Efficiency and selectivity of (E)-5-(2-bromovinyl)-2'-deoxyuridine and some other 5-substituted 2'-deoxypyrimidine nucleosides as anti-herpes agents.

A number of novel 5-substituted 2'deoxypyrimidine nucleosides exhibited antiviral activity against herpes simplex virus type 1 strain V3 (HSV-1-V3) when assayed under one-step conditions in primary human lung fibroblast j(PHLF) cell cultures, and compared with the reference compounds cytosine arabinoside (ara-C), 5-iodo-2'-deoxyuridine (IUdR), and 5-iodo-5'amino-2',5'-dideoxyuridine (AIU). The most effective of these were (in order of decreasing activity): (E)-5-(2-bromovinyl)-UdR (BrVUdR) greater than ara-C greater than IUdR greater than 5-azidomethyl-UdR (AMeUdR) greater than 5-formyl-UdR (fUdR) greater than 5-hydroxymethyl-UdR (HMeUdR) greater than AIU greater than 5-mercaptomethyl-UdR (MMeUdR) = 5-hydroxymethyl-2'-deoxy-cytidine (HMeCdR) greater than 5-benzyloxymethyl-UdR (BOMeUdR). In a multistep virus replication experiment (plaque reduction assay on Vero cells) the order of decreasing activity was as follows: BrVUdR = ara-C greater than HMeUdR greater than fUdR IUdR greater than HMeCdR greater than BOMeUdR greater than AMeUdR greater than AIU greater than MMeUdR. BrVUdR effected a 50% reduction in plaque formation of different strains of HSV-1 at a concentration of 0.06-0.22 microM, of pseudorabies virus (PRV) at 0.02-0.23 microM, and of herpes simplex virus type 2 (HSV-2) at 8 microM, whereas the ID50 values for adenovirus type 2 and type 5 were 100 and 50-100 microM, respectively. The growth of synchronied baby hamster kidney cells in suspension cultures was inhibited by 50% at concentrations of 100, 70, 20, 4, 8, and 0.2 microM for BrVUdR, HMeCdR, IUdR, fUdR, BOMeUdR, and HMeUdR, respectively.

Antiherpes activity of (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil and some other 5-substituted uracil arabinosyl nucleosides in two different cell lines.

Of a series of 5-substituted 1-beta-D-arabinofuranosyluracil (5-X-araU) analogues, (E)-5-(2-bromovinyl)-araU(BrVaraU) and 5-vinyl-araU (VaraU) were the most potent inhibitors of plaque formation by two herpes simplex virus type 1 (HSV-1) strains in human embryonic lung fibroblast (HELF) cell cultures. They were not only more active than 5-methyl-araU (MaraU, araT) and 5-ethyl-araU (EaraU), but even more than 1000 times more potent than the 5-fluoro, 5-iodo, 5-formyl and 5-trifluoromethyl (FaraU, IaraU, faraU, CF3araU) analogues. BrVaraU and VaraU were superior to 9-(2-hydroxyethoxymethyl)guanine (Acyclovir, ACV) and comparable in potency with 2'-fluoro-5-iodoaracytosine (FIAC) and 2'-fluoro-5-methylarauracil (FMAU). Their anti-HSV-1 potency was surpassed only by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVUdR). Surprisingly, in a HSV-1 plaque inhibition assay in African green monkey kidney (Vero) cells, BrVaraU and VaraU were nearly 100 times active or even inactive. In contrast, the antiherpes activity of ACV, FIAC, FMAU and BrVUdR differed only marginally in the two cell lines. The following order of (decreasing) activity against HSV-2 in HELF cells was found: FIAC = FMAU greater than MaraU (araT) greater than ACV greater than VaraU greater than BrVUdR greater than CF3araU greater than IaraU greater than FaraU = Eara U greater than BrVaraU greater than araU greater than faraU. When deoxyribose is replaced by arabinose in 5-X-UdR analogues, a slight increase in anti-HSV-1-77 activity was observed for the 5-vinyl or 5-ethyl substituent, whereas the other 5-X-araU nucleosides were two to more than 100 times less active than their deoxyribosyl counterparts. However, the sugar exchange led to a strong reduction in anti-HSV-2 activity regardless of the 5-substituent.

Efficacy of (E)-5-(2-bromovinyl)-2'-deoxyuridine against different herpes simplex virus strains in cell culture and against experimental herpes encephalitis in mice.

(E)-5-(2-Bromovinyl-2'-deoxyuridine (BrVUdR) showed strong antiviral activity against different laboratory strains and clinical isolates of herpes simplex virus type 1 (HSV-1) on primary rabbit testes (PRT) cells with a 50% inhibition of plaque formation (ID50) at 0.01-0.02 microM. One laboratory strain (HSV-1-S), however, was completely refractory even at concentrations as high as 100 microM. In contrast, the ID50S for all herpes simplex virus type 2 (HSV-2) strains were about 10(2) - 10(3) times higher (8-25 microM) than for the HSV-1 strains. No toxicity in mice treated with 140 mg BrVUdR/kg/day for 14 days was observed, and successful treatments of herpes encephalitis in mice induced experimentally by intracerebral infection with one laboratory strain (HSV-1-Kupka) and one clinical isolate (HSV-1-64) were achieved. Treatment of encephalitis in mice induced by the strain HSV-1-S insensitive to BrVUdR in cell culture failed to be effective. Similar antibody titers against HSV-1 were found in surviving mice of the control and of the BrVUdR-treated groups.

Differential antiherpes activity of the (E)-and (Z)-isomers of 5-(2-fluorovinyl)-2'-deoxyuridine (FVUdR).

Four novel analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine [(E)-BrVUdR]--the (E)- and (Z)-isomer of 5-(2-fluorovinyl)-UdR (FVUdR), (Z)-5-(2-carboxy-2-fluorovinyl)-UdR [(Z)-COOH-FVUdR], and (E)-5-(2-ethoxyvinyl)-UdR [(E)-EOVUdR] were compared with the reference compounds (E)-BrVUdR and 5-vinyl-UdR (VUdR) for their inhibitory effects on plaque formation of herpes simplex virus type 1 (HSV-1 strain 77) and type 2 (HSV-2 strain 82) in human embryonic lung fibroblast (HELF) cell cultures. (Z)-FVUdR and (Z)-COOH-FVUdR were completely inactive against HSV-1 and HSV-2 (ID50 greater than 500 microM). For the other analogues the following order of decreasing potency was found: (E)-BrVUdR greater than VUdR greater than (E)-FVUdR much greater than (E)-EOVUdR (against HSV-1) and VUdR much greater than (E)-BrVUdR greater than (E)-FVUdR much greater than (E)-EOVUdR (against HSV-2).

Antiherpes activity of some novel analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (E-BrVUdR) in two different cell lines.

In a series of 5-vinyl-2'-deoxyuridine (VUdR) analogues (5-(2-X-vinyl)-UdRs) the (E)-5-(2-bromovinyl)-UdR (E-BrVUdR) proved the most potent inhibitor of plaque formation of two herpes simplex virus type 1 (HSV-1) strains in human embryonic lung fibroblast (HELF) and African green monkey kidney (Vero) cell cultures. The (Z)-5-(2-bromovinyl)-UdR (Z-BrVUdR) isomer and the 5-(2,2-dibromovinyl)-UdR (Br2VUdR) analogue were 10-20 times less efficient, whereas the (E)-5-(2-cyanovinyl)-UdR (CNVUdR) and the (E)-5-(2-carboxyvinyl)-UdR (COOHVUdR) derivative were only marginally active (10(3)-10(4) times less than E-BrVUdR). The antiherpes potential of the 5-(2-X-vinyl)-UdRs was compared with that of 5-iodo-, 5-fluoro-, 5-formyl- and 5-ethyl-UdR (IUdR, FUdR, fUdR, EUdR) as well as of 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV), 2'-fluoro-5-iodo-1-beta-D-arabinofuranosyl(ara)-cytosine (FIAC), 2'-fluoro-5-methylarauracil (FMAU), arabinosylthymine (araT) and (E)-5-(2-bromovinyl)- and 5-vinyl-araU (BrVaraU, VaraU). In HELF cells the following order of decreasing activity against HSV-1-77 was found: E-BrVUdR greater than greater than BrVaraU greater than VaraU greater than FIAC greater than FMAU = VUdR = = Z-BrVUdR = ACV = araT = FUdR greater than Br2VUdR greater than greater than IUdR greater than fUdR greater than EUdR greater than CNVUdR greater than COOHVUdR. The inhibition of HSV-1 replication by most of the investigated compounds was somewhat weaker in the plaque inhibition assay on Vero than on HELF cells, but, in the case of the 5-X-araU reference compounds the activity was strongly reduced in Vero cells. In HELF cells the order of decreasing potential against HSV-2 strain 42/78 (HSV-2-42/78) was: FIAC = FMAU greater than greater than araT greater than IUdR = VUdR greater than ACV = FUdR = greater than fUdR = EUdR = VaraU greater than E-BrVUdR greater than Z-BrVUdR greater than Br2VUdR greater than greater than BrVaraU; CNVUdR and COOHVUdR were nearly inactive.

[(E)-5-(2-bromovinyl)-2'-desoxyuridine--a new nucleoside analog with selective inhibitory action against herpesviruses. Studies in cell culture and animal experiments]. / (E)-5-(2-Bromvinyl)-2'-desoxyuridin--ein neues Nucleosidanalogon mit selektiver Hemmwirkung gegenüber Herpesviren. Untersuchungen in der Zellkultur und im Tierexperiment.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (1; BrVUdR) inhibits the replication of herpes simplex virus type 1 (HSV-1) and of varicella-zoster virus (VZV) in vitro at concentrations of 0.01 to 0.23 mumol/l, whereas herpes simplex virus type 2 (HSV-2) is influenced only at 5.5 to 27 mumol/l. In comparison to some classical and newly developed antiherpetics, i. e. 5-iodo-2'-desoxyuridine (2; idoxuridine, IDU), 9-beta-D-arabinofuranosyladenine (4; vidarabine Ara-A), 9-(2-hydroxyethoxymethyl) guanine (5; acyclovir, ACV) and 2'-fluoro-5-iodo-1-beta-D-aracytosine (6;FIAC) the following order of decreasing activity was found:1 greater than 6 greater than 5 greater than 2 greater than 4 (against HSV-1) and 6 greater than 2 greater than 5 greater than 1 greater than 4 (against HSV-2). The high selectivity of the antiviral effect of BrVUdR towards HSV-1 and TZV is based on the fact, that proliferation of different mammalian cell lines is inhibited by 50% only at concentrations as high as 90 to 170 mumol/l, resulting in a therapeutical index of 1000 to 10,000. Successful treatment of an HSV-1 encephalitis in mice as well as an HSV-1 keratitis of rabbits confirmed the efficiency of 1 in experimental animal infections. No toxic side effects in both local and systemic applications were observed. Promising data from cell culture and animal experiments recommend 1 as a potential candidate for the local and systemic treatment of HSV-1 and VZV infections in man.

[5-(2-bromovinyl)-2'-deoxyuridine therapy of herpes zoster diseases in patients with malignant primary diseases]. / BVDU-Therapie von Zostererkrankungen bei Patienten mit malignem Grundleiden.

BVDU [(E)-5-(2-bromovinyl)-2'-deoxyuridine] has proved effective in the treatment of varicella-zoster virus diseases in patients with malignancies. In 13 out of 20 patients a prompt cessation of new vesicle formation accompanied by rapid resolution of fever, crusting and complete epithelialisation of cutaneous lesions were noted. Only in few cases a prolonged recovery from the severe zoster occurred. When starting the treatment later than 48 hours after the onset of initial rash the dissemination of epithelial lesions could not be prevented. BVDU was well tolerated. Laboratory abnormalities were observed only in some cases and did not result in interruption of treatment.

Synthesis and cloning of a human beta-casomorphin-5 gene.

As so far a singularity among opioid peptides, bovine beta-casomorphin-5 has been shown to display pronounced cardiatic activities. The gene sequence of a human beta-casomorphin-5, including plasmid insertion and fusion protein expression and cleavage instrumentary, has been synthesized by a manual phosphoramidite procedure and cloned in a bacterial system.

Inhibition of hepatitis B virus DNA polymerase by 3'-fluorothymidine triphosphate and other modified nucleoside triphosphate analogs.

The 3'-fluoromodified nucleotide analogs 3'-fluorothymidine triphosphate (FdTTP), 2',3'-dideoxy-3'-fluoro-5-chlorouridine triphosphate (F-5CldUTP), 2',3'-dideoxy-3'-fluoro-5-ethyluridine triphosphate (F-5EtdUTP), 2',3'-dideoxy-3'-fluorouridine triphosphate (FdUTP), and 2',3'-dideoxy-3'-fluoro-5-fluorouridine triphosphate (F-5FdUTP) as well as 2',3'-dideoxythymidine triphosphate (ddTTP), 2',3'-didehydro-2',3'-dideoxythymidine triphosphate (ddeTTP), 3'-chlorothymidine triphosphate (CldTTP), and 3'-rhodanothymidine triphosphate (SCNdTTP) were tested for their ability to inhibit hepatitis B virus (HBV)-associated DNA polymerase activity in vitro. The ID50 values of the most potent inhibitors were 0.15 microM for FdTTP, 0.2 microM for ddeTTP, 0.45 microM for ddTTP, and 0.8 microM for F-5CldUTP. SCNdTTP, CldTTP, and F-5EtdUTP were less efficient (ID50 = 3-5 microM), and FdUTP and F-5FdUTP were the least efficient inhibitors (ID50 = 25 microM) of the enzyme activity. Kinetic analysis revealed a competitive type of inhibition for FdTTP and ddeTTP. The Ki values were estimated to be 0.04 microM and 0.08 microM, respectively, compared with a Km value for dTTP of about 0.18 microM.

5-Formyl-2'-deoxyuridine: cytostatic and antiviral properties and possible modes of action.

5-Formyl-2'-deoxyuridine (fdUrd) was prepared by a new method starting from thymidine and investigated for its influence both on proliferation of cultured mammalian cells and virus replication in vitro. The compound was found to have strong cytostatic and antiviral properties: 50% inhibition of proliferation of BHK 21/C13 cells or Ehrlich ascites tumour cells (EAT) was obtained at 4 - 10(-6) and 6 - 10(-6) M, respectively, while the treatment of pseudorabies virus with the same concentration resulted in about 1.5 log reduction of virus yield. A concentration of 1 - 10(-4) M inhibited cell proliferation by 80 to 100% while the virus yield was reduced by more than 3 orders of magnitude. All inhibitions can be prevented by thymidine.--DNA synthesis of EAT cells in vitro, as estimated by incorporation of [32P]-phosphate or low concentrations of [3H]-thymidine, was inhibited. Further biochemical experiments have provided indirect evidence that the compound is phosphorylated by thymidine and thymidylate phosphorylating enzymes. An inhibition of cell free DNA synthesis was found to be depending on a given period of preincubation with the compound (supposed to be needed for the formation of fdUrd 5'-triphosphate). This suggests that the 5'-triphosphate of fdUrd is an inhibitor of DNA polymerases and--by analogy with experiments with 5-formyluridine-5'-triphosphate and RNA polymerases [14]--may be used as an affinity label for this group of enzymes. It is concluded that the described cytostatic and antiviral effects of fdUrd are due to an intracellular "lethal" synthesis of the relevant phosphates which inhibit thymidylate synthetase (as had been found earlier to occur with the chemically prepared nucleotide in cell free extracts [1, 2]) and DNA synthesizing enzymes.