Synthesis and biological evaluation of 2- and 7-substituted 9-deazaadenosine analogues

A series of 2-halogen and 7-alkyl substituted analogues of 9-deazaadenosine and 2'-deoxy-9-deazaadenosine was synthesized by new efficient methodology involving transformation of corresponding 9-deazaguanosine and 2'-deoxyguanosine, which in turn were synthesized by direct C-glycosylation of 1-benzyl-9-deazaguanine with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose and methyl 2-deoxy-3,5-di-O-(p-toluoyl)-D-ribofuranoside, respectively. Deoxychlorination of C6 and diazotization/chloroor fluoro-dediazoniation of the sugar-protected 9-deazaguanosine, followed by selective ammonolysis at C6 and deprotection of the sugar moiety, gave 2-chloro- and 2-fluoro-9-deazaadenosine (6 and 9). Substitution of the 7-position of the dihalogen-intermediate with alkyl groups, followed by ammonolysis and deprotection, provided 2-chloro-7-alkyl-9-deazaadenosines (13a-e) and 2-fluoro-7-benzyl-9-deazaadenosine (13f). Catalytic hydrogenation of 13a-e gave 7-alkyl-9-deazaadenosines 14a-e. Similarly, 2-chloro-2'-deoxy-9-deazaadenosine (21), 2-chloro-2'-deoxy-7-methyl-9-deazaadenosine (25), 2'-deoxy-9-deazaadenosine (22), and 2'-deoxy-7-methyl-9-deazaadenosine (26) were prepared from sugar-protected 2'-deoxy-9-deazaguanosine. Among these compounds, 7-benzyl-9-deazaadenosine (14b) showed the most potent cytotoxic activity, with IC50 values of 0.07, 0.1, 0.2 and 1.5 microM, while both 7-methyl-9-deazaadenosine (14a) and 2-fluoro-9-deazaadenosine (9) also demonstrated significant cytotoxic activity with IC50 values of 0.4, 0.7, 0.3, and 1.5 microM, and 1.5, 0.9, 0.3, and 5 microM against L 1210 leukemia, P388 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma cells, respectively.     

Synthesis of 2'-methylene-substituted 5-azapyrimidine, 6-azapyrimidine, and 3-deazaguanine nucleoside analogues as potential antitumor/antiviral agents

2'-Deoxy-2'-methylene-6-azauridine (5) and 2'-deoxy-2'-methylene-6-azacytidine (8) have been synthesized via a multi-step procedure from 6-azauridine. 2'-Deoxy-2'-methylene-5-azacytidine (14a) and 2'-deoxy-2'-methylene-3-deazaguanosine (19a) and their corresponding alpha-anomers (14b and 19b) have been synthesized by the transglycosylation of 3',5'-O-(1,1,3,3- tetraisopropyldisiloxane-1,3-diyl)-2'-deoxy-2'-methyleneu ridine (12) with silylated 5-azacytosine and silylated N2-palmitoyl-3-deazaguanine, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by separation of the isomers and deprotection of the blocking groups. These compounds were tested for cytotoxicity against B16F10, L1210, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1, HSV-1, and HSV-2.     

Synthesis of oligodeoxynucleotides containing 2-thiopyrimidine residues--a new protection scheme.

A method is described for the incorporation of 2'-deoxy-2-thiouridine (dS2U) and 2'-deoxy-2-thiothymidine (dS2T) into oligodeoxynucleotides at predetermined positions. This requires N3 or O4-acylation of dS2U and dS2T with toluoyl chloride. These base-protected thiopyrimidines are completely stable toward the aqueous iodine oxidation reagent used in the phosphoramidite DNA synthesis method. The toluoyl protecting group is removed during the standard post-synthetic ammonia treatment. This novel protection strategy allows dS2U and dS2T to be efficiently incorporated into oligodeoxynucleotides at predetermined sites without the usual problem of desulfurization and decomposition. Several 14-mers containing the Eco-RI recognition site (dGGCGGAAXXCCGCC and dGGCGGAAXXCGCGG, where X represents dT, dS2U or dS2T) have been synthesized and characterized by base composition, thermal denaturation, CD spectroscopy and endonuclease substrate activity.     

Synthesis and in vitro anti-HCV activity of beta-D- and 1-2'-deoxy-2'-fluororibonucleosides

Based on the discovery of beta-D-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of beta-D- and L-2'-deoxy-2'-fluoroibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2'-fluoro group was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely beta-D-2'-deoxy-2',5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As beta-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2'-fluoro were combined into one molecule, yielding beta-D-2'-deoxy-2'-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

G-quadruplex induced stabilization by 2'-deoxy-2'-fluoro-D-arabinonucleic acids (2'F-ANA)

The impact of 2'-deoxy-2'-fluoroarabinonucleotide residues (2'F-araN) on different G-quadruplexes derived from a thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), an anti-HIV phosphorothioate aptamer PS-d(T2G4T2) and a DNA telomeric sequence d(G4T4G4) via UV thermal melting (T(m)) and circular dichroism (CD) experiments has been investigated. Generally, replacement of deoxyguanosines that adopt the anti conformation (anti-guanines) with 2'F-araG can stabilize G-quartets and maintain the quadruplex conformation, while replacement of syn-guanines with 2'F-araG is not favored and results in a dramatic switch to an alternative quadruplex conformation. It was found that incorporation of 2'F-araG or T residues into a thrombin-binding DNA G-quadruplex stabilizes the complex (DeltaT(m) up to approximately +3 degrees C/2'F-araN modification); 2'F-araN units also increased the half-life in 10% fetal bovine serum (FBS) up to 48-fold. Two modified thrombin-binding aptamers (PG13 and PG14) show an approximately 4-fold increase in binding affinity to thrombin, as assessed via a nitrocellulose filter binding assay, both with increased thermal stability (approximately 1 degrees C/2'F-ANA modification increase in T(m)) and nuclease resistance (4-7-fold) as well. Therefore, the 2'-deoxy-2'-fluoro-d-arabinonucleic acid (2'F-ANA) modification is well suited to tune (and improve) the physicochemical and biological properties of naturally occurring DNA G-quartets.     

Synthesis and anti-HIV activity of 4'-cyano-2',3'-didehydro-3'-deoxythymidine

A new anti-HIV agent 4'-cyano-2',3'-didehydro-3'-deoxythymidine (9) was synthesized by allylic substitution of the 3',4'-unsaturated nucleoside 14, having a leaving group at the 2'-position, with cyanotrimethylsilane in the presence of SnCl4. Evaluation of the anti-HIV activity of 9 showed that this compound is much less potent than the recently reported 2',3'-didehydro-3'-deoxy-4'-(ethynyl)thymidine (1).     

The synthesis of some branched-chain-sugar nucleoside analogues.

1-(2,3-Epoxy-5-O-trityl-beta-D-lyxofuranosyl)uracil was treated with a number of carbon nucleophiles. Ethynyl lithium gave 3'-deoxy-3'-ethynyl-5'-O-trityl-ara-uridine, which was reduced to the corresponding 3'-ethenyl compound. Sodium cyanide gave 3'-cyano-3'-deoxy-5'-O-trityl-ara-uridine which upon alkaline hydrolysis gave the corresponding 3'-carboxamido compound. 1,3-Dithian-2-yl lithium gave 3'-deoxy-3'-(1,3-dithian-2-yl)-5'-O-trityl-ara-uridine. The trityl group was removed from each of these compounds by mild acidic hydrolysis. Treatment of 2 with 0.1M H2sO4 and mercury (II) acetate afforded 3'-acetyl-3'-deoxy-ara-uridine which upon reduction with NaBH4 gave 3'-deoxy-3'-(1-hydroxyethan-1-yl)-ara-uridine. Acetylation of 6 yielded 5'-O-acetyl-3'-acetyl-2',3'-didehydro-2',3'-dideoxyuridine which upon reduction with NaBH4 produced a mixture of 5'-O-acetyl-2',3'-didehydro-2',3'-dideoxy-3'-(1-hydroxyethan -1-yl)uridine and 1-(R)[5-(S)-acetoxymethyl-4-(1-hydroxyethan-1-yl)-tetrahydrofuran- 2-yl]- uracil. Reduction of 14 with Raney nickel followed by removal of the trityl group gave 3'-deoxy-3'-methyl-ara-uridine.     

Synthesis and anti-viral activity of a series of d- and l-2'-deoxy-2'-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2'R)-d-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2'-deoxy-2'-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2'-fluoro group, was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2'R)-d-2'-deoxy-2',5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N(4)-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N(4)-hydroxyl and the 2'-fluoro into one molecule, resulting (2'R)-d-2'-deoxy-2'-fluoro-N(4)-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.     

Convenient preparation of L-arabino-hexos-5-ulose derivatives from lactose

2,6-di-O-benzyl- (9), 2-O-benzyl-3,4-O-isopropylidene- (19), and 2-O-benzyl-6-O-m-chlorobenzoyl-L-arabino-hexos-5-ulose (20) have been prepared using 4'-deoxy-4'-eno- and 6'-deoxy-5'-eno lactose dimethyl acetal derivatives 7 and 14 as key intermediates. The synthesis of enol ethers 7 and 14 has been performed with good yields by base-promoted elimination of acetone or p-toluenesulfonic acid from 2',6'-di-O-benzyl-, and 6'-O-p-toluenesulfonyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal, respectively. The epoxidation with MCPBA of 7 and 14 in methanol or dichloromethane furnishes C-5'-methoxy and C-5'-m-chlorobenzoyloxy derivatives, easily transformed with good yields into L-arabino 5-ketoaldohexoses 9, 19 and 20.     

New method for the preparation of 3'- and 2'-O-phosphoramidites of 2'- and 3'-difluoromethyluridine derivatives

Hydrogenation of 2'-deoxy-2'-difluoromethylene-5'-O-dimethoxytrityluridine (1) and 3'-deoxy-3'-difluoromethylene-5'-O-dimethoxytrityluridine (7), gave the corresponding 2'- and 3'-difluoromethyluridine derivatives 2a and 8a. Detritylation of compounds 2a, 2b and 8a, 8b resulted in the formation of 1-(2-deoxy-2-C-difluoromethyl-beta-D-arabino-pentofuranosyl)uracil (3a) and 1-(3-deoxy-3-C-difluoromethyl-beta-D-xylo-pento furanosyl)- uracil (9a) as well as corresponding minor isomers 3b and 9b. Compounds 3a and 3b were also obtained from 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (4). Finally, phosphitylation of 2a and 8a provided the title 2'- and 3'-O-phosphoramidites 6 and 10.     

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Synthesis of 2'-modified nucleotides and their incorporation into hammerhead ribozymes.

Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.     

Kilo-scale synthesis process for 2'-O-(2-methoxyethyl)-pyrimidine derivatives

We describe an improved process to produce 2'-O-(2-methoxyethyl)-pyrimidines. Starting with commercially available O-2,2'-anhydro-5-methyluridine and tris-(2-methoxyethyl)borate, we modified the ring-opening reaction conditions and changed to a continuous extraction purification method to give 2'-O-(2-methaxyethyl)-5-methyluridine. The dimethoxytritylation 5'/3' ratios and yield were improved by the use of 2,6-lutidine as the base. Conditions to convert to the 5'-methylcytidine analog and its isolation by crystallization were optimized. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities.     

Crystal structures of B-DNA with incorporated 2'-deoxy-2'-fluoro-arabino-furanosyl thymines: implications of conformational preorganization for duplex stability.

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2' position, such as in the 2'-deoxy-2'-fluoro ribo furanosyl analogue, will drive the conformational equilibrium towards the C3'- endo type (north). Conversely, the 2'-deoxy-2'-fluoro arabino furanosyl modification with opposite stereochemistry at C2' appears to have a preference for a C2'- endo type pucker (south). Incorporation of 2'-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2and [d(CGCGAASTCGCG)]2with incorporated 2'-deoxy-2'-fluoroarabinofuranosyl thymines S (south) at 1.55 A resolution. In the crystal structures, all S residues adopt an O4'- endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2'-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2'-deoxy-2'-fluoroarabino thymidines.     

Synthesis and biological activities of 2'-deoxy-2'-fluoro-4'-thioarabinofuranosylpyrimidine and -purine nucleosides

As part of our ongoing investigation of the synthesis of biologically interesting 2'-modified-4'-thionucleosides, we synthesized 2'-deoxy-2'-fluoro-4'-thioarabinofuranosylpyrimidine and -purine nucleosides, and evaluated their antiviral and antitumor activities. In the pyrimidine series, beta-anomers of 5-ethyluracil, 5-iodouracil, 5-chloroethyluracil, and 5-iodocytosine derivatives showed potent and selective anti-HSV-1 and HSV-2 activities in vitro. In the purine series, guanine and 2,6-diaminopurine derivatives showed prominent antiviral activities with slight cytotoxicity. On the other hand, the 5-fluorocytosine derivative (5F-4'-thioFAC) showed potent antitumor activity against both leukemia and solid tumor. Its antitumor spectrum against 14 human solid tumor and one leukemic cell lines was compared with that of 4'-thioFAC. The results showed that 5F-4'-thioFAC had an antitumor spectrum similar to that of 4'-thioFAC. However, 5F-4'-thioFAC was about 10 times less active than 4'-thioFAC.     

Synthesis and biological properties of 2''-deoxy-5-vinyluridine and 2''deoxy-5-vinylcytidine.

Rapid and efficient syntheses for the preparation of 2'-deoxy-5-vinyluridine and 2'-deoxy-5-vinylcytidine are described starting from nucleoside precursors. Contrary to some previous reports, 2-deoxy-5-vinyluridine has be found to be quite stable under normal laboratory conditions but when tested in animals shows neither toxicity nor anti-leukemic (L1210 cells) or anti-parasitic (Plasmodium berghei) activity. 2'-Deoxy-5-vinylcytidine appears to polymerise readily. It is much less toxic to cell cultures than 2'-deoxy-5'vinyluridine but is almost as active against herpes virus replication (ID50:0.2 microgram/ml) for both type 1 and type 2 herpes virus (selectivity index:225).     

The use of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for regiospecific cleavage of RNA by RNase H from Escherichia coli.

Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.     

Solid-phase synthesis of 5'-deoxy-5'-amino-clitocine analogues

Several 6-substituted-amino-5'-deoxy-5'-amino-clitocine analogues were synthesized in a parallel fashion in solid phase. The desired scaffold was generated by coupling 2,3-O-bis-(t-butyldimethylsilyl)-5-N-(monomethoxytrityl-polystyrene-resin)-1,5-diamino-5-deoxy-beta-D-ribofuranose and 4, 6-dichloro-5-nitropyrimidine. The scaffold was then reacted with a variety of amines to generate a small library of 14 analogues of 5'-deoxy-5'-amino-clitocine following a protocol developed earlier.     

DNA polymerase recognition of 2'-deoxy-2'-fluoroarabinonucleoside 5'-triphosphates (2'F-araNTPs)

We examined the ability of 2'-deoxy-2'-fluroarabinonucleoside 5'-triphosphates (2'F-araNTPs) to serve as substrates of various DNA polymerases. In addition, we also examined the ability of these polymerases to accept DNA-FANA (2'-deoxy-2'-fluoroarabinonucleic acids) chimeras as template strands while synthesizing a DNA or FANA-DNA complementary strand. We provide preliminary data demonstrating that 2'F-araNTPs are indeed substrates of several DNA polymerases, and that FANA-DNA chimeric templates are generally well recognized by these polymerase enzymes.