Pyrimidine Nucleoside Analogues Research Articles

Resistance to purine and pyrimidine nucleoside and nucleobase analogs by the human MDR1 transfected murine leukemia cell line L1210/VMDRC.06

Overexpression of human MDR1 P-glycoprotein [Pgp] is associated with cellular resistance to bulky amphipathic drugs, such as taxol, anthracyclines, vinca alkaloids, and epipodophyllotoxins by actively effluxing drugs from cells. We have found that human MDR1 transfected murine L1210/VMDRC.06 leukemia cells exhibit relatively large amounts of Pgp and high levels of resistance to 6-mercaptopurine [6-MP] and other purine and pyrimidine nucleobase and nucleoside analogs. L1210/VMDRC.06 cells accumulated 6-MP as the nucleotide in vitro at only about one-third of that formed by parental L1210 cells in normal medium; however, under conditions of ATP-depletion, the amount of 6-MP nucleotide formed was essentially the same in both cell lines. The findings support active efflux of 6-MP in L1210 cells, suggesting involvement of Pgp in 6-MP resistance even though it is generally believed that Pgp does not transport such agents. The resistance pattern observed in L1210/VMDRC.06 cells was not duplicated in P388/VMDRC.04 leukemia cells transfected with the same MDR1 cDNA, even though a similar amount of Pgp was present in both cell lines. Immunofluorescent staining of surface membrane Pgp showed that L1210/VMDRC.06 cells contained at least three-fold more surface Pgp than P388/VMDRC.04, implying that P388/VMDRC.04 cells are unable to actively efflux 6-MP and other antimetabolites as effectively as L1210/VMDRC.06, because of significantly lower membrane Pgp. The findings suggest that the exceedingly large concentration of overexpressed Pgp in the surface membrane of L1210/MDRC.06 cells is responsible for resistance to 6-MP and other purine and pyrimidine analogs, even though these agents usually are not considered to be substrates for Pgp.

A TOPS-MODE approach to predict adenosine kinase inhibition

The TOPological Sub-Structural Molecular Design (TOPS-MODE) approach has been applied to the study of the adenosine kinase inhibitory activity of pyrrolo[2,3-d]pyrimidine nucleoside analogues. A model capable of describing around 77% of the variance in the experimental activity of 32 analogues of these compounds was developed with the use of the mentioned approach. In contrast, no one of nine different approaches, including the use of Constitutional, Topological, BCUT, 2D autocorrelations, geometrical, RDF, 3D Morse, WHIM, and GETAWAY descriptors were able to explain more than 70% of the variance in the mentioned property with the same number of descriptors. Although, statistically significant models were derived containing other descriptors than spectral moments still the best one fitted out model was find with these variables.

A TOPS-MODE approach to predict adenosine kinase inhibition

The TOPological Sub-Structural Molecular Design (TOPS-MODE) approach has been applied to the study of the adenosine kinase inhibitory activity of pyrrolo[2,3- d]pyrimidine nucleoside analogues. A model capable of describing around 77% of the variance in the experimental activity of 32 analogues of these compounds was developed with the use of the mentioned approach. In contrast, no one of nine different approaches, including the use of Constitutional, Topological, BCUT, 2D autocorrelations, geometrical, RDF, 3D Morse, WHIM, and GETAWAY descriptors were able to explain more than 70% of the variance in the mentioned property with the same number of descriptors. Although, statistically significant models were derived containing other descriptors than spectral moments still the best one fitted out model was find with these variables.

In vitro assessment of nucleoside analogs in multiple myeloma.

To identify nucleoside analogs that may be effective for multiple myeloma (MM), we tested fludarabine, clofarabine, arabinosylguanine, cytarabine, troxacitabine, and gemcitabine in MM cell lines. We employed biologic and biochemical assays in MM cell lines to evaluate the clinical potential of these nucleoside analogs. Among these purine and pyrimidine nucleoside analogs, fludarabine, clofarabine and gemcitabine were the most potent. MM cell lines, resistant to commonly used chemotherapeutic agents for this disease, were more sensitive to gemcitabine with an IC50 in the nanomolar range. The greater cytotoxicity of gemcitabine in MM cells was consistent with greater accumulation of gemcitabine triphosphate, the major cytotoxic metabolite of this drug. MM.1S cells accumulated >100 microM gemcitabine triphosphate but accumulated <20 microM of the other analogs as the respective triphosphates. In addition incubation with gemcitabine resulted in inhibition of DNA synthesis. Incubation with 25, 50 or 100 nM gemcitabine resulted in a dose- and time-dependent increase in the cell population with a subG1 DNA content indicative of apoptosis. These results suggest that gemcitabine is a potent nucleoside analog in MM cell lines including cell types resistant to other chemotherapeutic agents. The greater activity of gemcitabine compared to other analogs seems to be due to favorable metabolism of this agent.

An Efficient Route to Pyrimidine Nucleoside Analogues by [4 + 2] Cycloaddition Reaction.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Inactivity of the bicyclic pyrimidine nucleoside analogues against simian varicella virus (SVV) does not correlate with their substrate activity for SVV-encoded thymidine kinase

Simian varicella virus (SVV) and human varicella-zoster virus (VZV) are closely related viruses that share many structural and functional properties. 5-Substituted 2 ′-deoxyuridine derivatives (e.g., BVDU, BVaraU) and acyclic guanine nucleoside derivatives (i.e., ACV and GCV) show comparable antiviral efficacy against VZV and SVV in cell culture. In contrast, the novel bicyclic nucleoside analogues (BCNAs) are exquisitely inhibitory to VZV (EC 50 in the lower nanomolar range) but completely inactive against SVV. The VZV-encoded thymidine kinase (TK) appeared to be essential for BCNA activation (phosphorylation) and anti-VZV activity. Also SVV TK is able to recognize the BCNAs as substrate, although with a different structure–affinity relationship. Thus, viral TK-catalyzed phosphorylation is necessary but not sufficient for the BCNAs to display antiviral activity. Our data suggest that the eventual target of the BCNAs against VZV is either absent in SVV or, alternatively, is insensitive for the (phosphorylated) BCNAs.

Hepatology highlights: Viral hepatitis

Hepatology highlights: Viral hepatitis

Synthesis of the Novel Bicyclic Oxepinopyrimidine and Fluorinated Pyrrolidinopyrimidines

The new 5,6-disubstituted pyrimidine nucleoside analogues (5-8) were synthesized by addition of the C-6 lithiated pyrimidine derivative to oxirane as key reaction step. The bicyclic tetrahydrooxepinopyrimidine (9) and fluorinated pyrrolidinopyrimidines (12 and 15) were obtained by intramolecular linkage of acyclic chain to the CH 2 -5 and N-1 of the pyrimidine ring. The structure of compound (9) containing pyrimidine and tetrahydrooxepine skeleton was determined unequivocally by its X-Ray crystal structure analysis.

Efficient Pyrimidine N‐1‐Alkylation via Activation of Electron Rich Olefins with Oxoammonium Salts: Synthesis of Methoxy TEMPO Substituted Pyrimidine Nucleoside Analogs

Our work outlines the use of oxoammonium salts in a formal 1,2 addition process to olefins giving nucleoside analogs as products. Specifically, oxoammonium salts can be added to a solution of olefin and silylated heterocycle to give Methoxy TEMPO substituted nucleoside analogs after hydrolytic workup and chromatographic purification.

The role of thymidine kinases in the activation of pyrimidine nucleoside analogues.

Deoxynucleoside analogues need activation by deoxynucleoside kinases to serve as antiviral or anticancer agents. Here we review the properties of cellular cytoplasmic thymidine kinase 1, mitochondrial thymidine kinase 2, the multisubstrate deoxynucleoside kinase from Drosophila melanogaster and Herpes virus 1 thymidine kinase. Important substrate activity relationships will be discussed.

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth.

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.

Thiosugars. X. Novel Nucleoside Analogues Derived from 4-Thio-L-lyxofuranose#

1-O-Acetyl-2,3,5-tri-O-benzyl-4-thio-L-lyxofuranose 1 was transformed into O-benzyl- and O-acetyl-protected 1-(4-thio-L-lyxofuranosyl) nucleoside derivatives by use of the TMSOTf method. Debenzylation with boron tribromide or deacetylation with sodium methoxide yielded the corresponding pyrimidine (7–11, 17, 18, 26 and 27) and purine (29 and 34) nucleoside analogues. The anomeric configurations were determined by NMR spectroscopy and, in the case of the 5-halo- (7–9) and nitrouridine derivative 11 and the 6-methylcytidine derivative 27, by X-ray structural analyses. – The unprotected nucleosides were not antivirically inhibitory at 250 µM.

An efficient route to pyrimidine nucleoside analogues by [4 + 2] cycloaddition reaction.

We report here an efficient synthesis for pyrimidine nucleoside analogues by [4 + 2] cycloaddition reaction. These compounds were obtained by convergent chemistry from glycosyl isothiocyanates 3a-f (pyranoses, furanoses, and dissaccharides) and diazadienium salt 5. In fact, diazapentadienium iodide 5 prepared from vinylthioamide 4 is an efficient intermediate in heterocyclic synthesis and reacts with isothiocyanates 3a-f affording beta-D-uracil analogues 7a-f in good yields and with total regiocontrol. All compounds were fully characterized by IR, HRMS, and 13C and 1H NMR (COSY and HMQC).

Electrospray tandem mass-spectrometric analysis of diastereo- and stereoisomeric pyrimidine nucleoside analogues based on the 1,3-dihydrobenzo[ c]furan core

Electrospray mass spectrometry and tandem mass spectrometry have aided the structural characterization of the diastereoisomeric cis- and trans-1-(3-benzoyloxymethyl-1,3-dihydrobenzo[ c]furan-1-yl)thymines and the four enantiomerically pure stereoisomers of uracil analogues. Low-energy collision-induced dissociation MS/MS analysis of the various precursor molecular and cluster ions confirmed the characteristic fingerprint pattern obtained in the conventional electrospray spectra and allowed a convenient method for the characterization of novel 1,3-dihydrobenzo[ c]furan nucleosides.

Highly Potent and Selective Inhibition of Varicella—Zoster Virus Replication by Bicyclic Furo[2,3‐d]pyrimidine Nucleoside Analogues

AbstractFor Abstract see ChemInform Abstract in Full Text.

Identification of a novel human uridine phosphorylase

Uridine phosphorylase catalyzes the reversible phosphorylytic cleavage of uridine and deoxyuridine to uracil and ribose- or deoxyribose-1-phosphate. The enzyme has an important role in the metabolism of pyrimidine analogs used in cancer chemotherapy. The cDNA of a novel 317 amino acid human uridine phosphorylase ≈60% identical to the previously identified human uridine phosphorylase was cloned. The novel enzyme, named uridine phosphorylase-2 (UPase-2), showed broad substrate specificity and accepted uridine, deoxyuridine, and thymidine as well as the two pyrimidine nucleoside analogs 5-fluorouridine and 5-fluoro-2 ′-deoxyuridine. The human UPase-2 gene was mapped to chromosome 2q24.1 and the 2.2-kb mRNA was predominantly expressed in kidney. The mouse UPase-2 cDNA was also identified and shown to be predominantly expressed in liver. The identification of a novel uridine phosphorylase with broad substrate specificity is important for studies on both nucleoside metabolism as well as for studies on the pharmacological mechanisms of therapeutic pyrimidine nucleoside analogs.

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.

Highly potent and selective inhibition of varicella-zoster virus replication by bicyclic furo[2,3-d]pyrimidine nucleoside analogues.

The bicyclic furo[2,3-d]pyrimidine nucleoside analogues represent an entirely new class of fused furopyrimidine derivatives with unprecedented selectivity for varicella-zoster virus (VZV). From extensive structure-activity relationship (SAR) studies, the 6-(p-alkylphenyl)substituted furopyrimidine derivatives Cf 1742 and Cf 1743 emerged as the most potent inhibitors of VZV replication: they were found to inhibit both laboratory VZV strains and clinical VZV isolates at subnanomolar concentrations, while not being toxic to the host cells at 100,000-fold higher concentrations. Although the precise mechanism of action of these compounds remains to be elucidated, it is clear that for their antiviral activity they depend on phosphorylation by the VZV-encoded thymidine kinase. The furo[2,3-d]pyrimidine nucleoside analogues are not susceptible to degradation by human or bacterial thymidine phosphorylase, which may otherwise release the free aglycone. Also, the latter is not inhibitory to dihydropyrimidine dehydrogenase, an enzyme involved in the degradation of thymine, uracil, and the anticancer agent 5-fluorouracil. Further development of the furo[2,3-d]pyrimidine nucleoside analogues as new therapeutic modalities for the treatment of VZV infections (i.e., varicella and herpes zoster) seems highly justified.

Comparative Study of Three Methods for Non-radioactive In Vivo DNA Labeling in Escherichia coli Using Nucleoside Analogs

In the present work, a comparative study of 5-FdUrd, thy -, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5α cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labelled DNA (MDLD) was 312 pg with the 5-FdUrd and thy - methods for 5-BrdUrd labelled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy - and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.

Lamivudine in renal transplant candidates with chronic hepatitis B infection

CHRONIC hepatitis B virus (HBV) is one of the most common and serious infectious diseases throughout the world, currently affecting 400 million persons globally. Although the incidence of chronic HBV infection among patients on hemodialysis has declined, the illness remains an important problem in renal transplant recipients. HBV infection is a frequent and major risk factor for acute and chronic liver disease, causing increased morbidity and mortality both before and after transplantation. Treatment of HBV in kidney recipients is difficult but probably necessary to reduce morbidity and mortality. Interferon has been associated with an increased incidence of rejection and limited success. Recent reports have noted promising results with the antiviral agent lamivudine, a pyrimidine nucleoside analogue. Lamivudine therapy significantly decreases serum HBV-DNA levels and improves the histologic findings, reduces serum aminotransferase levels, and slows progression to cirrhosis in patients with chronic HBV. In renal transplant patients with recurrent hepatitis B, it stabilizes liver enzymes and decreases HBV replication. Our aim in this study was to assess the efficacy and safety of lamivudine in renal transplant candidates with replicative HBV.