NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4-7), 837-840 (2001)
`
`UNNATURAL ENANTIOMERS OF 5-AZACYTIDINE
`ANALOGUES: SYNTHESES AND ENZYMATIC
`PROPERTIES
`
`G. Gaubert,1 G. Gosselin,1 S. Eriksson, 2 A. Vita,3
`and G. Maury 1•*
`
`1 UMR 5625 du CNRS, Departement de Chimie, Universite Montpellier II,
`Place Bataillon, 34095 Montpellier Cedex 5, France
`2Swedish University of Agricultural Sciences, Department of Veterinary
`Medical Chemistry, the Biomedical Centre, Box 575,
`S-75 I 23 Uppsala, Sweden
`3Dipartimento di Scienze Veterinarie, Universita di Camerino,
`62024 Matelica, Italy
`
`ABSTRACT
`
`2' -Deoxy-/J-L-5-azacytidine (L-Decitabine), /J-L-5-azacytidine, and derivatives
`were stereospecifically prepared starting from L-ribose or L-xylose. D- and
`L-enantiomers of 2' -deoxy-/J-5-azacytidine were weak substrates of human
`recombinant deoxycytidine kinase (dCK), whereas both enantiomers of /3-5-
`azacytidine or the L-xylo-analogues were not substrates of the enzyme. None
`of the reported derivatives of /3-L-5-azacytidine was a substrate of human
`recombinant cytidine deaminase (CDA).
`
`,B-D-5-Azacytidine (,B-D-5-azaC), 1, and 2' -deoxy-,B-D-5-azacytidine (,B-D-
`5-azadC, Decitabine), 2, are important antileukemic agents used in clinical treat(cid:173)
`ment [1,2]. The use of ,B-D-5-azaC or ,B-D-5-azadC suffers however from several
`drawbacks. The inclusion of an extra nitrogen atom into the cytosine base increases
`its chemical sensitivity with respect to nucleophiles and accounts for the instability
`of the compounds in aqueous solution [3]. Another major cause of resistance is
`
`*Corresponding author.
`
`Copyright © 2001 by Marcel Dekker, Inc.
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`837
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`CELGENE 2017
`APOTEX v. CELGENE
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`GAUBERT ET AL.
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`NH 2
`
`NAN AJ ko-J'OH
`y
`
`R2
`
`R4
`
`1 (RI= R3 = H,R2 = R4= OH)
`
`2 (RI = R2 = R3 = H, R4 = OH)
`
`3 (RI = R3 = H, R2 = R4 = OH)
`4 (RI = R2 = R3 = H, R4 = OH)
`
`S(RI =R4=H,R2=R3=0H)
`
`6(RI =R2=R4=H,R3=0H)
`
`Figure 1.
`CDA.
`
`f/-D- and fl-L-5-azacytidine analogs studied as substrates of ·human dCK and human
`
`induced by the ubiquitous cellular enzyme cytidine deaminase (CDA) since deami(cid:173)
`nation of 1 or 2 results in total Joss of activity [4,5). We have previously shown that
`a number of cytidine analogues having the unnatural L stereochemistry are both
`substrates of human deoxycytidine kinase (dCK) and resistant to human cytidine
`deaminase [6]. We therefore stereospecifically prepared the L-enantiomers of 1, 2
`and other analogues (Fig. 1 ), and we studied their enzymatic properties with respect
`to dCK and CDA in the hope of getting phosphorylation, a Jack of deamination of
`these compounds and possibly an efficient incorporation into DNA.
`
`Chemistry
`
`Most previous syntheses of 5-azacytidine analogues yield both a- and {J(cid:173)
`anomers often difficult to separate. For this reason, we used regio-and stereospe(cid:173)
`cific methods to prepare the L-enantiomers 3-6 starting from L-ribose or L-xylose
`(Fig. 1). 1-0-Acetyl-2,3,5-tri-O-benzoyl-{J-L-ribofuranose was coupled with sily(cid:173)
`Jated 5-azacytidine in the presence of trimethylsilyltriflate. The 2'-benzoyl group
`controls the stereochemistry of the substitution [7] and only the ,8-anomeric deriva(cid:173)
`tive was obtained. Deprotection catalysed by sodium methanolate in methanol af(cid:173)
`forded fJ-L-ribofuranosyl-5-azacytosine,3, in good yield. The 3'- and 5' -positions of
`3 were then protected using dichlorotetraisopropyldisiloxane. A Barton-McCombie
`elimination of the 2' -hydroxyl group of the 3' ,5' -diprotected compound followed
`by deprotection gave L-Decitabine, 4, in 42% overall yield from 3. l,2-Di-O(cid:173)
`acetyl-3,5-di-O-benzoyl-L-xylo-furanose was similarly condensed with silylated 5-
`azacytosine giving exclusively the {J-anomeric derivative. Deprotection with sodium
`
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`

`UNNATURAL ENANTIOMERS OF 5-AZACYTIDINE ANALOGUES
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`839
`
`methanolate afforded /3-L-xylofuranosyl-5-azacytosine, 5, whereas deacetylation
`followed by a Barton-McCombie elimination of the 2'-hydroxyl group and deben(cid:173)
`zoylation yielded 2' -deoxy-/3-L-threo-pentofuranosyl-5-azacytosine, 6.
`
`Biological Results and Discussion
`
`Several studies of the action of dCK on 1 and 2 have been previously re(cid:173)
`ported, and evaluations of the substrate character of /3-D-5-azaC with respect to
`dCK have shown that it is either low or non-existent (8]. Concerning /3-D-5-azadC
`(Decitabine), all existing studies indicate that this compound is an average sub(cid:173)
`strate of human or mammal dCK, with Km ranging from 29 to 71 µM depending
`on the origin of the enzyme and on the conditions [9,10]. We used HPLC to follow
`the kinetics of phosphorylation of 1-6 in the presence of human dCK. Under our
`conditions, substrate reversible decomposition was held below 2% for the dura(cid:173)
`tion of the kinetics. With 5 mM ATP as phosphate donor, only 2'-deoxy-/3-D-5-
`azacytidine, 2, and 2'-deoxy-/3-L-5-azacytidine, 4, were phosphorylated (Km: 94
`and 17 µM, respectively). The efficiencies of phosphorylation were similar for the
`two enantiomers (Vm/Km: 0.075 and 0.05, respectively, compared to 2'-deoxy-/3-
`D-cytidine). The value of the Km constant for 2'-deoxy-/3-D-5-azacytidine is sim(cid:173)
`ilar to the constants previously determined [9,10]. ln contrast, no phosphoryla~
`tion was observed in the case of both enantiomers of /3-5-azacytidine, 1 and 3.
`The /3-L-xylo- or 2'-deoxy-xylo-derivatives 5 and 6 did not display any substrate
`properties.
`Numerous studies have evaluated the sensitivity of /3-D-5-azacytidine and 2' -
`deoxy-/3-D-5-azacytidine to deamination catalysed by cytidine deaminase [11,12].
`Using a low temperature (25°C) and short kinetic durations allowed us to limit
`the decomposition of the substrates as observed from HPLC analysis. Only the
`D-enantiomers 1 and 2 were substrates of human CDA (Km values 225 and 690 µ,M,
`respectively) whereas no deamination occurred for the L-enantiomers 3-6, under
`the same conditions and in the presence of increased concentrations of enzyme.
`The prepared L-nucleoside analogues 3-6 were tested as inhibitors of HIV
`replication in CEM-SS and MT-4 cell systems or as inhibitors of HB V replication in
`HepG2 cells following standard protocols. None of the compounds displayed any
`significant antiviral or cytotoxic effect. Our study only shows that L-Decitabine
`may be monophosphorylated in cells and is resistant to enzymatic deamination.
`The substrate properties and enantioselectivities of cellular nucleotide kinases, vi(cid:173)
`ral DNA polymerases or other concerned enzymes with respect to the ·reported
`compounds are not known and could be unfavourable.
`
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