Mild and efficient functionalization at C6 of purine 2'-deoxynucleosides and ribonucleosides

[reaction: see text] Treatment of sugar-protected inosine and 2'-deoxyinosine derivatives with a cyclic secondary amine or imidazole and I(2)/Ph(3)P/EtN(i-Pr)(2)/(CH(2)Cl(2) or toluene) gave quantitative conversions into 6-N-(substituted)purine nucleosides. S(N)Ar reactions with 6-(imidazol-1-yl) derivatives gave 6-(N, O, or S)-substituted products. The 6-(benzylsulfonyl) group underwent S(N)Ar displacement with an arylamine at ambient temperature.     

2-aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligonucleotides

2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.     

O6-(benzotriazol-1-yl)inosine derivatives: easily synthesized, reactive nucleosides

A novel class of O6-(benzotriazol-1-yl)inosine as well as the corresponding 2'-deoxy derivatives can be conveniently prepared by a reaction between sugar-protected or -unprotected inosine or 2'-deoxyinosine nucleosides and 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The reaction appears to proceed via a nucleoside phosphonium salt, and in the absence of any additional nucleophile, the released 1-hydroxybenzotriazole undergoes reaction with the formed phosphonium salt leading to the requisite O6-(benzotriazol-1-yl)inosine or 2'-deoxyinosine derivatives. Isolation and characterization of the phosphonium salt as well as analysis by 31P{1H} NMR appear to be consistent with this reaction pathway. The resulting O6-(benzotriazol-1-yl)inosine derivatives are effective as electrophilic nucleosides, undergoing facile reactions with a variety of nucleophiles such as alcohols, phenols, amines, and a thiol. Unusual and challenging nucleoside derivatives such as an aryl-bridged dimer, a nucleoside-amino acid conjugate, and a nucleoside-nucleoside dimer have also been synthesized from the O6-(benzotriazol-1-yl)-2'-deoxyinosine derivative. Finally, a fully protected DNA building block, the O6-(benzotriazol-1-yl)-2'-deoxyinosine 5'-O-DMT 3'-O-phosphoramidite, has been prepared and a preliminary evaluation of its use for DNA modification has been performed. Results from these studies indicate several important facts: A single, simple methodological approach provides a class of stable, isolable ribo and 2'-deoxyribonucleoside derivatives that possess excellent reactivity for SNAr chemistry with a wide range of nucleophiles. Also, a benzotriazolyl nucleoside phosphoramidite appears to be a suitable reagent for incorporation into DNA for purposes of site-specific DNA modification.     

Palladium catalysis for the synthesis of hydrophobic C-6 and C-2 aryl 2'-deoxynucleosides. Comparison of C-C versus C-N bond formation as well as C-6 versus C-2 reactivity

Suzuki-Miyaura cross-coupling of haloaromatic compounds with arylboronic acids provides a simple entry to biaryl systems. Despite its ease, to date, there are no detailed investigations of this procedure for deoxynucleoside modification. As shown in this study, a wide variety of C-6 arylpurine 2'-deoxyriboside (C-6 aryl 2'-deoxynebularine analogues) and C-2 aryl 2'-deoxyinosine analogues can be conveniently prepared via the Pd-mediated cross-coupling of arylboronic acids with the C-6 halonucleosides, 6-bromo- or 6-chloro-9[2-deoxy-3,5-bis-O-(tert-butyldimethylsilyl)-beta-D-erythro-pentofuranosyl]purine (1 and 2), and the C-2 halonucleoside, 2-bromo-O(6)-benzyl-3',5'-bis-O-(tert-butyldimethylsilyl)-2'-deoxyinosine (3). Although bromonucleoside 1 proved to be a good substrate for the Pd-catalyzed Suzuki-Miyaura cross-couplings, we have noted that for several C-6 arylations, the chloronucleoside 2 provides superior coupling yields. Also described in this study is a detailed evaluation of catalytic systems that led to optimal product recoveries. Finally, a comparison of the C-C and C-N bond-forming reactions of deoxynucleosides is also reported. On the basis of this comparison, we provide evidence that C-N bond formation at the C-6 position, leading to N-aryl 2'-deoxyadenosine analogues, is more sensitive to the ligand used, whereas C-C bond-forming reactions at the same position are not. In contrast to the ligand dependency exhibited in C-N bond formation at the C-6 position, comparable reactions at the C-2 position of purine deoxynucleosides proceed with less sensitivity to the ligand used.     

N,1-(15)N2]-2'-deoxyadenosines

[reaction: see text] An efficient route to deoxyadenosine derivatives labeled on both the amino group and nitrogen 1 is uncovered. First, 3',5'-di-O-acetyl-2'-deoxy-1-(2-nitrobenzenesulfonyl)inosine (2a) and only 1.1 equiv of (15)NH4Cl are used for labeling position 1 (1a) through the isolation of the open intermediate and its cyclization with DBU in anhydrous CH3CN. Inosine 1a is then converted to [N,1-(15)N2]-3',5'-di-O-acetyl-N6-benzoyl-2'-deoxyadenosine (5a, the precursor of 6a) via a Pd/dppf-catalyzed chloride-to-benzamide replacement, by using again only 1.1 equiv of the labeling source.     

SNAr iodination of 6-chloropurine nucleosides: aromatic Finkelstein reactions at temperatures below -40 degrees C

Mesitoyl or toluoyl esters of inosine and 2'-deoxyinosine were deoxychlorinated at C6 to give the crystalline 6-chloropurine nucleoside derivatives, which underwent quantitative conversion to the 6-iodo analogues with NaI/TFA/butanone at -50 to -40 degrees C. The 6-iodo compounds were efficient substrates for SNAr, Sonogashira, and Suzuki-Miyaura reactions, in contrast with the 6-chloro analogues, and gave good to high yields of C-N and C-C coupled products.     

Separate deamination mechanisms for isomeric styrene oxide induced N1-adenine adducts

[formula: see text] Styrene 7,8-oxide (SO) induced N1-2'-deoxyadenosine 5'-phosphate (AMP) adducts deaminate to corresponding inosine derivatives. For the beta-isomer of N1-SO-AMP, the chiral alpha-carbon was found to be involved in the hydrolytic deamination, suggesting formation of an oxazolinium ring as an intermediate and that a water molecule attacks the benzylic carbon. The mechanism differs from the one suggested for the alpha-isomer of N1-SO-AMP, for which deamination occurs by direct attack of water at the 6-position of purine ring.     

Palladium-catalyzed synthesis of nucleoside adducts from bay- and fjord-region diol epoxides

Palladium-catalyzed C-N bond formation has been utilized to synthesize covalent 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) adducts of benzo[a]pyrene (BaP) series 1 (syn) and benzo[c]phenanthrene (BcPh) series 2 (anti) diol epoxides. For this, (+/-)-10 alpha-amino-7 beta,8 alpha,9 beta-trisbenzoyloxy-7,8,9,10-tetrahydro BaP and (+/-)-1 beta-amino-2 alpha,3 alpha,4 beta-trisbenzoyloxy-1,2,3,4-tetrahydro BcPh were coupled with 6-halo-9-[3,5-bis-O-(tert-butyldimethylsilyl)-beta-D-erythro-pentofuranosyl]purine and O6-benzyl-3',5'-bis-O-(tert-butyldimethylsilyl)-2-bromo-2'-deoxyinosine, using a (+/-)-BINAP-Pd complex and Cs2CO3. For the synthesis of the dA adducts, both the 6-chloro- as well as the 6-bromopurine nucleoside derivatives were analyzed for the C-N coupling reaction with the hydrocarbon amino tribenzoates. With the BaP amino tribenzoate, the 6-chloronucleoside provided satisfactory results, whereas the 6-bromo analogue proved to be superior with the BcPh amino tribenzoate. Overall, lower yields of the dA adducts were obtained with the more hindered fjord-region BcPh amino tribenzoate as compared to the bay-region BaP amino tribenzoate. In contrast to reactions leading to the dA adducts, the C-N reactions of both BaP and BcPh amino tribenzoates with the 2-bromo-2'-deoxyinosine derivative proceeded in comparable yields. This seems to indicate that such Pd-catalyzed adduct forming reactions at the C-6 position may be influenced by steric constraints of the amine component, whereas those at the C-2 position are less sensitive. Diastereomeric adduct pairs were separated and characterized by spectral methods and by comparisons to adducts produced by direct displacement reactions as well as those formed from DNA alkylation by diol epoxides.     

N6-Arylation of 2'-deoxyadenosine via copper-catalyzed direct coupling with aryl halides

[reaction: see text] A general method for efficient N(6)-arylation of 2'-deoxyadenosine via copper-catalyzed direct coupling with aryl iodides and bromides is described. The method is useful for aryl halides with either electron-donating or electron-withdrawing groups.     

Unusual deoxygenation and reactivity studies related to O6-(benzotriazol-1-yl)inosine derivatives

New and unusual developments related to the chemistry of O6-(benzotriazol-1-yl)inosine derivatives are reported. First, a simple, scalable method for their syntheses via the use of PPh3/I2/HOBt has been developed and has been mechanistically investigated by 31P(1H) NMR. Studies were then conducted into a unique oxygen transfer reaction between O6-(benzotriazol-1-yl)inosine nucleosides and bis(pinacolato)diboron (pinB-Bpin) leading to the formation of C-6 (benzotriazol-1-yl)purine nucleoside derivatives and pinB-O-Bpin. This reaction has been investigated by 11B(1H) NMR and compared to pinB-O-Bpin obtained by oxidation of pinB-Bpin. The structures of the C-6 (benzotriazol-1-yl)purine nucleosides have been unequivocally established via Pd-mediated C-N bond formation between bromo purine nucleosides and 1H-benzotriazole. Finally, short and extremely simple synthesis of 1,N6-ethano- and 1,N6-propano-2'-deoxyadenosine are reported in order to demonstrate the synthetic versatility of the O6-(benzotriazol-1-yl)inosine nucleoside derivatives for the assembly of relatively complex compounds.     

Synthesis of pyrene and benzo[a]pyrene adducts at the exocyclic amino groups of 2'-deoxyadenosine and 2'-deoxyguanosine by a palladium-mediated C-N bond-formation strategy

Single-electron oxidation of the carcinogenic hydrocarbon benzo[a]pyrene (BaP) is thought to result in a radical cation intermediate and this species has been proposed to cause alkylation at the nitrogens of the purine nucleobases. Although several different nucleoside adducts have been isolated as arising from this mode of metabolic activation, there are no selective, total syntheses of the stable exocyclic amino group adducts formed by the single-electron oxidation of any hydrocarbon with the purine 2'-deoxynucleosides to date. In this paper we disclose the synthesis of the model adducts N(6)-(1-pyrenyl)-2'-deoxyadenosine and N(2)-(1-pyrenyl)-2'-deoxyguanosine as well as the first synthesis of the carcinogen-linked nucleoside derivatives N(6)-(6-benzo[a]pyrenyl)-2'-deoxyadenosine and N(2)-(6-benzo[a]pyrenyl)-2'-deoxyguanosine via a palladium-mediated C-N bond formation. Two different coupling strategies were attempted: coupling of an aryl bromide with a suitably protected nucleoside and the coupling of an arylamine with a suitable halonucleoside. The former had somewhat limited applicability in that only N(6)-(1-pyrenyl)-2'-deoxyadenosine was prepared by this method; on the other hand, the latter was more general. However, there are noteworthy differences in the amination reactions at the C-6 and C-2 positions. Reactions at the C-6 resulted in the competing formation of a 1:2 amine-nucleoside adduct in addition to the desired monoaryl nucleoside. Such a dimer formation was not observed at the C-2. The C-2 adducts, however, displayed an interesting conformational behavior.     

Studies on the synthesis of compounds related to adenosine 3',5'-cyclic phosphate. VII. Synthesis and cardiac effects of N6,N6-dialkyl adenosine 3',5'-cyclic phosphates

A series of novel N6,N6-dialkyl adenosine 3',5'-cyclic phosphates N6,N6-dialkyl cAMPs) was synthesized from 2'-O-p-toluenesulfonyl cAMP (2'-O-tosyl cAMP, 2) and tested for inotropic and chronotropic activities in vitro. Treatment of 2 with excess alkyl halides and sodium hydride followed by detosylation with aqueous NaOH readily gave N6,N6-dialkyl cAMPs (3) in good yields. Various N6,N6-dialkyl cAMPs having different alkyl groups at the N6-position (9-12) were prepared by alkylation followed by detosylation of N6-alkyl-2'-O-tosyl cAMPs (4) which were obtained by the reductive alkylation of 2 with aldehydes in the presence of sodium cyanoborohydride in acetic acid or tosylation of N6-methyl cAMP. The mechanism of the detosylation is briefly discussed. Among the N6,N6-dialkylated derivatives, N6,N6-dipentyl (3f) and N6-ethyl-N6-heptyl (10e) derivatives were found to exhibit a potent positive inotropic effect and a weak positive chronotropic effect. The structure-activity relationships for the position and the length of alkyl residue are discussed.     

Highly diastereoselective synthesis of nucleoside adducts from the carcinogenic benzo[a]pyrene diol epoxide and a computational analysis

A diastereoselective synthesis of the nucleoside adducts corresponding to a cis ring-opening of the carcinogen (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE-2) by 2'-deoxyadenosine and 2'-deoxyguanosine is described. The key intermediate (+/-)-10alpha-amino-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene was synthesized by a highly diastereoselective dihydroxylation wherein phenylboronic acid was a water surrogate. The resulting boronate ester was converted to a tetraol derivative in which two of the four hydroxyl groups (trans 7, 8) were protected as benzoate esters while the remaining two (cis 9, 10) were free. The cis glycol entity was then subjected to a reaction with 1-chlorocarbonyl-1-methylethylacetate to yield an intermediate chloro monoacetoxy dibenzoate. Displacement of the halide with azide, complete cleavage of the esters, and catalytic reduction of the azide yielded the requisite amino triol. Fluoride displacement from appropriately protected nucleoside derivatives, 6-fluoropurine 2'-deoxyribonucleoside and 2-fluoro-2'-deoxyinosine, by the amino triol then yielded diastereomeric pairs of diol epoxide-adducted 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) nucleosides. Small aliquots of these adducts were separated for characterization purposes. The present approach provides the first diastereoselective synthesis of the cis adducts of BaP DE-2 with 2'-deoxyguanosine as well as the first synthesis of both dA and dG adducts from a common intermediate. An informative analysis of the 1H NMR spectra of the cis adducts synthesized and comparisons to the trans adducts are reported. To gain insight into the diastereoselectivity in the key dihydroxylation step, a computational analysis, including molecular mechanics (MMFF94) and semiempirical AM1 geometry optimizations, yielded results that are in fairly good agreement with the experimental observations.     

Regiospecific and highly stereoselective coupling of 6-(substituted-imidazol-1-yl)purines with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride. Sodium-salt glycosylation in binary solvent mixtures: improved synthesis of cladribine

Glycosylation of 6-(substituted-imidazol-1-yl)purine sodium salts with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride proceeds with regiospecific formation of the N9 isomers. Base substrates with lipophilic substituents on the C6-linked imidazole moiety are more soluble in organic solvents, and the solubility is further increased with binary solvent mixtures. Selective solvation also diminishes the extent of anomerization of the chlorosugar. Stirred reaction mixtures of the modified-purine sodium salts generated in a polar solvent and cooled solutions of the protected 2-deoxysugar chloride in a nonpolar solvent give 2'-deoxynucleoside derivatives with N9 regiochemistry and enhanced beta/alpha configuration ratios. Application of the binary-solvent methodology with 2-chloro-6-(substituted-imidazol-1-yl)purine salts in cold acetonitrile and the chlorosugar in cold dichloromethane gives essentially quantitative yields of the N9 isomers of beta-anomeric 2'-deoxynucleoside intermediates. Direct ammonolysis (NH(3)/MeOH) of such intermediates or benzylation of the imidazole ring followed by milder ammonolysis of the imidazolium salt gives high yields of the clinical anticancer drug cladribine (2-chloro-2'-deoxyadenosine).     

Fluorinase mediated C-(18)F bond formation, an enzymatic tool for PET labelling

The fluorinase enzyme from S. cattleya is applied as a catalyst for the efficient incorporation of [18F]-fluoride into [18F]-5'-fluoro-5'-deoxyadenosine, [18F]-5'-fluoro-5'-deoxyinosine and [18F]-5-fluoro-5-deoxyribose for positron emission tomography (PET) applications.     

6-bromopurine nucleosides as reagents for nucleoside analogue synthesis.

Surprisingly facile direct substitution reactions with acetyl-protected 6-bromopurine nucleosides are described. Included in the series of bromonucleosides studied is the guanosine derivative N(2)-2',3',5'-tetraacetyl-6-bromopurine ribonucleoside, the synthesis of which is reported here for the first time. Brominated nucleosides had not previously been considered optimal substrates for S(N)Ar reactions given the general reactivity trend for halogenated aromatic systems (i.e. F > Cl > Br > I). However, even weakly nucleophilic aromatic amines give high yields of the substitution products in polar solvents with these 6-bromopurine nucleosides. For primary aromatic amines, secondary aliphatic amines, and imidazole, reaction takes place only at C6, with no effect on the acetyl-protected ribose. In addition, we report the first synthesis of 3',5'-di-O-acetyl-6-bromopurine-2'-deoxyribonucleoside and its reaction with an arylamine in MeOH in the absence of added metal catalyst. Thus, C6-arylamine derivatives of both adenosine and 2'-deoxyadenosine can be prepared via simple S(N)Ar reactions with the corresponding 6-bromo precursor. We also describe high yielding and C6-selective substitution reactions with 6-bromonucleosides using alcohol and thiol nucleophiles in the presence of added base (DBU). Finally, C6-bromonucleosides are shown to be readily hydrogenated to give purine or 2-aminopurine products in good yield. This work increases the arsenal of reactions and strategies available for the synthesis of nucleoside analogues as potential biochemical tools or new therapeutics.     

Central depressant effects of N3-substituted 6-azauridines in mice.

Central depressant effects in mice of N3-substituted 6-azauridines (6-AzUd) (1) were examined by intracerebroventricular (i.c.v.) injection. Eleven derivatives including alkyl-, benzyl-, xylyl- and phenylethyl-substitution onto the N3-position of 1 were synthesized and their pharmacological effects were evaluated using hypnotic activity, locomotor activity, motor incoordination and pentobarbital-induced sleep prolongation as indices. Six of 12 compounds showed the hypnotic activity. At a dose of 2 mumol/mouse, the mean sleeping time induced by 1, N3-benzyl-6-AzUd (7), N3-o-xylyl-6-AzUd (8), N3-m-xylyl-6-AzUd (9), N3-p-xylyl-6-AzUd (10) and N3-alpha-phenylethyl-6-AzUd (11) was 14, 11, 45, 12, 9 and 16 min, respectively. These derivatives and N3-beta-phenylethyl-6-AzUd (12) (1.5 mumol/mouse) significantly prolonged pentobarbital-induced (40 mg/kg, i.p.) sleeping time, whereas none of the N3-alkylated derivatives (methyl-, ethyl-, n-propyl-, n-butyl- and allyl-substitution) exerted the hypnotic activity or pentobarbital-induced sleep prolongation. Nucleoside 1 and its xylyl-derivatives (1.5 mumol/mouse) significantly decreased locomotor activity of mice, their effects paralleled the hypnotic activity. These compounds (1.5 mumol/mouse) also produced motor incoordination and potentiated the effect of diazepam-induced motor incoordination. These results indicate that 1 and its benzyl-related derivatives, but not alkyl-derivatives have a depressant effect on the central nervous system.     

Regiospecific N9 alkylation of 6-(heteroaryl)purines: shielding of N7 by a proximal heteroaryl C-H1

Purine alkylations have been plagued with formation of mixtures of N9 (usually desired), N7, and other regioisomers. We have developed methods for synthesis of 6-(azolyl)purine derivatives whose X-ray crystal structures show essentially coplanar conformations of the linked azole-purine rings. Such ring orientations position the C-H of the azole above N7 of the purine, which results in protection of N7 from alkylating agents. Treatment of 6-(2-butylimidazol-1-yl)-2-chloropurine (9) with sodium hydride in DMF followed by addition of ethyl iodide resulted in exclusive formation of 6-(2-butylimidazol-1-yl)-2-chloro-9-ethylpurine (10), whereas identical treatment of 2-chloro-6-(4,5-diphenylimidazol-1-yl)purine (11) produced a regioisomeric mixture 12/13 (N9/N7, approximately 5:1). The linked imidazole and purine rings are coplanar in 9 (the butyl side chain is extended away from the purine ring and C-H is over N7) but are rotated approximately 57 degrees in 11, and the more bulky azole substituent in 11 did not prevent formation of the minor N7 regioisomer 13. Access to various regioisomerically pure 9-alkylpurines is now readily available.     

Syntheses and antitumor activities of N6,N6-dimethyladenosine carboxylate analogues

Several analogues substituted with fatty acid at the 2'-, 3'-, or 5'-position of the ribose moiety of N6,N6-dimethyladenosine were synthesized and tested for antitumor activity against cultured cells of L1210 leukemia and/or Ehrlich ascites. The cytotoxicity and increase of life span obtained with congeners in the N6,N6-dimethyladenosine 3'- or 5'-substituted series were comparable to in vitro or several times better in vivo than those of the mother compound.     

Structure and synthesis of 6-(substituted-imidazol-1-yl)purines: versatile substrates for regiospecific alkylation and glycosylation at N9

X-ray crystal structures of several 6-(azolyl)purine base and nucleoside derivatives show essentially coplanar conformations of the purine and appended 6-(azolyl) rings. However, the planes of the purine and imidazole rings are twisted approximately 57 degrees in a 2-chloro-6-(4,5-diphenylimidazol-1-yl)purine nucleoside, and a twist angle of approximately 61 degrees was measured between the planes of the purine and pyrrole rings in the structure of a 6-(2,5-dimethylpyrrol-1-yl)purine nucleoside derivative. Shielding "above" N7 of the purine ring by a proximal C-H on the 6-azolyl moiety is apparent with the coplanar compounds, but this effect is diminished in those without coplanarity. Syntheses of 6-(azolyl)purines from both base and nucleoside starting materials are described. Treatment of 2,6-dichloropurine with imidazole gave 2-chloro-6-(imidazol-1-yl)purine. Modified Appel reactions at C6 of trityl-protected hypoxanthine and guanine derivatives followed by detritylation gave 6-(imidazol-1-yl)- and 2-amino-6-(imidazol-1-yl)purines. Imidazole was introduced at C6 of 2',3',5'-tri-O-acetylinosine by a modified Appel reaction, and solvolysis of the glycosyl linkage gave 6-(imidazol-1-yl)purine. Guanosine triacetate was transformed into the protected 2,6-dichloropurine nucleoside, which was subjected to S(N)Ar displacement with imidazoles at C6 followed by glycosyl solvolysis to provide 2-chloro-6-(substituted-imidazol-1-yl)purines. Potential applications of these purine derivatives are outlined.