5-Fluorouracil derivatives. III. A simple method to prepare 5-fluoro-2'-deoxyuridine derivatives

3',5'-Diacyl-2'-bromo-5-fluoro-2'-deoxyuridine (4) was obtained by the reaction of 5, 6-dihydro-6-hydroxy-5-fluorouridine (2) and acyl bromide. Because the route from uridine (1) to 2, the route from 4 to 3',5'-diacyl-5-fluoro-2'-deoxyuridine (5), and the route from 5 to 5-fluoro-2'-deoxyuridine (FUDR, 6) are known reactions, the three step synthesis from uridine to 5 and four step synthesis from uridine to FUDR have been accomplished.     

Antiviral activity of some 5-arylethynyl 2'-deoxyuridine derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by cross-linking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

Antiviral Activity of Some 2'-Deoxyuridine 5-Arylethynyl Derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by crosslinking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

Glycosidic bond cleavage of 5-fluoro-2'-deoxyuridine and 5-fluorouridine by amino acyl-tRNA synthetases

Lysyl-tRNA synthetase isolated from rat liver cleaves glycosidic bond of 5-Fluorouridine and 5-Fluoro-2'-deoxyuridine to generate 5-Fluorouracil. The generation of 5-Fluorouracil was monitored by cellulose thin layer chromatography and by spectrophotometry. The enzyme was found to be 1.4 fold more efficient in cleaving the glycosidic bond of 5-Fluoro-2'-deoxyuridine than 5-Fluorouridine.     

Measurement of 5-fluoro-2'-deoxyuridine serum levels by gas chromatography chemical ionization mass spectrometry

Serum levels of 5-fluoro-2'-deoxyuridine in cancer treated patients were measured by gas chromatography mass spectrometry under chemical ionization conditions; 1-(2-deoxy-beta-D-lyxofuranosyl)-5-fluorouracil (3'-epi-5-fluoro-2'-deoxyuridine) was used as an internal standard. The drug and internal standard were quantitatively isolated from the serum sample by a mini-column anion exchange method and the extract permethylated using potassium-tert-butoxide in dimethylsulphoxide and methyl iodide. The derivatized nucleosides were then re-extracted from the reaction mixture and analysed on a glass capillary column coated with Superox-4. The column was coupled directly to the chemical ionization source of the mass spectrometer; NH3 was used as the reagent gas. The gas chromatographic effluent was monitored at m/z 289, the [MH]+ ion of the N,O-permethyl derivatives of 5-fluoro-2'-deoxyuridine and the internal standard. Recovery of 5-fluoro-2'-deoxyuridine from serum in the 0-1 microgram ml-1 concentration range averaged 93 +/- 2% (SD); a linear detector response was observed up to 50 ng 5-fluoro-2'-deoxyuridine ml-1. At the 10 ng ml-1 level, a within-run assay precision of 10% (CV) (n = 5) was found, while a detection limit of about 1 ng 5-fluoro-2'-deoxyuridine ml-1 of serum was attained. The method was applied to the measurement of disappearance curves of 5-fluoro-2'-deoxyuridine in the serum of treated patients.     

The preparation of 5-cyanouracil and 5-cyano-2''-deoxyuridine from the corresponding 5-iodo derivative and cuprous cyanide.

5-Cyanouracil has been prepared in high yield from cuprous cyanide and 5-iodouracil. The deoxynucleoside has been similarly prepared form 5-iodo-2'-deoxyuridine and this has enabled these compounds to be labelled with (14-C) cyanide. Attempts have been made to incorporate 5-cyanouracil into Escherichia coli 15T and into Mycoplasma mycoides var. capri DNA under conditions in which several other 5-substituted uracils have been incorporated, but without success. Similarly 5-cyano-2'-deoxyuridine could not be incorporated into the DNA of T3 phage under conditions in which 5-bromo-2'-deoxyuridine is easily incorporated. These results suggest that the criteria for a 5-substituted uracil to be incorporated into DNA in vivo depends on some factor other than the size of the substituent.     

New synthesis of 5-carboxy-2'-deoxyuridine and its incorporation into synthetic oligonucleotides

5-Carboxy-2'-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2'-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2'-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

Mechanism-based inactivation of thymidylate synthase by 5-(3-fluoropropyn-1-yl)-2'-deoxyuridine 5'-phosphate

5-Fluoropropynyl-2'-deoxyuridine 5'-phosphate (3) was designed as a mechanism-based inactivator of thymidylate synthase (TS). The inhibitor was synthesized from 5-iodo-2'-deoxyuridine and propargyl alcohol by palladium-catalyzed coupling, followed by fluorination and selective phosphorylation. Incubation of TS with 3, in the presence or absence of the CH2H4folate cofactor, caused rapid, irreversible inactivation of the enzyme.     

The synthesis and biological properties of some 5-substituted-2'-deoxyuridines

The reaction of various nucleophiles with 5-vinyl-2'-deoxyuridine has been studied in an attempt to explain the in vitro toxicity of the compound. Attempts to synthesise pro-drugs of 5-vinyl- and 5-ethynyl-2'-deoxyuridine are described.     

The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Nucleoside conjugates. 8. The preparation of 5-fluoro-2'-deoxyuridine conjugates of corticosteroids

Three 5'-(steroid-21-phosphoryl)-5-fluoro-2'-deoxyuridines (VI-VIII) have been prepared and characterized by uv, ir, 1H-nmr, elemental analysis, chemical and enzymatic hydrolyses. These new compounds are 5-fluoro-2'-deoxyuridine conjugates of cortisol (VI), cortico-sterone (VII), and prednisolone (VIII). Besides the physical and analytical data, all of the conjugates were demonstrated to be enzymatically hydrolyzed to the corresponding steroid and 5-fluoro-2'-deoxyuridine 5'-monophosphate (III), and the latter was further shown to be hydrolyzed to 5-fluoro-2'-deoxyuridine (II) by phosphodiesterase I, 5'-nucleotidase, and acid phosphatase. However, they were shown to be resistant to hydrolysis by bacterial alkaline phosphatase.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

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.     

5-bromo-2''-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells.

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.     

5'-O-Palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine--novel lipophilic analogues of 5'-fluoro-2'-deoxyuridine: synthesis, incorporation into liposomes and preliminary biological results

5'-O-palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine were prepared by the reaction of 5-fluoro-2'-deoxyuridine in dimethylacetamide with palmitic acid chloride. The incorporation of the synthesized prodrugs into liposomes composed of egg phosphatidylcholine/stearylamine/cholesterol/alpha-tocopherol at a molar ratio of 10:1:2:0.05 was nearly quantitative; homogeneous bilayer vesicles (75 nm diameter) were obtained. Preliminary tolerance studies revealed that the prodrug-liposome preparations are about 20-60 times more toxic than the parent drug. The prodrugs incorporated into liposomes were 10 to 30 times more active against murine colon 38 carcinoma compared to the free drug. In comparison to the administration of the prodrugs in peanut oil the liposomal preparations seem to exert improved effects and represent a valuable drug delivery system for parenteral applications.     

A comparison of the conformations adopted by some 5-bromovinyl-2''-deoxyuridines and a correlation with their antiviral properties: an X-ray study.

Crystal structures of (Z)-5-(2-bromovinyl)-2'-deoxyuridine, 3',5'-di-O-acetyl-(E)-5-(2-bromovinyl)-2'-deoxyuridine and 3',5'-di-O-p-chlorobenzoyl-5-(2-dibromovinyl)-2'-deoxyuridine are compared with each other and with that of the most potent antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (E-BVDU) reported earlier. A comparison of the conformation of 3',5'-di-O-acetyl-pyrimidine nucleoside structures in which intermolecular hydrogen bond network formation is minimized, with those of their parent compounds has shown that the greatest change in rotation about the glycosyl bond and in the sugar ring pucker is exhibited by E-BVDU. Upon acylation this molecule changes from C2'-endo/C3'-exo conformation to C3'-endo/C4'-exo conformation. The relevance of these structures upon the biological activity of the nucleosides and in particular to their ability to be a substrate for thymidine kinase is discussed.     

Synthesis of a novel aldehyde: 4-O-methyl-5-formylmethyl-2'-deoxyuridine

The synthesis of the blocked nucleoside 3',5'-di-O-p-toluoyl-4-O-methyl-5-formylmethyl-2'-deoxyuridine was accomplishied in eleven steps from gamma-butyrolactone. This aldehyde, which should facilitate the synthesis of nucleosides containing 18F. was converted to the corresponding blocked dithianyl nucleoside, and also to 5-(2,2-difluoroethyl)-substituted derivatives of 2'-deoxyuridine and 2'-deoxycytidine.     

2'',3''=Carbonates in the synthesis of uridine 5''-deoxy and 2'',5''-dideoxy derivatives.

Detritylation of 2',3'-O-carbonyl-5'-O-trityluridine (Ia) with ethereal hydrogen chloride affords 2',3'-O-carbonyluridine (Ib; 83%) which is converted by mesylation to the 5'-mesylcarbonate Ic (75%). Reaction of compound, Ic with tetrabutylammonium bromide in DMF affords the 5'-bromo carbonate Id (77%) which is reduced with tributyltin hydride to the 5'-deoxyuridine 2',3'-cyclic carbonate Ie (70%). When heated with imidazole, compound Ie affords the 2,2'-anhydro derivative IIa (76%) which is converted to the 2'-chloro derivative IIIa (88%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIa gives 2',5'-dideoxyuridine (IIIb; 68%). When heated with NaHCO3 in DMF, the 5'-bromo carbonate Id affords the anhydro bromo derivative IIb (50%) which is converted to the 2',5'-dichloro derivative IIIc (86%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIc affords the 2',5'-dideoxy derivative IIIb (59%). Alkaline hydrolysis of the 2,2'-anhydro derivative IIa affords the arabinosyl derivative IVa which is converted to the diacetyl derivative IVb (34%) by acetylation. When refluxed in water, the 2',3'-cyclic carbonates Ib, Id, and Ie are hydrolysed to the parent nucleosides, namely, uridine (Va; 81%), 5'-bromo-5'-deoxyuridine (Vb; 78%), and 5'-deoxyuridine (Vc; 83%). Hydrolysis of carbonates Ib and Ie is accompanied by the formation of the 2,2'-anhydro derivatives IIc (10%) and IIa (5%) as by-products.     

Synthesis, conversion and biological activity of 5-substituted 2'-deoxyuridines modified by carbohydrates]

5-Benzyloxymethyl(Bom)-2'-deoxyuridine and its alpha-anomer were used as the key compounds for syntheses of thymidine analogues or 3'-derivatives. Anomeric 5-Bom-2'-deoxyuridines were synthesized from 5-Bom-uracil and 2-deoxy-3,5-di-O-p-toluyl-alpha-D-ribo-furanosyl chloride by means of the silyl method. 5-Bom-2'-deoxyuridine was transformed successively to 3',5'-di-O-mesyl derivative, 2,3'-anhydro-1-(2-deoxy-5-O-p-toluyl-beta-D-xylofuranosyl)-5-Bom-uracil and 3'-azido-2',3'-dideoxy-5-Bom-uridine. Treatment of the last with SnCl4 in methylene dichloride--methanol led to 3'-azido-2',3'-dideoxy-5-methoxymethyluridine. Under the same conditions the 5-methoxymethyl derivative was obtained from 3',5'-di-O-p-toluyl-5-Bom-2'-deoxyuridine. Interaction of 1-(2-deoxy-alpha-D-ribofuranosyl)-4-Bom-uracil with SnCl4 in methylene dichloride as well as the hydrogen transfer hydrogenolysis in the presence of cyclohexene and Pd(OH)2/C in ethanol led to 1-(2-deoxy-alpha-D-ribofuranosyl)-5-hydroxymethyluracil. Only 3'-azido-2',3'-dideoxy-5-Bom-uridine showed a cytotoxic activity against CaOv cells in vitro: in 10(-5)-10(-4) M concentrations it inhibits the thymidine incorporation into DNA by 78.8-95.1%. Elucidation of antitumor activity in vivo showed that this nucleoside inhibits growth of solid tumours, Ca755 and LLC, by 79 and 79-83%, respectively, but has no therapeutic effect against lympholeukemia P388.     

5-Alkynyl-2'-deoxyuridines: chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells

Starting with 5-iodo-2'-deoxyuridine, a series of 5-alkynyl-2'-deoxyuridines (with n-propyl, cyclopropyl, 1-hydroxycyclohexyl, p-tolyl, p-tert-butylphenyl, p-pentylphenyl, and trimethylsilyl alkyne substituents) have been synthesized via the palladium-catalyzed (Sonogashira) coupling reaction followed by a simplified isolation protocol (76-94% yield). The cytotoxic activity of modified nucleosides against MCF-7 and MDA-MB-231 human breast cancer cells has been determined in vitro. 5-Ethynyl-2'-deoxyuridine, the only nucleoside in the series containing a terminal acetylene, is the most potent inhibitor with IC(50) (microM) 0.4+/-0.3 for MCF-7 and 4.4+/-0.4 for MDA-MB-231.