Inhibition of B Virus in Cell Culture by 5-Iodo-2′-Deoxyuridine

The antimetabolite 5-iodo 2′-deoxyuridine (IDU) interferes with B virus replication in monolayers of cynomolgous monkey kidney, KB and HEp 2 cells. To determine the effect of this clinically active nucleoside in laboratory antiviral screening systems, several methods were compared. Most of the methods used were capable of detecting the activity of IDU at low levels.     

Mechanism of Antiviral Activity of 5-Ethyl-2′-Deoxyuridine

Abstract

5-Ethyl-2′-deoxyurldine (EDU) is phosphorylated to a much greater extent by herpes simplex virus (HSV)-infected Vero cells than by mock-infected cells. Within the infected cells, EDU is preferentially incorporated into viral DNA and more inhibitory to viral than cellular DNA synthesis     

Novel Enamine Derivatives of 5,6-Dihydro-2′-Deoxyuridine Formed in Reductive Amination of 5-Formyl-2′-Deoxyuridine

Reductive amination of 5-formyl-3′,5′-di-O-acetyl-2′-deoxyuridine with primary amines and sodium triacetoxyborohydride (NaBH(OAc)₃) afforded novel enamine derivatives of 5,6-dihydro-2′-deoxyuridine as a result of unexpected 1,4-conjugate reduction of intermediate Schiff bases in addition to the secondary amine derivatives of 2′-deoxyuridine, typical 1,2-reduction products.     

Effect of 2-Amino Substitution on the Antiviral Effects of 5-Ethyl-2′-Deoxyuridine and (E)-5-(2-bromovinyl)-2′-Deoxyuridine and Their Incorporation into DNA

Abstract

The 2-amino derivatives of 5-ethyl-2′-deoxyuridine (EDU) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) have been synthesized and evaluated for anti-herpesvirus activity. They were at least 1000-fold less effective against herpes simplex virus replication than the parent compounds EDU and BVDU. The 5′-triphosphates of the 2-amino substituted EDU, BVDU and thymidine derivatives were also synthesized and examined on their substrate/inhibitor properties against different DNA polymerases. None of the compounds proved markedly inhibitory to HSV-1 DNA polymerase or cellular DNA polymerase a. Nor were they incorporated into the growing DNA chain.     

Facile Post-Synthetic Derivatization of Oligodeoxynucleotide Containing 5-Methoxycarbonylmethyl-2′-Deoxyuridine

Abstract

5-Methoxycarbonylmetyl-2′-deoxyuridine residue was incorporated into oligoDNAs containing either an exclusive thymidine residue (dT) or all four natural deoxynucleoside residues (dA, dG, dC, dT) via a phosphoramidite method. The treatment of the fully protected oligomer bound to controlled pore glass (CPG), with a variety of polyamine resulted in the release of the oligomer from CPG and the incorporation of the polyamine at the 5-position of the uracil component, simultaneously and in good yields.     

Highly Efficient and Facile Synthesis of 5-Azido-2′-Deoxyuridine

Previously reported syntheses of the photoaffinity label 5-azido-2′-deoxyuridine are rather inefficient and involve the tedious preparation of a 5-nitro intermediate. To overcome these inconveniences, we have developed a new approach from the commercially available 5-bromo-2′-deoxyuridine nucleoside. Our synthetic route makes use of a benzylamination reduction sequence. Using this strategy, the 5-azido-2′-deoxyuridine photolabel is prepared in three steps and quantitative yields.     

The Structure of 5-Cyclohexyl-2′-Deoxyuridine as Studied by X-Ray Crystallography and NMR Spectroscopy

Abstract

5-Cyclohexyl-2′-deoxyuridine (I) is an example of a 5-substituted pyrimidine 2′-deoxynucleoside which exhibits no antiviral activity and which is not a substrate for either cellular or viral (herpes) kinases. Despite the fact that a cursory inspection of NMR spectra of the compound, taken in DMSO-d ₆ solution, suggested that the compound had a normal conformation, we here show that in the crystal and in aqueous solution (analysed by 2D NMR techniques), the conformation of this nucleoside has a syn-glycosidic and C4′-exo (₄E) sugar pucker conformation.     

Synthesis and Antiviral Activity of New 5-Substituted 2′-Deoxyuridine Derivatives

New 5-azole- and 5-oxime-substituted analogues of 2′-deoxyuridine are synthesized. The analogues with azole ring manifest low toxicities and antiherpetic activities on Vero cell culture, the imidazole derivative being the most active. The inhibitory effects of oximes of 5-formyl-deoxyuridine are comparable with those of the azole-containing nucleoside analogues, although their cytotoxicities are found to be higher; oxime of 5-formyldeoxyuridine is particularly toxic. The nucleoside analogues synthesized exhibit no marked activity on cell cultures infected with various variants of poxvirus.     

3′-O- and 5′-O-Propargyl Derivatives of 5-Fluoro-2′-Deoxyuridine: Synthesis,Cytotoxic Evaluation and Conformational Analysis

A series of new 3′-O- and 5′-O-propargyl derivatives of 5-fluoro-2′-deoxyuridine (1–4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2′-deoxyuridine analogues (1–4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3′-O-propargyl-5-fluoro-2′-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.     

Synthesis and Biological Activity of BIS(Pivaloyloxymethyl) Ester of 2′-Azido-2′-Deoxyuridine 5′-Monophosphate

Abstract

Bis(pivaloyloxymethyl) ester of 2′-azido-2′-deoxyuridine 5′-monophosphate was prepared as a prodrug to generate 2′-azido-2′-deoxyuridine 5′-diphosphate inside the cell. A synthetic route utilizing stannyl phosphate was adopted in the preparation. The prodrug was evaluated for cell growth inhibition against a variety of tumor cell lines along with 2′-azido-2′-deoxyuridine and 2′-azido-2′-deoxycytidine.     

Syntheses and Interactions of Oligodeoxyribonucleotides Containing 2′-Amino-2′-Deoxyuridine

Abstract

Oligodeoxyribonucleotides containing 2′-amino-2′-deoxy-uridine (dU) were synthesized and their ability to form duplexes with complementary DNA or RNA oligonucleotides was studied. Substitution of dU with dU in these oligomers results in lowered Tms of the duplexes.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[¹²⁵I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac₂[¹²⁵I]IUdR), 5-[¹²⁵I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv₂[¹²⁵I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac₂[¹²⁵I]IUdR, Piv₂[¹²⁵I]IUdR and [¹²⁵I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [¹²⁵I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv₂[¹²⁵I]IUdR>Ac₂[¹²⁵I]IUdR>[¹²⁵I]IUdR. For Ac₂[¹²⁵I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [¹²⁵I]IUdR, Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.     

Biological Activity and Phosphorylation of 2′-Azido-2′-Deoxyuridine and 2′-Azido-2′-Deoxycytidlne

Abstract

2′-Azido-2′-deoxyuridine and 2′-azido-2′-deoxycytidine were evaluated for their inhibitory activity against ribonucleotide reductase and for subsequent cell growth inhibition. Their mono-and di-phosphates were synthesized and their inhibitory activities against the reductase were also determined in a permeabilized cell system, along with the two nucleosides. The results of the present study identify the first phosphorylation step involved in the conversion of the two azidonucleosides to the corresponding diphosphates to be rate-limiting in the overall activation.     

Synthesis of 2′-Deoxyuridine Nucleosides with Appended 5-Position Carbonyl Cross-Linking Groups

Abstract

A simple modification of Stille type carbonylative coupling conditions resulted in high yield reactions giving new carbonyl appended 2′-deoxyuridine derivatives useful for chemical cross-linking.     

C1′ Acylated Derivatives of 2′-Deoxyuridine. Photolabile Precursors of 2′-Deoxyuridin-1′-yl

Abstract

ABSTRACT

C1′ acylated derivatives of 2′-dcoxyuiidinc (1a-c) were synthesised from 1-[3-deoxy-β-D-psieofiiraiiosylliii.acil (6). The acyl group is introduced via the C1′ aldehyde (11). Following nucleophilic addition, the ketones (1a-c) are obtained via periodinane oxidation and desilylation with NH₄F.     

Isolation of Chlamydia trachomatis by Use of 5-Iodo-2-Deoxyuridine—Treated Cells

Irradiated McCoy cells have provided a useful technique for the isolation of Chlamydia trachomatis strains, among which are found the etiological agents of trachoma, inclusion conjunctivitis, and lymphogranuloma venereum. Because irradiation is not always readily available, 5-iodo-2-deoxyuridine (IUDR) treatment of cells was investigated as a substitute procedure. IUDR-treated cells were found to be as sensitive to C. trachomatis infection as were irradiated McCoy cells. Stock chlamydial strains gave similar titers of iodine-stained inclusions in either system. When cells treated with IUDR were compared with irradiated cells for the isolation of C. trachomatis from clinical specimens, 5 of 138 specimens yielded isolates in IUDR-treated cells not found in irradiated ones, and one isolate was obtained from irradiated but not from IUDR-treated cells. In those 56 cases where inclusions were seen in both systems, there were significantly more inclusions in IUDR-treated than in irradiated cells. Although this series of cultures is too small to determine whether IUDR-treated cells are superior to irradiated ones for the isolation of C. trachomatis, the data indicate that IUDR treatment is at least equally effective.     

Synthesis and Antiviral Activities of Some New 5-Heteroaromatic Substituted Derivatives of 2′-Deoxyuridine

Abstract

Eight new 5-heteroaromatic substituted analogues of 2′-deoxyuridine have been synthesized and evaluated for their inhibitory properties against a panel of different viruses. Several analogues containing a substituted thiophene moiety proved to be highly selective against herpes simplex virus type 1 (HSV-1).     

Synthesis,Cytostatic Activity and Inhibition of Ribonucleotide Reductase by 5′-Phosphoramidates and 5′-Diphosphates,of 2′-O-Allyl-arabinofuranosyl Nucleosides

Abstract

The diphosphates of a series of 2′-O-allyl-1-β-D-arabinofuranosyl derivatives, previously obtained by us, have been prepared and tested for their inhibitory activity in an in vitro assay using R1 and R2 subunits of the purified recombinant mouse ribonucleotide reductase (RNR). 2′-O-Allyl-araU diphosphate proved to be inhibitory, with an IC₅₀ of 100 μM. The 5′-phosphoramidate pronucleotide of 2′-O-allyl-araU was also prepared and tested for inhibition of tumor cell proliferation.     

Cytomegalovirus Replication in Cells Pretreated with 5-Iodo-2′-Deoxyuridine

The replication of human cytomegalovirus (CMV) in cells pretreated with 5-iodo-2′-deoxyuridine (IUdR) was studied. Pretreatment of cells with IUdR enhanced several parameters of virus replication. Virus grown in drug-treated cells exhibited a shorter eclipse period and the cells produced more infectious virus sooner than did untreated cells. There was an approximate fivefold increase in virus yield per cell in the drug-treated samples when compared to control cultures. The time required for plaque development was shortened by 6 days in drug-treated cultures. Pretreatment of cells with IUdR also increased plaquing efficiency of the virus by approximately 10-fold. The enhancement of virus replication by IUdR was further demonstrated by varying the multiplicity of infection. In a 7-day period there was a 100-fold increase in sensitivity of the cultures for virus detection when the cells had been previously exposed to IUdR. The data presented indicate the possibility that IUdR interferes with the production of a cellular product inhibitory for CMV replication.