expected, the 1H NMR spectrum of 7 showeda large coupling between protons H-10 and H-20 (J = 10.6 Hz),indicating an axial orientation of both protons and an equatorially

OH

OH

FOAcS S

HOF

OH1 2

SAcO

FOR

5: R=H6: R=Bz

SRO

FOBz

NH

N

O

O

7: R=Ac8: R=H

9

SF

OAc

ONH

N

O

O

i

v

vi

vii

15

SF

O

AcO

viii

Scheme 2. Reagents: (i) Ammonia/MeOH; (ii) (CH3)2C(OCH3)2)/p-TsOH/DMF; (iii) Ac2O/NaOH/EtOH/pyridine; (viii) PDC/Ac2O/60 �C/15 min.

oriented thymine ring. When selective deprotection of the fullyprotected thionucleoside 7 with NaOH–ethanol–pyridine24 wasemployed, partially benzoyl analog of thymine 8 was formed. Thecrucial step of this synthetic pathway proved to be the oxidation ofthe suitably protected precursor 8. However, the target ketone 9was obtained, only when the oxidation reaction was performedby pyridinium dichromate (PDC)/Ac2O at 60 �C for 15 min, whilesurprisingly, a simultaneous replacement of the benzoyl group byan acetyl was observed. The IR spectrum measured immediatelyafter completion of the reaction workup revealed a characteristicabsorption of a carbonyl group at 1712 cm�1, confirming thepresence of the desired ketonucleoside 9. It is of interest to men-tion that when the duration of the aforementioned oxidation reac-tion was extended, a mixture of untreatable products wasobtained. Furthermore, when oxidation of 8 was performed by amodified Albright–Goldman reaction, using the DMSO/EtOAc/Ac2O system,32 Pfitzner–Moffatt,33 Garegg–Samuelsson,34 Swern,35

Dess–Martin,36 and pyridinium dichromate (PDC)/3E molecularsieves37 methods, resulted in a mixture of intractable and unsepa-rable materials. The IR spectra of the crude reaction mixturesexhibited characteristic absorptions of S@O and O@S@O groupsat 1055 and 1140 cm�1, respectively, convincing the instability ofsulfur versus oxidative step.

For the synthesis of the 30-deoxy-30-fluoro-20-keto-5-thio-b-D-xylopyranonucleoside (15), a synthetic route starting from theisopropylidene 5-thioxylopyranose 3 was devised (Scheme 2).

OH

SRO

FO OCMe2

3: R=H4: R=Ac

10: R=Bz

OR

ii

iii

iv

viii

SBzO

F OROR

11: R=H12: R=Ac

SBzO

FOR

NH

N

O

O

13: R=Ac14: R=H

vii

NH

N

O

O

vi

iii

v

pyridine; (iv) 90% TFA; (v) BzCl/pyridine; (vi) silylated thymine/SnCl4/CH3CN; (vii)

E. Tsoukala et al. / Carbohydrate Research 346 (2011) 2011–2015 2013

The benzoyl derivative 10 was prepared by protection of the freehydroxyl group in the 4-position of the key isopropylidene thio-sugar 3, in the presence of BzCl in pyridine. The fully protectedthiosugar 12 was readily available from derivative 10 throughhydrolysis with 90% aqueous trifluoroacetic acid (TFA) at roomtemperature followed by acetylation of the resulting compound11. 5-Thioxylopyranose 12 was then coupled to silylated thy-mine using tin chloride (IV) as activator31 to give the protectedb-nucleoside, 1-(40-J-benzoyl-20-O-acetyl-30-deoxy-30-fluoro-50-thio-b-D-xylopyranosyl)thymine (13), in 66% yield. The 1H NMRspectrum of 13 showed large JH,H coupling value of J = 10.6 Hz,indicative for the b-configuration of the sugar moiety. Selectivedeacetylation of the protected thionucleoside 13 with NaOH–ethanol–pyridine24 afforded the 40-J-benzoyl analog of thymine14. In the final step, oxidation of the fluoro benzoyl thionucleo-side precursor 14, performed by PDC/Ac2O at 60 �C for 15 min,and concomitant displacement of the benzoyl group by an acetylgave the desired 1-(40-O-acetyl-30-deoxy-30-fluoro-b-D-xylopyr-anosyl-20-ulose)thymine (15), well characterized by NMR, massand IR spectroscopy.

It is noteworthy that, when oxidation is performed on fluoro-thioxylopyranosyl nucleosides, simultaneous b-elimination reac-tion does not occur, as it happens in the case of the correspondingfluoro-pyranonucleosides. This may be due to the smaller dihedralangle J4–C4–C3–H3 in thiopyranonucleosides 9 and 15, comparedto that in ketopyranosyl analogs (<35� vs >50�), as shown bymolecular modeling studies.

In conclusion, we present herein a convenient synthesis of anovel class of keto-fluoro-thioxylopyranosyl nucleosides, reliedon suitable and selective protection/deprotection sequences.Our synthesis highlighted the efficient installation of keto groupin the sugar moiety and construction of keto-thiopyranose skel-eton, by overcoming sulfur sensitivity during oxidation reaction.The preparation of the new keto-thiopyranonucleosides is aninteresting and primary approach in the field of keto-thiosugaranalogues.

1. Experimental

1.1. General procedure

Melting points were recorded in a Mel-Temp apparatus andare uncorrected. Thin layer chromatography (TLC) was per-formed on Merck precoated 60F254 plates. Reactions were moni-tored by TLC on silica gel, with detection by UV light (254 nm)or by charring with sulfuric acid. Flash column chromatographywas performed using silica gel (240–400 mesh, Merck). 1H and13C NMR spectra were obtained at room temperature with aBrucker 400 spectrometer at 400 and 100 MHz, respectively,using CDCl3 and MeOH-d4 (CD3OD) with internal tetramethylsil-ane (TMS).

The chemical shifts are expressed in parts per million (d) andthe following abbreviations were used: s = singlet, d = doublet,dd = doublet doublet, dtr = doublet triplet and m = multiplet.UV–vis spectra were recorded on a PG T70 UV–VIS spectrometerand mass spectra were obtained with a Micromass Platform LC(ESI-MS). Optical rotations were measured using an Autopol Ipolarimeter. Infrared spectra were obtained with a ThermoScientific Nicolet IR100 FT-IR spectrometer.

All reactions were carried out in dry solvents. CH2Cl2 wasdistilled from phosphorous pentoxide and stored over 4T molec-ular sieves. Acetonitrile was distilled from calcium hydride andstored over 3E molecular sieves. DMF was also stored over 3Emolecular sieves, and pyridine was stored over potassiumhydroxide pellets.

1.2. Synthesis of 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-thio-D-xylopyranose (3)

1.2.1. 3-Deoxy-3-fluoro-5-thioxylopyranose (2)A mixture of methanolic ammonia (382 mL) and compound 117

(9.2 mmol, 1.95 g) was stirred for 2 h at room temperature. Thereaction mixture was evaporated to dryness to give 1.40 g (72%)of compound 2 as a viscous oil, and it was used without furtherpurification (EtOAc, Rf 0.4).

1.2.2. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-5-thio-a-D-xylopyranose (3)

To a stirred suspension of 2 (7.79 mmol, 1.31 g) in anhydrousDMF (98.5 mL) and 2,2-dimethoxypropane (31.15 mL) was addedp-toluenesulfonic acid monohydrate (1.56 mmol, 0.029 g). After20 h the resulting solution was neutralized with Et3N so that pHdid not exceed 7. The solution was concentrated and the residuewas purified by flash chromatography (4:6 hexane–EtOAc, Rf

0.35) to give 1.13 g (70%) of 3 as a yellow syrup: Anal. Calcd forC8H13FO3S: C, 46.14; H, 6.29. Found: C, 46.32; H, 6.37. ESIMS: m/z 209.3 [M+H+].

1.3. Synthesis of 1-(30-deoxy-30-fluoro-20-O-acetyl-b-D-xylopyranosyl-40-ulose)thymine (9)

1.3.1. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-4-O-acetyl-5-thio-a-D-xylopyranose (4)

Compound 3 (5.45 mmol, 1.13 g) was dissolved in a mixture ofpyridine (27 mL) and Ac2O (4.9 mmol, 0.46 mL). The reaction wascarried out at room temperature for 1 h, then was quenched withMeOH at 0 �C and was concentrated in vacuum. The residue wasdiluted with EtOAc, washed with saturated sodium bisulfate, so-dium bicarbonate, and water. The organic extract was dried overanhydrous sodium sulfate, filtered, and evaporated to dryness.The residue was purified by flash chromatography (6:4 hexane–EtOAc, Rf 0.30) to give 1.21 g (89%) of compound 4 as a yellow syr-up: ½a�22

D +26 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 5.24 (d,1H, J = 5.0 Hz, H-1), 4.49 (dtr, 1H, J = 49.8 Hz, J = 9.0 Hz, H-3),4.40–4.31 (m, 2H, H-2 and H-4), 2.91–2.85 (m, 1H, H-5b), 2.68–2.64 (m, 1H, H-5a), 2.10 (s, 3H, OAc), 1.58 (s, 3H, CH3), 1.42 (s,3H, CH3). Anal. Calcd for C10H15FO4S: C, 47.99; H, 6.04. Found: C,48.07; H, 6.17. ESIMS: m/z 251.3 [M+H+].

1.3.2. 3-Deoxy-3-fluoro-4-O-acetyl-5-thio-D-xylopyranose (5)A soln of thioxylopyranose 4 (4.85 mmol, 1.21 g) in 90% TFA

(24.2 mL) was stirred for 10 min at room temperature. The mixturewas concentrated under diminished pressure to leave a residue,which was purified by flash chromatography (3:7 hexane–EtOAc,Rf 0.20) to give 0.92 g (90%) of diol 5 as a colorless syrup: Anal.Calcd for C7H11FO4S: C, 39.99; H, 5.27. Found: C, 40.08; H, 5.12.ESIMS: m/z 211.2 [M+H+].

1.3.3. 3-Deoxy-3-fluoro-1,2-O-benzoyl-4-O-acetyl-5-thio-D-xylopyranose (6)

Compound 5 (4.36 mmol, 0.92 g) was dissolved in a mixture ofpyridine (16 mL) and BzCl (10.9 mmol, 1.27 mL). The reaction wascarried out at room temperature for 1 h, and then was concen-trated in vacuum. The residue was purified by flash chromatogra-phy (6:4 hexane–EtOAc, Rf 0.36) to give 1.51 g (83%) ofcompound 6 as a yellow syrup: ½a�22

D +21 (c 0.1, CHCl3); 1H NMR(CDCl3, 400 MHz): d 8.20–7.96 and 7.72–7.34 (m, 5H, Yarom benzoylgroup), 5.93 (br s, 1H, H-1), 5.75–5.70 (m, 1H, H-2), 5.46–5.39 (m,1H, H-4), 5.09 (dtr, 1H, J = 50.9 Hz, J = 9.4 Hz, H-3), 3.22–3.02 (m,

2014 E. Tsoukala et al. / Carbohydrate Research 346 (2011) 2011–2015

1H, H-5a), 2.95–2.84 (m, 1H, H-5b), 2.19 (s, 3H, OAc). Anal. Calcdfor C21H19FO6S: C, 60.28; H, 4.58. Found: C, 60.17; H, 4.37. ESIMS:m/z 419.4 [M+H+].

1.3.4. 1-(30-Deoxy-30-fluoro-20-O-benzoyl-40-O-acetyl-5-thio-b-D-xylopyranosyl)thymine (7)

A mixture of thymine (5.05 mmol, 0.63 g), hexamethyldisilaz-ane (HMDS) (6.26 mmol, 1.3 mL), and saccharin (0.23 mmol,40.1 mg) in dry CH3CN (18 mL) was refluxed for 30 min undernitrogen. To this were added 3-deoxy-3-fluoro-1,2-O-benzoyl-4-O-acetyl-5-thio-D-xylopyranose (6) (3.61 mmol, 1.51 g) and SnCl4

(5.05 mmol, 0.59 mL). The reaction mixture was refluxed for 3 h,neutralized with saturated sodium bicarbonate, and then ex-tracted with TtOAc (2 � 250 mL). The organic extract was driedover anhydrous sodium sulfate, filtered, and evaporated to dry-ness. The residue was purified by flash chromatography (4:6hexane–EtOAc, Rf 0.32) to give 1.04 g (68%) of compound 7 asa white foam: ½a�22

D +38 (c 0.1, CHCl3); kmax (CHCl3) 260 nm (e9700); 1H NMR (CDCl3, 400 MHz): d 8.05–7.15 (m, 6H, Ythymine

and benzoyl group), 5.92 (d, 1H, J = 10.6 Hz H-10), 5.73 (m, 1H,H-20), 5.31–5.24 (m, 1H, H-40), 4.62 (dtr, 1H, J = 49.8 Hz,J = 9.1 Hz, H-30), 3.11–3.02 (m, 1H, H-50b), 2.92–2.83 (m, 1H, H-50a), 2.13 (s, 3H, OAc), 1.95 (s, 3H, 5-CH3). Anal. Calcd forC19H19FN2O6S: C, 54.02; H, 4.53; N, 6.63. Found: C, 54.27; H,4.73; N, 6.38. ESIMS: m/z 423.4 [M+H+].

1.3.5. 1-(30-Deoxy-30-fluoro-20-O-benzoyl-5-thio-b-D-xylopyranosyl)thymine (8)

Compound 7 (2.46 mmol, 1.04 g) was dissolved in EtOH–pyr-idine (25 + 7.4 mL), 2 M NaOH (4.9 mL) was added and the mix-ture stirred at 0 �C for 30 min. Amberlite IR-120 (H+) was addedto neutralize the base. The suspension was filtered, the resin waswashed with EtOH and pyridine (50 + 50 mL), and the filtratewas evaporated. The solid residue was purified by flash chroma-tography (3:7 hexane–EtOAc, Rf 0.30) to give 0.54 g (58%) ofcompound 8 as a white foam: ½a�22

D +29 (c 0.1, CHCl3); kmax

(CHCl3) 260 nm (e 9137); 1H NMR (CDCl3, 400 MHz): d 8.13–7.21 (m, 5H, Yarom benzoyl group), 7.19 (s, 1H, H-6), 5.93 (d,1H, J = 10.5 Hz, H-10), 5.74–5.65 (m, 1H, H-20), 4.45 (dtr, 1H,J = 50.0 Hz, J = 8.9 Hz, H-30), 4.24–4.17 (m, 1H, H-40) 2.96–2.88(m, 2H, H-50a and Y-50b), 1.92 (s, 3H, 5-CH3). Anal. Calcd forC17H17FN2O5S: C, 53.68; H, 4.50; N, 7.36. Found: C, 53.47; H,4.33; N, 7.08. ESIMS: m/z 381.4 [M+H+].

1.3.6. 1-(30-Deoxy-30-fluoro-20-O-acetyl-b-D-xylopyranosyl-40-ulose)thymine (9)

To a solution of 8 (1.42 mmol, 0.54 g) in dry CH2Cl2 (7.3 mL) wasadded PDC (2.13 mmol, 0.8 g) and Ac2O (7.1 mmol, 0.67 mL). Themixture was heated at 60 �C for 15 min, then cooled to room tem-perature, EtOAc (1.6 mL) was added and the mixture was evapo-rated to dryness. The residue was purified by flashchromatography (4:6 hexane–EtOAc, Rf 0.4) to give 0.22 g (50%) ofcompound 9 as a foam: ½a�22

D +20 (c 0.1, CHCl3); kmax (CHCl3)260 nm (e 14548); IR (Neat); 1712 cm�1 (keto group); 1H NMR(CDCl3, 400 MHz): d 7.26 (s, 1H, H-6), 6.37 (d, 1H, J = 10.7 Hz H-10), 5.97 (m, 1H, J = 9.2 Hz, H-20), 5.03 (dtr, 1H, J = 48.1 Hz, H-30),3.69–3.58 (m, 2H, H-50a and Y-50b), 2.18 (s, 3H, OAc), 1.97 (s, 3H,5-CH3); 13C NMR (CDCl3, 100 MHz): d 193.4, 166.33, 163.01,147.45, 139.17, 109.22, 95.32, 67.50, 62.55, 40.85, 20.66, 12.36;19F NMR: d �65.0. Anal. Calcd for C12H13FN2O5S: C, 45.57; H, 4.14;N, 8.86. Found: C, 45.74; H, 4.38; N, 8.78. ESIMS: m/z 317.4 [M+H+].

1.4. Synthesis of 1-(30-deoxy-30-fluoro-40-O-acetyl-b-D-xylopyranosyl-20-ulose)thymine (15)

1.4.1. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-4-O-benzoyl-5-thio-a-D-xylopyranose (10)

Compound 3 (6.24 mmol, 1.3 g) was dissolved in a mixture ofpyridine (30.8 mL) and BzCl (12.48 mmol, 1.46 mL). The reactionwas carried out at room temperature for 1 h and the mixturewas concentrated in vacuum. The residue was purified by flashchromatography (6:4 hexane–EtOAc, Rf 0.32) to give 1.7 g (87%)of compound 10 as a yellow syrup: ½a�22

D +8 (c 0.1, CHCl3); kmax

(CHCl3) 242 nm (e 8757); 1H NMR (CDCl3, 400 MHz): d 8.07 (d,1H, Yarom benzoyl group), 7.58 and 7.45 (2 tr, 4H, Yarom benzoylgroup), 5.43–5.33 (m, 1H, H-4), 5.20 (d, 1H, J = 5.0 Hz, H-1), 4.68(dtr, 1H, J = 49.8 Hz, J = 8.8 Hz, H-3), 4.47–4.40 (m, 1H, H-2),3.02–2.97 (m, 1H, H-5a), 2.86–2.81 (m, 1H, H-5b), 1.63 (s, 3H,CH3), 1.45 (s, 3H, CH3). Anal. Calcd for C15H17FO4S: C, 57.68; H,5.49. Found: C, 57.47; H, 5.27. ESIMS: m/z 313.3 [M+H+].

1.4.2. 3-Deoxy-3-fluoro-4-O-benzoyl-5-thio-D-xylopyranose(11)

Thioxylopyranose 11 was synthesized from 10 by the similarprocedure as described for 5. It was purified by flash chromatogra-phy (3:7 hexane–EtOAc, Rf 0.23) to give 1.32 g (89%) of diol 11 as acolorless syrup: Anal. Calcd for C12H13FO4S: C, 52.93; H, 4.81.Found: C, 52.82; H, 4.62. ESIMS: m/z 273.3 [M+H+].

1.4.3. 3-Deoxy-3-fluoro-1,2-O-acetyl-4-O-benzoyl-5-thio-D-xylopyranose (12)

Compound 11 (4.77 mmol, 1.3 g) was dissolved in a mixture ofpyridine (23 mL) and Ac2O (4.3 mmol, 0.4 mL). The reaction wascarried out at room temperature for 1 h, and then was concen-trated in vacuum. The residue was purified by flash chromatogra-phy (6:4 hexane–EtOAc, Rf 0.35) to give 1.46 g (86%) ofcompound 12 as a yellow syrup: ½a�22

D +5 (c 0.1, CHCl3); kmax (CHCl3)244 nm (e 9147); 1H NMR (CDCl3, 400 MHz): d 8.20–7.96 and 7.72–7.34 (m, 5H, benzoyl group), 6.14 (br s, 1H, H-1), 5.49–5.45 (m, 1H,H-4), 5.41–5.37 (m, 1H, H-2), 4.90 (dtr, 1H, J = 51.1 Hz, J = 9.4 Hz,H-3), 3.07–2.95 (m, 2H, H-50a and Y-50b), 2.19 (s, 3H, OAc), 2.09(s, 3H, OAc). Anal. Calcd for C16H17FO6S: C, 53.93; H, 4.81. Found:C, 53.87; H, 4.67. ESIMS: m/z 357.4 [M+H+].

1.4.4. 1-(30-Deoxy-30-fluoro-40-O-benzoyl-20-O-acetyl-5-thio-b-D-xylopyranosyl)thymine (13)

A mixture of thymine (5.72 mmol, 0.72 g), HMDS (6.26 mmol,1.3 mL) and saccharin (0.26 mmol, 50 mg) in dry CH3CN (19 mL)was refluxed for 30 min under nitrogen. To this were added 3-deoxy-3-fluoro-1,2-O-acetyl-4-O-benzoyl-5-thio-D-xylopyranose(12) (4.09 mmol, 1.46 g) and SnCl4 (5.73 mmol, 1.02 mL). The reac-tion mixture was refluxed for 3 h, neutralized with saturated so-dium bicarbonate, and then extracted with TtOAc (2 � 250 mL).The organic extract was dried over anhydrous sodium sulfate, fil-tered, and evaporated to dryness. The residue was purified by flashchromatography (4:6 hexane–EtOAc, Rf 0.34) to give 1.59 g (66%)of compound 13 as a white foam: ½a�22

D +18 (c 0.1, CHCl3); kmax

(CHCl3) 260 nm (e 10087); 1H NMR (CDCl3, 400 MHz): d 8.13–8.04 and 7.62–7.19 (m, 6H, Ythymine and benzoyl group), 5.82 (d,1H, J = 10.6 Hz, H-10), 5.55 (m, 1H, H-20), 5.48–5.41 (m, 1H, H-40),4.67 (dtr, 1H, J = 49.8 Hz, J = 9.1 Hz, H-30), 3.18–3.13 (m, 1H, H-50b), 2.94–2.89 (m, 1H, H-50a), 2.06 (s, 3H, OAc), 1.96 (s, 3H, 5-CH3). Anal. Calcd for C19H19FN2O6S: C, 54.02; H, 4.53; N, 6.63.Found: C, 54.31; H, 4.27; N, 6.82. ESIMS: m/z 423.4 [M+H+].

E. Tsoukala et al. / Carbohydrate Research 346 (2011) 2011–2015 2015

1.4.5. 1-(30-Deoxy-30-fluoro-40-O-benzoyl-5-thio-b-D-xylopyranosyl)thymine (14)

Compound 13 (3.31 mmol, 1.4 g) was dissolved in EtOH–pyri-dine (33 + 9.9 mL), 2 M NaOH (6.7 mL) was added and the mixturestirred at 0 �C for 30 min. Amberlite IR-120 (H+) was added to neu-tralize the base. The suspension was filtered, the resin was washedwith EtOH and pyridine (65 + 65 mL), and the filtrate was evapo-rated. The solid residue was purified by flash chromatography(3:7 hexane–EtOAc, Rf 0.32) to give 0.72 g (57%) of compound 14as a white foam: ½a�22

D +11 (c 0.1, CHCl3); kmax (CHCl3) 260 nm (e9960); 1H NMR (CDCl3, 400 MHz): d 8.13–7.21 (m, 6H, Ythymine

and benzoyl group), 5.93 (d, 1H, J = 10.5 Hz, H-10), 5.75–5.62 (m,1H, H-40), 4.41 (dtr, 1H, J = 49.8 Hz, J = 9.0 Hz, H-30), 4.19–4.11(m, 1H, H-20), 2.96–2.88 (m, 2H, H-50a and Y-50b), 1.97 (s, 3H, 5-CH3). Anal. Calcd for C17H17FN2O5S: C, 53.68; H, 4.50; N, 7.36.Found: C, 53.72; H, 4.64; N, 7.53. ESIMS: m/z 381.4 [M+H+].

1.4.6. 1-(30-Deoxy-30-fluoro-40-O-acetyl-b-D-xylopyranosyl-20-ulose)thymine (15)

To a solution of 14 (1.42 mmol, 0.54 g) in dry CH2Cl2 (7.3 mL)was added PDC (2.13 mmol, 0.8 g) and Ac2O (7.1 mmol, 0.67 mL).The mixture was heated at 60 �C for 30 min, then cooled to roomtemperature, EtOAc (1.6 mL) was added and the mixture was evap-orated to dryness. The residue was purified by flash chromatogra-phy (4:6 hexane–EtOAc, Rf 0.38) to give 0.28 g (52%) of compound15 as a foam: ½a�22

D +17 (c 0.1, CHCl3); kmax (CHCl3) 260 nm (e15609); IR (Neat); 1720 cm�1 (keto group); 1H NMR (CDCl3,400 MHz): d 7.21 (s, 1H, H-6), 6.07 (d, 1H, J = 10.6 Hz, H-10), 5.75(m, 1H, J = 9.1 Hz, H-40), 4.83 (dtr, 1H, J = 48.0 Hz, H-30) 3.58–3.43(m, 2H, H-50a and Y-50b), 2.17 (s, 3H, OAc), 1.96 (s, 3H, 5-CH3);13C NMR (CDCl3, 100 MHz): d 195.05, 171.60, 163.00, 147.22,137.54, 112.23, 98.57, 66.39, 64.61, 30.31, 20.84, 12.36; 19F NMR:d �63.2. Anal. Calcd for C12H13FN2O5S: C, 45.57; H, 4.14; N, 8.86.Found: C, 45.69; H, 4.08; N, 8.97. ESIMS: m/z 317.4 [M+H+].

Acknowledgments

This work was supported in part by the Postgraduate Pro-grammes ‘Biotechnology-Quality assessment in Nutrition and theEnvironment’, ‘Application of Molecular Biology-Molecular Genet-ics-Molecular Markers’, Department of Biochemistry and Biotech-nology, University of Thessaly.

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