3′-DEOXYRIBOFURANOSE DERIVATIVES OF 1-DEAZA AND 3-DEAZA-ADENOSINE AND THEIR ACTIVITY AS ADENOSINE DEAMINASE INHIBITORS

2,6-Dichloro-1-deazapurine and 2,6-dichloro-3-deazapurine were coupled with 1,2-O-diacetyl-5-O-benzoyl-3-deoxy-β-D-ribofuranose. Deprotection of the obtained compounds and reaction with liquid ammonia gave the desired 2-chloroadenine nucleosides, which were dechlorinated to afford the corresponding 1-deaza and 3-deazaadenosine derivatives. Biological studies performed on ADA from calf intestine showed that these new nucleosides are inhibitors of the enzyme.     

Coupling of 2,6-Dichloropurine and 2,6-Dichlorodeazapurines with Ribose and Ribose Modified Sugars

Abstract

Substituted purine and deazapurine nucleosides are of great interest in medicinal chemistry. Furthermore, 3′-deoxynucleosides exhibit a number of biological activities. In this research the coupling of 2,6-dichloro-1- or 3-deazapurine with protected 3′-deoxyribose is reported. Depending upon coupling conditions and base structure, different anomeric and isomeric mixtures have been obtained. Extensive studies, utilizing chemical and physical methods, have been performed to assign the correct configuration to the resulting nucleosides.     

Inhibitory Effects of 1-Deazaadenosine Analogues on HIV Replication and Adenosine Deaminase

Abstract

A series of 2′,3′-dideoxy-N⁶-(cyclo)alkyl-1-deazaadenosine derivatives were synthesized starting from 2,6-dichloro-1-deazapurine (9). The new nucleosides proved to be good inhibitors of HIV-1 replication, the most active being the 2′,3′-dideoxy-2-chloro-N⁶-cycloctyl-1-deazaadenosine (14h, ED₅₀ = 0.4 μ).     

Impact of 3-deazapurine nucleobases on RNA properties

Deazapurine nucleosides such as 3-deazaadenosine (c³A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c³A and the thus far underinvestigated 3-deazaguanosine (c³G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c⁷G, c⁷A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c³A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.     

X-ray crystallographic study of several 2'-deoxy-beta-D-ribonucleosides with 1-deazapurine-derived aglycones

The 2'-deoxy-beta-D-ribonucleosides of 1,3-deazapurine (benzimidazole (1)), 1-deazapurine (both 1H-imidazo[4,5-b]pyridine (2) and 3H-imidazo[4,5-b]pyridine (3)), and 6-benzoylamino-1-deazapurine (7-benzoylamino-3H-imidazo[4,5-b]pyridine (4)) have been prepared and structurally characterized by X-ray crystallography. Especially compounds 1-3 can serve as artificial nucleosides that may substitute 2'-deoxy adenosine because they lack the exocyclic amino group and one or two of the endocyclic nitrogen atoms and hence have a much smaller potential to engage in hydrogen bonds. In the latter respect, they are candidates for nucleosides in metal-ion mediated base pairs. The unit cell of compound 3 contains two crystallographically independent molecules. Compound 4 was crystallized from methanol and water, respectively, giving rise to two different solvates. Despite the closely related aglycones, the sugar conformations in 1-4 are found to be highly variable (1: (2)T(1); 2: (3)T(2); 3: (3)E and E(4); 4: (2)E and (2)T(3)). The structures reported here confirm that there is no simple correlation between the sugar conformation and the character of the nucleoside, and they will hopefully contribute to a better understanding of the complex interplay of different effects that are in control of the conformational equilibrium.     

Propynyl groups in duplex DNA: stability of base pairs incorporating 7-substituted 8-aza-7-deazapurines or 5-substituted pyrimidines

Oligonucleotides incorporating the 7-propynyl derivatives of 8-aza-7-deaza-2′-deoxyguanosine (3b) and 8-aza-7-deaza-2′-deoxyadenosine (4b) were synthesized and their duplex stability was compared with those containing the 5-propynyl derivatives of 2′-deoxycytidine (1) and 2′-deoxyuridine (2). For this purpose phosphoramidites of the 8-aza- 7-deazapurine (pyrazolo[3,4-d]pyrimidine) nucleosides were prepared and employed in solid-phase synthesis. All propynyl nucleosides exert a positive effect on the DNA duplex stability because of the increased polarizability of the nucleobase and the hydrophobic character of the propynyl group. The propynyl residues introduced into the 7-position of the 8-aza-7-deazapurines are generally more stabilizing than those at the 5-position of the pyrimidine bases. The duplex stabilization of the propynyl derivative 4b was higher than for the bromo nucleoside 4c. The extraordinary stability of duplexes containing the 7-propynyl derivative of 8-aza-7- deazapurin-2,6-diamine (5b) is attributed to the formation of a third hydrogen bond, which is apparently not present in the base pair of the purin-2,6-diamine 2′-deoxyribonucleoside with dT.     

Total synthesis of certain 2-, 6-mono- and 2,6-disubstituted-tubercidin derivatives. Synthesis of tubercidin via the sodium salt glycosylation procedure.

Direct glycosylation of the sodium salt of 4,6-dichloro- or 4,6-dibromo-2-methylthiopyrrolo[2,3-d]pyrimidine with 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide gave good yield of the corresponding N7-glycosylated pyrrolo [2,3-d]pyrimidine. The intermediate 4-amino-6-chloro-2-methylthio-7-beta-D-ribofuranosylpyrrolo[2,3-d] pyrimidine provided a new synthetic route to tubercidin, via 6-chlorotubercidin. 6-Chloro-2-methoxytubercidin was also obtained from 10 via the methylsulfone. Application of this glycosylation procedure to 4,6-dichloro- or 4,6-dibromo-2-methylpyrrolo [2,3-d]-pyrimidine also furnished the corresponding N7-glycosyl derivatives with beta-configuration. Dehalogenation of gave 2-methyl-tubercidin and bromination with bromine in a buffered solution gave 5,6-dihalo-2-methyltubercidin. Several new 2,6-disubstituted tubercidin derivatives were prepared from these glycosyl intermediates. This new sodium salt glycosylation procedure was found to be superior to other procedures for the total synthesis of these halogenated 7-deazapurine nucleosides.     

Studies on the Chemical Synthesis of Potential Antimetabolites. 35.1 Synthesis of 2-Chloro-1-deazaadenosine and 2-Chloro-1-deazainosine as Candidate Inhibitors for S-Adenosylhomocysteinases and Methyltransferases

Abstract

The syntheses of 2-chloro-1-deazaadenosine (2) and 2-chloro-1-deazainosine (3) are described. Conversion of 7-ribosylated 6-chloro-1-deazapurine 3-oxide to the desired 2,6-disubstituted 9-ribosyl-1-deazapurines was effected by a series of reactions involving “deoxygenative chlorination” and transglycosylation in satisfactory yields.     

Phosphoramidate pronucleotides of cytostatic 6-aryl-7-deazapurine ribonucleosides

A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2′,3′-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.     

Biological activity of 1-deazapurine nucleosides: role of deoxycytidine kinase?

Several 1-deazapurine nucleosides were tested for their biological activity, anti-HIV-1, cytotoxicity and inhibition of adenosine deaminase (ADA). A2780 human ovarian cancer cells and the deoxycytidine kinase (dCK) deficient variant AG6000, used to determine whether dCK plays a role in their activation, showed a similar sensitivity to the analogs. This is in line with substrate specificity tests, which revealed a very low affinity of dCK.     

Biological Activity of 1-Deazapurine Nucleosides: Role of Deoxycytidine Kinase?

Abstract

Several 1-deazapurine nucleosides were tested for their biological activity; anti-HIV-1, cytotoxicity and inhibition of adenosine deaminase (ADA). A2780 human ovarian cancer cells and the deoxycytidine kinase (dCK) deficient variant AG6000, used to determine whether dCK plays a role in their activation, showed a similar sensitivity to the analogs. This is in line with substrate specificity tests, which revealed a very low affinity of dCK.     

A remarkable stabilization of complexes formed by 2,6-diaminopurine oligonucleotide N3'-->P5' phophoramidates

2'-Deoxyribo- and ribo-oligonucleotide N3'-->P5'phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 degrees C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3'-->P5' phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.     

Efficient synthesis of the tRNA nucleoside preQ0, 7-cyano-7-deazaguanosine, via microwave-assisted iodo→carbonitrile exchange

The naturally occurring tRNA nucleoside preQ(0), 7-cyano-7-deazaguanosine, which is a central intermediate for other natural occurring 7-deazapurine nucleosides was synthesized via a copper(I)-ion-mediated iodo→carbonitrile exchange. The reaction was performed on the easily accessible 7-iodo-7-deazaguanosine under microwave conditions. The overall reaction yield was 30% starting with the glycosylation reaction of the nucleobase. Corresponding 2'-deoxyribonucleosides were prepared following the same route.     

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.     

A Remarkable Stabilization of Complexes Formed by 2,6-Diaminopurine Oligonucleotide N3′→P5′ Phosphoramidates

Abstract

2′-Deoxyribo- and ribo-oligonucleotide N3′→P5′phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 °C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3′→5′ phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.     

8-Aza-7-deazapurine DNA: Synthesis and Duplex Stability of Oligonucleotides Containing 7-Substituted Bases

Abstract

The 7-substituted 8-aza-7-deazapurine phosphoramidites 1a – 3c as well as the phosphoramidite 4a were synthesized. In comparison to the parent purine oligonucleotide duplexes, the 7-substituted 8-aza-7-deazapurine residues lead to a significant duplex stabilization.     

Convergent syntheses and cytostatic properties of 2-chloro-2′-deoxy-2′-fluoroadenosine and its N-isomer

Glycosylation of trimethylsilylated 2,6-dichloropurine 2 with acetate 1 in anhydrous MeCN was investigated. In the presence of SnCl₄, the reaction was regio- and stereoselective affording N⁷-β-glycoside 3 (86%). The use of TMS-Tfl instead of SnCl₄ afforded a ≈ 9:1 mixture of the N⁹-β- and --glycosides 5 and 6 (90%, combined). The title nucleosides were tested for their cytotoxicity.     

Cytostatic evaluations of nucleoside analogs related to unnatural base pairs for a genetic expansion system

The introduction of an unnatural base pair into DNA enables the expansion of genetic information. To apply unnatural base pairs to in vivo systems, we evaluated the cytostatic toxicity of several nucleoside analogs by an MTT assay. Several nucleoside analogs based on two types of unnatural base pairs were tested. One is a hydrogen-bonded base pair between 2-amino-6-(2-thienyl)purine (s) and pyridin-2-one (y), and the other is a hydrophobic base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa). Among the nucleoside analogs, the ribonucleoside of 6-(2-thienyl)purine possessed the highest cytostatic activity against CCRF-CEM and especially HT-1080, as well as the normal fibroblast cell line, WI-38. The other analogs, including its 2'-deoxy, 2-amino, and 1-deazapurine nucleoside derivatives, were less active against CCRF-CEM and HT-1080, and the toxicity of these nucleosides toward WI-38 was low. The nucleosides of y and Pa were inactive against CCRF-CEM, HT-1080, and WI-38. In addition, no cytostatic synergism was observed with the combination of the pairing nucleosides of s and y or Ds and Pa.     

Synthesis and anti-HIV activity of beta-D-3'-azido-2',3'-unsaturated nucleosides and beta-D-3'-azido-3'-deoxyribofuranosylnucleosides

Since the discovery of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2',3'-didehydro-2',3'dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Microbial synthesis of 2,6-diaminopurine nucleosides

2,6-Diaminopurine nucleosides are used as pharmaceutical drugs or prodrugs against cancer and viral diseases.

The synthesis of 2,6-diaminopurine riboside, -2′-deoxyriboside, -2′,3′-dideoxyriboside and -arabinofuranoside was efficiently carried out by transglycosylation using bacterial whole cells as biocatalysts. The preparation of 2,6-diaminopurine-2′,3′-dideoxyriboside catalysed by whole cells is here reported for the first time.