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1.  Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists 
Journal of medicinal chemistry  2000;43(11):2196-2203.
Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A1 and A3 receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A1, A2A, and A3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A1 or A3 receptors, respectively, were synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.
PMCID: PMC3471159  PMID: 10841798
2.  4′-C-Methyl-2′-Deoxyadenosine and 4′-C-Ethyl-2′-Deoxyadenosine Inhibit HIV-1 Replication▿ 
It is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine. In vitro experiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.
PMCID: PMC3088259  PMID: 21343443
3.  Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability 
Tetrahedron  2010;66(34):6707-6717.
Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in “fixed” relationships, either antiperiplanar where the AE is permanently “on”, or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values.
PMCID: PMC2967253  PMID: 21052524
In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4’) oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold.
PMCID: PMC2910114  PMID: 20183605
Adenosine deaminase; cytidine deaminase; carbocyclic nucleosides; bicyclo[3.1.0]hexane nucleosides
5.  Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2’-deoxyzebularine 
Journal of medicinal chemistry  2008;51(23):7593-7601.
We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2’-deoxyzebularine-5’-triphosphate (dZTP). Because 2’-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine-5’-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
PMCID: PMC2659950  PMID: 19006382
6.  Comprehensive Structural Studies of 2′,3′-Difluorinated Nucleosides: Comparison of Theory, Solution, and Solid State 
The conformations of three 2′,3′-difluoro uridine nucleosides were studied by X-ray crystallography, NMR spectroscopy, and ab initio calculations in an attempt to define the roles that the two vicinal fluorine atoms play in the puckering preferences of the furanose ring. Two of the compounds examined contained fluorine atoms in either the arabino or xylo dispositions at C2′ and C3′ of a 2′,3′-dideoxyuridine system. The third compound also incorporated fluorine atoms in the xylo configuration on the furanose ring but was substituted with a 6-azauracil base in place of uracil. A battery of NMR experiments in D2O solution was used to identify conformational preferences primarily from coupling constant and NOE data. Both 1H and 19F NMR data were used to ascertain the preferred sugar pucker of the furanose ring through the use of the program PSEUROT. Compound-dependent parameters used in the PSEUROT calculations were newly derived from complete sets of conformations calculated from high-level ab initio methods. The solution and theoretical data were compared to the conformations of each molecule in the solid state. It was shown that both gauche and antiperiplanar effects may be operative to maintain a pseudodiaxial arrangement of the C2′and C3′ vicinal fluorine atoms. These data, along with previously reported data by us and others concerning monofluorinated nucleoside conformations, were used to propose a model of how fluorine influences different aspects of nucleoside conformations.
PMCID: PMC2739414  PMID: 18558684
7.  2’-Deoxy-N4-[2-(4-nitrophenyl) ethoxycarbonyl]-5-azacytidine: a novel inhibitor of DNA methyltransferase that requires activation by human carboxylesterase 1 
Cancer letters  2008;266(2):238-248.
2’-Deoxy-N4-[2-(4-nitrophenyl) ethoxycarbonyl]-5-azacytidine (NPEOC-DAC), decitabine with a modification of the N4 position of the azacitidine ring can be used to inhibit DNA methyltransferase. This modification protects the azacitidine ring and can be cleaved by carboxylesterase to release decitabine. NPEOC-DAC was 23-fold less potent at low doses (<10 µM) than decitabine at inhibiting DNA methylation, and was also associated with a 3-day delay in its effect. However, at doses ≥ 10 µM NPEOC-DAC was more effective at inhibiting DNA methylation. Theses differences between decitabine and NPEOC-DAC are dependent on the cleavage of the carboxylester bond, and could be potentially exploited pharmacologically.
PMCID: PMC2528876  PMID: 18499340
DNA methylation; decitabine; NPEOC-DAC; carboxylesterase; DNA methyltransferase
8.  What are the consequences of freezing the anomeric effect in nucleosides? 
The consequences of freezing the orientation of the oxygen’s lone pair orbitals —which determines whether the anomeric effect is operative or not— were studied theoretically and experimentally in two oxobicyclo-[3.1.0]hexane nucleosides (1 and 2). The results showed significant differences in the properties of these molecules, which correlated with the magnitude of the n2 → σ* delocalization.
PMCID: PMC2700545  PMID: 18776494
9.  The Triphosphate of β-D-4’-C-Ethynyl-2’,3’-Dideoxycytidine is the Preferred Enantiomer Substrate for HIV Reverse Transcriptase 
Bioorganic & medicinal chemistry  2006;15(1):283-287.
The enantioselective synthesis of the β-D (1) enantiomer of 4’-C-ethynyl-2’,3’-dideoxycytidine confirms an earlier stereochemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate between the D- and L-configuration of nucleoside 5’-triphosphates.
PMCID: PMC1764207  PMID: 17046266

Results 1-9 (9)