Synthesis and antiviral activity of 5-[(cyanomethylene)oxy]-2'-deoxyuridine

To study the influence of substitution of CN for C identical to CH in the anti-herpes virus nucleoside 5-(propynyloxy)-2'-deoxyuridine (1), 5-[(cyanomethylene)oxy]-2'-deoxyuridine (2) was prepared. When the potassium salt of 5-hydroxy-2'-deoxyuridine was reacted with iodoacetonitrile in dry DMF, the bisalkylated product 3-(cyanomethyl)-5-[(cyanomethylene)oxy]-2'-deoxyuridine (3) was the major product with a lesser amount of 3-(cyanomethyl)-5-hydroxy-2'-deoxyuridine (5) and only a trace amount of the desired product (2). In contrast, when 5-hydroxy-2'-deoxyuridine was alkylated in water in the presence of 1 equiv of KOH, compound 2 was the major product. In cultures of primary rabbit kidney (PRK) cells, compound 2 showed an anti-herpes virus activity that was comparable to that of 1 and ara-A. Compound 2 did not inhibit incorporation of [Me-3H]dThd or [1',2'-3H]dUrd into DNA of PRK cells; however, its anti-herpes virus activity was completely prevented upon the addition of either dThd or dUrd.     

Comparative metabolism of E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in herpes simplex virus-infected cells

The antiviral activities and metabolic fates of E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BrVaraUra) were compared in a dThd kinase-deficient human fibroblast cell line, infected with parental strains of herpes simplex virus, and other strains expressing no viral dThd kinase activity. Metabolic experiments were performed at concentrations well above the ID50 for each compound because radiolabeled agents were not available. BrVaraUra and its nucleotides qualitatively displayed chromatographic and anabolic characteristics which closely paralleled those of BrVdUrd and its nucleotides. Monophosphorylation of both drugs was dependent upon the presence of viral dThd kinase activity except in the case of one dThd kinase-negative type 1 mutant (SC16R5C1) which retained BrVdUrd/BrVaraUra kinase activity. Intracellular uptake of either parent compound was absent during mock-infection and minimal in the cases of infection with mutants unable to phosphorylate the parent compound. Parental type 1 strains were able to induce diphosphorylation and triphosphorylation of both compounds to a similar, dose-dependent degree. Extracts of type 2-infected cells contained greater quantities of BrVdUrd and its monophosphate compared with BrVaraUra and its monophosphate, after identical drug exposure and infection conditions. As previously observed for BrVdUrd, diphosphorylated and triphosphorylated nucleotides of BrVaraUra were not detected after type 2 infection. BrVdUrd and BrVaraUra metabolic breakdown pathways differed, however, as evidenced by the formation of E-5-(2-bromovinyl)uracil (BrVUra). Unlike BrVdUrd, BrVaraUra formed no BrVUra in infected cells, suggesting that replacement of 2'-deoxyribose with arabinose makes the compound biologically more stable, presumably because of resistance to enzymatic breakdown by pyrimidine nucleoside phosphorylases. In this dThd kinase-negative cell line, BrVdUrd and BrVaraUra displayed qualitatively similar susceptibility profiles in that activities were type 1 selective and dThd kinase dependent. Antiviral activities against dThd kinase-positive type 1 strains were similar with both compounds. These data would suggest that BrVdUrd and BrVaraUra have identical type-specific dThd-dTMP kinase-dependent mechanisms of cellular uptake and phosphorylation, but that the latter is not subjected to phosphorolysis and resultant formation of an inactive metabolite. Furthermore, the absence of presence of phosphorolysis of the parent nucleoside does not apparently adversely affect in vitro antiviral activity.     

Biotransformation of 5-(2-chloroethyl)-2'-deoxyuridine in male NMRI mice

When the selective anti-herpes agent [2-14C]-5-(2-chloroethyl)-2'-deoxyuridine [( 14C]CEDU) was administered as a single oral dose to mice, 87.9% of the radioactivity was excreted in the urine and 11.2% in the feces within 72 hr. Compounds accounting for 84% of the 14C radioactivity in the 0- to 24-hr urine were isolated by various chromatographic techniques and identified by MS, NMR, IR, and CD analysis. Approximately 25% of the radioactivity found in the urine was the parent compound (CEDU). According to the 14C-metabolites detected in the urine, one may infer that [14C]CEDU is metabolized, first, by cleavage of its N-glycosidic bond, resulting in the formation of 5-(2-chloroethyl)uracil (38.7%) and, second, by stereoselective hydroxylation of the alpha carbon atom of the haloalkyl side chain of 5-(2-chloroethyl)uracil, resulting in the formation of 5-(1-hydroxy-2-chloroethyl)uracil (29.6%). CEDU was absorbed rapidly from the gastrointestinal tract and the bloodstream, and did not show any particular accumulation in mouse tissues, as revealed by whole body autoradiography.     

Synthesis and antiviral properties of (Z)-5-(2-bromovinyl)-2'-deoxyuridine

(Z)-5-(2-Bromovinyl)uracil was obtained by photoisomerization of the E. isomer. Similarly, (E)-5-(2-bromovinyl)-2'-deoxyuridine gave the required Z isomer. (Z)-5-(2-Bromovinyl)-2'-deoxyuridine is much less active against herpes simplex virus type 1 (HSV-1) and somewhat less active against herpes simplex virus type 2 than is the E isomer. Both isomers show similar activity against vaccinia virus. Therefore, the highly potent and selective activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against HSV-1 is due to its E configuration.     

Phosphorolysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted-2'-deoxyuridines by purified human thymidine phosphorylase and intact blood platelets

Various 5-substituted-2'-deoxyuriclines (dUrd), inclucling 5-ethyl-,5-propyl-, 5-trifluoromethyl-, 5-hydroxymethyl-, 5-formyl-, 5-vinyl-, (E)-5-(2-chlorovinyl)-, (E)-5-(2-bromovinyl)-, 5-fluoro-. 5-chloro-. 5-bromo-. 5-iodo-, 5-cyano-, 5-thiocyano-, 5-nitro- and 5-amino-dUrd. were shown to be effective substrates for the thymidine (dThd) phosphorylase isolated from human blood platelets. Some of dUrd analogs, i.e. the highly potent and selective antiherpes agent (E)-5-(2-bromovinyl)-dUrd. were degraded more rapidly than the natural substrates, dUrd and dThd. All dUrd analogs were also readily catabolised by intact human blood platelets. The potent inhibitors of thymicline phosphorylase, 6-amino-thymine and 6-amino-5-bromo-uracil, strongly inhibited the phosphorolysis of (E)-5-(2-bromovinyl)-dUrd by both purified enzyme and intact platelets.     

Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene

Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.     

Metabolism of anti-herpes agent 5-(2-chloroethyl)-2'-deoxyuridine in mice and rats

The enzymatic splitting and metabolic elimination of anti-viral agent 5-(2-chloroethyl)-2'-deoxyuridine [CEDU] have been studied. For elucidation of structures of metabolites, several different kinds of extraction, purification and spectroscopic methods were used (Extrelut LC, TLC, HPLC, MS, NMR, IR, UV and CD). For mass spectral analysis, various ionization techniques (EI, CI and FAB-MS) were performed as complementary methods. After oral administration of [14C]-CEDU to mice and rats, the parent compound, 5-(2chloroethyl) uracil [CEU] and hydroxylated CEU metabolites were isolated and identified from urine and faeces by the above mentioned methods. The CEDU showed rapid phosphorolysis in vitro with thymidine phosphorylase Km 41.0 +/- 5.0; and uridine phosphorylase Km 10.0 +/- 1.5. The cleavage of the N-glycosidic bond of the nucleoside analogue and a new metabolic pathway of CEDU [stereoselective oxidation of 5-(2-chloroethyl) uracil] was observed in both species.     

Synthesis and antiviral activity of (E)-5-(2-bromovinyl)uracil and (E)-5-(2-bromovinyl)uridine

(E)-5-(2-Bromovinyl)uracil (BVU) and (E)-5-(2-bromovinyl)uridine (BVRU) were synthesized starting from 5-formyluracil via (E)-5-(2-carboxyvinyl)uracil or starting from 5-iodouridine via (E)-5-(2-carbomethoxyvinyl)uridine and (E)-5-(2-carboxyvinyl)uridine, respectively. Depending on the choice of the cell system, BVU and BVRU exhibited a marked activity against herpes simplex virus type 1 (HSV-1) in vitro. Although BVU and BVRU were less potent than the reference compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), their antiviral activity spectrum was remarkably similar to that of BVDU. The latter findings suggest that BVU and BVRU are metabolically converted to BVDU or a phosphorylated product thereof. In vivo, BVU protected mice against a lethal disseminated HSV-1 infection.     

Antiherpes virus activity and effect on deoxyribonucleoside triphosphate pools of (E)-5-(2-bromovinyl)-2'-deoxycytidine in combination with deaminase inhibitors

The antiviral activity and cytotoxicity of (E)-5-(2-bromovinyl)-2'-deoxycytidine (BrVdCyd) against herpes simplex virus type 1 (HSV-1), singly and in combination with deaminase inhibitors was determined using rabbit kidney (RK-13), HEP-2, BHK-21 and VERO cells. BrVdCyd was a potent inhibitor of HSV-1 replication with ED50 values of 0.30 to 1.20 microM depending on the cell line used. In the presence of tetrahydrouridine or tetrahydrodeoxyuridine (H4dUrd), potency of BrVdCyd increased approximately two fold (ED50: 0.54 microM) in HSV-infected VERO cells. The combination of BrVdCyd and H4dUrd was also effective in decreasing virus yield. Dihydrodeoxyuridine (H2dUrd) reversed the activity of BrVdCyd (ED50: 6 to 7 microM). The effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd), BrVdCyd and BrVdCyd in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was assessed in VERO cells infected with a high multiplicity of infection (10 PFU/cell). Significant differences in dNTP poll sizes (pmol/10(6) cell) were observed with different treatments. BrVdUrd and BrVdCyd treatment resulted in marked expansion of the dTTP pool (greater than 1200 pmol) compared to HSV-infected VERO cells (303 pmol). Exposure to H4dUrd resulted in a 12-fold expansion of the dCTP pool (326 pmol) and barely detectable levels of dTTP (less than 1.0 pmol). BrVdCyd plus H4dUrd treatment resulted in a slight expansion of the dTTP pool (515 pmol). These results indicate: (i) H4dUrd inhibits de novo dCyd/dCMP deaminase pathway and (ii) exposure to BrVdCyd plus H4dUrd puts a strain on viral DNA synthesis to such an extent that even though dTTP is being formed from alternative pathways, its eventual utilization as a substrate is reduced and hence it builds up.     

Antiviral activity of 5-methylthiomethyl-2'-deoxyuridine and other 5-substituted 2'-deoxyuridines.

Of a series of eight 5-substituted 2'-deoxyuridine (dUrd) derivatives, which were evaluated for their antiviral and antimetabolic activities in primary rabbit kidney or human skin fibroblast cell cultures, five dUrd derivatives, 5-dimethylaminomethyl-dUrd, 5-chloroacetamidomethyl-dUrd, 5-iodoacetamidomethyl-dUrd, 5-pyrrolidinylmethyl-dUrd and 5-N-methylpiperazinylmethyl-dUrd, showed little, if any, activity. The three others, 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiom-ethyl-dUrd, were found to inhibit the replication of various HSV (herpes simplex virus) strains (whether type 1 or 2) at a concentration of approximately 1–10 μg/ml. The antiviral activity of 5-formyl-dUrd may be accounted for by an inhibition of thymidylate synthetase. The inhibitory effect of 5-formyl-dUrd on HSV multiplication was readily reversed by adddition of 2'-deoxythymidine (dThd), and, in analogy with other established thymidylate synthetase inhibitors, 5-formyl-dUrd blocked the incorporation of [2-¹⁴C]dUrd into cellular DNA to a markedly greater extent than the incorporation of [methyl-³H]dThd. Unlike 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiomethyl-dUrd did not preferentially inhibit the incorporation of [2-¹⁴C]dUrd. Antiviral indexes, defined as the id₅₀ for [2-¹⁴C]dUrd incorporation divided by the id₅₀ for HSV (type 1, strain KOS) replication, were 0.25, 43 and > 100 for 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiomethyl-dUrd, respectively. The latter two compounds may therefore be considered as rather selective anti-herpes agents.     

Synthesis and biological activities of 4-O-(difluoromethyl)-5-substituted-uracil nucleoside analogues

Various 4-O-difluoromethyl analogues of 5-substituted uridine (Urd), 2'-deoxyuridine (dUrd), and arabinofuranosyluracil (araU) nucleosides were prepared via a CF2-insertion reaction into 4-O-silylated nucleosides and evaluated for activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytotoxicity in human embryonic lung fibroblast (HELF) cell cultures. The introduction of the 4-substituent led to a strong reduction of antiviral activity for dUrd but not for araU analogues. Three of the 4,5-disubstituted uracil nucleoside derivatives, 4-O-(difluoromethyl)-5-bromo-araU (5c),-5-methyl-araU (5e), and -(E)-5-(2-bromovinyl)-araU (5g), displayed a high and selective inhibitory effect against HSV-1, but only 5e was effective against both HSV-1 and HSV-2 comparably with the antiherpes potential of the reference compounds 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and 1-beta-D-arabino-furanosylthymine (araT).     

Mutagenic and antimutagenic effects of 5-substituted 2'-deoxyuridines depending on the nature of the 5-substituent

Various 5-substituted pyrimidine 2'-deoxyribosides with anti-herpes activity were investigated for their genotoxic activity. 5-Iodo-2'-deoxycytidine (IDC), 5-(2-chloroethyl)-2'-deoxycytidine (CEDC), 5-(3-chloropropyl)-2'-deoxyuridine (CPDU), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-ethyl-2'-deoxyuridine (EDU), 2'-deoxyuridine (DU) and 2'-deoxythymidine (DT) were non-mutagenic in Salmonella typh. as well as in V79 Chinese hamster cells, 5-Iodo-2'-deoxyuridine (IDU) was moderately mutagenic and 5-(2-chloroethyl)-2'deoxyuridine (CEDU) was highly mutagenic in V79 cells; neither IDU nor CEDU were mutagenic in the bacterial assay. None of the compounds induced unscheduled DNA synthesis in primary rat hepatocytes. In addition, antimutagenic effects of 2'-deoxyuridines were discovered: in V79 cells, BVDU, EDU, DU and DT prevented the mutagenicity induced by CEDU; in these cells EDU also inhibited the mutagenicity induced by MNNG. In primary rat hepatocytes, IDU and EDU inhibited the induction of unscheduled DNA synthesis induced by MNNG, DMBA or UV-light. The compounds were inactive at inducing differentiation in hematopoietic cells. The significance of these data, particularly with regard to the use of 5-substituted 2'-deoxyuridines in anti-herpes therapy, is discussed.     

Potent activity of 5-fluoro-2'-deoxyuridine and related compounds against thymidine kinase-deficient (TK-) herpes simplex virus: targeted at thymidylate synthase

5-Fluorouracil, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorocytidine (FCyd), 5-fluoro-2'-deoxycytidine (FdCyd), 5-trifluoro-2'-deoxythymidine (F3dThd), and the 5'-monophosphates and 3',5'-cyclic monophosphates thereof were found to inhibit thymidine kinase-deficient (TK-) mutant strains of herpes simplex virus (HSV) at a much lower concentration than the wild-type (TK+) HSV strains. Other 5-substituted 2'-deoxyuridines that have previously been recognized as potent thymidylate synthase inhibitors behaved in a similar fashion. The activity of FdUrd, FdCyd, F3dThd, and their 3',5'-cyclic monophosphates against TK-HSV was readily reversed by 2'-deoxythymidine (dThd) but not by 2'-deoxyuridine (dUrd). These compounds also inhibited the incorporation of [6-3H]dUrd into DNA at a concentration which was up to 5 orders of magnitude lower than the concentration at which the incorporation of [methyl-3H] dThd was inhibited. Thus, while not being a target for the well established anti-HSV compounds in TK+HSV-infected cells, thymidylate synthase appears to be an important target in TK-HSV-infected cells. In addition to dTMP synthase, TK-HSV-infected cells appear to reveal other therapeutically exploitable targets such as OMP decarboxylase (towards pyrazofurin), CTP synthase (towards carbodine and its cyclopentenyl analogue), dihydrofolate reductase (towards methotrexate), and S-adenosylhomocysteine hydrolase (towards neplanocins).     

Differential binding affinities of sugar-modified derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine for herpes simplex virus-induced and human cellular deoxythymidine kinases

The affinity of a large number of sugar-modified derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was determined towards deoxythymidine (dThd) kinases (TK) of various origin, i.e. human cytosol and mitochondrial TK, as well as herpes simplex virus (HSV) type 1 and type 2 TK. Substitution at the 3'- and 5'-position had differential effects on the interaction of BVDU with TK from different sources. The binding affinity of the nucleoside analogs for these different TKs was also influenced by the nature of the 5-substituent (2-bromovinyl vs 2- chlorovinyl ). The 5'-azido and 5'-amino derivatives of BVDU showed affinity for HSV-1 TK only and may, therefore, be useful to differentiate HSV-1 TK from all other TKs . There was no stringent correlation between the antiviral effects of the compounds and their binding constants for viral TK, suggesting that phosphorylation by viral TK is an essential but not sufficient factor in determining the antiviral activity of these analogs.     

Role of thymidine kinase and thymidylate synthetase in the cytostatic, antimetabolic, and antitumor effects of the carbocyclic analogue of 5-nitro-2'-deoxyuridine. A comparison with 5-nitro-2'-deoxyuridine

The carbocyclic analogue of 5-nitro-2'-deoxyuridine (NO2dUrd), in which the sugar moiety is replaced by a cyclopentane ring and which was designated C-NO2dUrd, has been evaluated for its cytostatic, antimetabolic, and antitumor properties. The following findings are noted. C-NO2dUrd is about 500- to 2000-fold less inhibitory to tumor cell proliferation in vitro than NO2dUrd. Phosphorolysis of C-NO2dUrd by rabbit liver extracts was not observed under conditions where NO2dUrd was readily converted to 5-nitrouracil (NO2Ura). Also, C-NO2dUrd is converted to its 5'-monophosphate (C-NO2dUMP) by dThd kinase nearly as efficiently as the true nucleoside NO2dUrd. This metabolic conversion is necessary for the inhibitory effect of C-NO2dUrd on tumor cell proliferation in cell culture. The principal target enzyme for the cytostatic action of C-NO2dUrd is 2'-deoxythymidylate (dTMP) synthetase. C-NO2dUMP, the active metabolite of C-NO2dUrd, has a much lower affinity for dTMP synthetase than does NO2dUMP. This is the first demonstration of the interaction of a carbocyclic pyrimidine nucleotide analogue with dTMP synthetase. Neither NO2dUrd nor C-NO2dUrd exerts any significant antitumor activity in mice bearing L1210 or P388 leukemia; for NO2dUrd, this failure may be related to a rapid degradation to its inactive metabolite, NO2Ura; for C-NO2dUrd, it is most likely due to a decreased affinity of C-NO2dUMP for its target enzyme, dTMP synthetase.     

Differential metabolism of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) by equine herpesvirus type 1- and herpes simplex virus-infected cells

Thymidine kinase (TK) enzymes encoded by herpes simplex viruses types 1 and 2 (HSV-1, HSV-2), and equine herpesvirus type 1 (EHV-1) catalyze the phosphorylation of thymidine (dThd) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). The replication of HSV-1 is sensitive to BVDU, but the replication of HSV-2 and EHV-1 is not. To investigate the differential sensitivity of the viruses to halogenated vinyldeoxyuridine drugs, the phosphorylation of 125I-labeled (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) was studied. Cytosol enzymes from cells infected by HSV-2 and EHV-1 phosphorylated [125I]IVDU to the monophosphate, IVDUMP, but did not convert IVDUMP to higher di- plus triphosphates (IVDUDP plus IVDUTP) forms. In contrast, enzymes from HSV-1-infected cells converted [125I]IVDU to radioactive IVDUMP and IVDUDP plus IVDUTP. Experiments with mixtures of EHV-1- and HSV-1-induced enzymes showed that the EHV-1 enzyme did not inhibit formation of the IVDUDP plus IVDUTP by the HSV-1 enzyme. With [125I]IVDU as substrate, the Km values for the EHV-1 and HSV-1 TKs were 1.82 and 0.34 microM, respectively, and the Ki (dThd) value for the EHV-1 TK was 0.35 microM. In vivo experiments showed that HSV-1-infected cells converted IVDU to the mono- and the di- plus triphosphate forms. In contrast, EHV-1-infected cells converted IVDU to the monophosphate to a lesser extent than did HSV-1-infected cells, and did not produce the di- plus triphosphates. Thus, inefficient phosphorylation of the monophosphates probably contributes to the insensitivity of EHV-1 replication to IVDU, as it does to the insensitivity of HSV-2 replication to this drug.     

(E)-5-(2-bromovinyl)uridine requires phosphorylation by the herpes simplex virus (type 1)-induced thymidine kinase to express its antiviral activity

(E)-5-(2-Bromovinyl)uridine (BVUrd), the riboside counterpart of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd), effected a dose-dependent inhibition of viral progeny formation and viral DNA synthesis in herpes simplex virus type 1 (HSV-1, strain KOS)-infected human (E6SM) diploid fibroblast cells. BVUrd was directly phosphorylated in HSV-1-infected cells, presumably by the virus-encoded thymidine kinase (TK), since (i) BVUrd was not phosphorylated by extracts of cells infected with a HSV-1 strain deficient in TK expression and (ii) the phosphorylation was inhibited by a polyclonal anti-HSV-1 antibody. Within the HSV-1-infected cells, BVUrd was incorporated into the viral DNA as BVdUMP (BVdUrd 5'-monophosphate). This incorporation may account for the antiviral action of BVUrd, and implies that, following its initial phosphorylation by the viral TK, BVUrd is converted to its 2'-deoxy counterpart, most likely at the 5'-diphosphate level (BVUDP----BVdUDP).     

Synthesis and antiviral activity of the carbocyclic analogues of (E)-5-(2-halovinyl)-2'-deoxyuridines and (E)-5-(2-halovinyl)-2'-deoxycytidines

The carbocyclic analogues of the potent and selective antiherpes agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) were synthesized by conventional methods with use of carbocyclic 2'-deoxyuridine as starting material. C-BVDU, C-IVDU, and C-BVDC were equally selective, albeit slightly less potent, in their antiherpes action than BVDU, IVDU, and BVDC. Although resistant to degradation by pyrimidine nucleoside phosphorylases, C-BVDU did not prove more effective than BVDU in the systemic (oral, intraperitoneal) or topical treatment of HSV-1 infections in mice.     

Synthesis of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine and related analogues: potent and unusually selective antiviral activity of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine against herpes simplex virus type 1

Syntheses of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine (TFPe-dUrd) (1), 5-(3,3,3-trifluoro-1-propyl)-2'-deoxyuridine (11), 5-(3,3,3-trifluoro-1-methoxy-1-propyl)-2'-deoxyuridine (8), and 5-(3,3,3-trifluoro-1-hydroxy-1-propyl)-2'-deoxyuridine (10) from 5-chloromercuri-2'-deoxyuridine are described. The antiviral activity of TFPe-dUrd was determined in cell culture against herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and vaccinia virus and compared concurrently with 5-(1-propenyl)-2'-deoxyuridine, 5-(2-bromovinyl)-2'-deoxyuridine, 5-iodo-2'-deoxyuridine, and 5-(trifluoromethyl)-2'-deoxyuridine. TFPe-dUrd demonstrated a potent and unusually selective activity against HSV-1, with a 2-log reduction in virus yield at 0.03 micrograms/mL (0.09 microM); L-1210 cell growth was inhibited by 50% only at 290 micrograms/mL. Isopycnic centrifugation of 32P-labeled DNA indicated that if 0.5 or 2 microM TFPe-dUrd was present for 0-6 h postinfection, viral DNA synthesis was reduced by ca. 50 and 85%, respectively; concomitantly, a new DNA band appeared at lower density than normal cellular or viral DNA.     

Synthesis and biological activity of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine deoxyuridine diastereomers.

The synthesis of the two diastereomers (9 and 10) of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine are described. Their antiviral and cytotoxic activities were determined, in comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine (FDU), respectively. 5-[(1R)-2,2-Difluorocyclopropyl]-2'-deoxyuridine (10) was the most active antiviral agent against HSV-1 (IC50 = 5 micrograms/ml) relative to BVDU (IC50 = 0.082 micrograms/ml), and cytotoxic agent in the CCRF-CEM (IC50 = 230 microM) screen relative to FDU (IC50 = 4.7 x 10(-3) microM). The 5-[(1S)-2,2-difluorocyclopropyl] diastereomer was inactive in both screens. Partition coefficients (P) and affinity for the mouse erythrocyte nucleoside transporter (Ki) were not determinants of antiviral or cytotoxic activities. However, the (1R)-diastereomer (10) was more resistant to glycosidic bond cleavage by thymidine phosphorylase than the (1S)-diastereomer (9).