Mechanism and mode of action of 5-iodo-2-pyrimidinone 2'-deoxyribonucleoside, a potent anti-herpes simplex virus compound, in herpes simplex virus-infected cells.

The anti-herpes simplex virus type 2 (-HSV-2) action of 5-iodo-2-pyrimidinone deoxyribonucleoside (IPdR) was found to be exerted through inhibition of HSV DNA synthesis. The inhibition of viral DNA synthesis was not caused by inhibition of the synthesis of HSV-2-specified proteins or HSV-2 mRNA species involved with viral DNA synthesis or by depletion of deoxynucleotides. The inhibition of viral DNA synthesis may be due to damage to the DNA template in the nuclei or to an action at the DNA replication complex, because nuclei isolated from HSV-2-infected cells treated with IPdR could not support DNA synthesis in vitro. Moreover, the addition of exogenous template to the reaction enabled nuclear DNA synthesis to occur at the level of control. The major cellular metabolite of IPdR in HeLa S3 cells infected with HSV-2 was IPdR monophosphate, which was formed through virally specified kinase. Attempts to either identify or synthesize IPdR diphosphate and triphosphate were unsuccessful. The accumulation of IPdR monophosphate was dependent on the extracellular concentration of IPdR. IPdR monophosphate did not have any inhibitory effect on nuclear DNA synthesis, even at 200 microM. Thus, the action of IPdR could be due to an unidentified metabolite of IPdR or the depletion of a cellular metabolite that is essential for viral DNA synthesis.     

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.     

Conversion of 5-iodo-2-pyrimidinone-2'-deoxyribose to 5-iodo-deoxyuridine by aldehyde oxidase. Implication in hepatotropic drug design.

5-Iodo-2-pyrimidinone-2'-deoxyribose (IPdR) can be converted into 5-iodo-deoxyuridine (IUdR), a clinical radiosensitizer, by aldehyde oxidase in the liver. This conversion does not require exogenous cofactors and cannot be catalyzed by mixed-function oxidases, xanthine oxidase or many other oxido-reductases. This "IPdR oxidase" activity is enriched in the liver; thus, extensive conversion of IPdR to IUdR could be anticipated in the liver and the therapeutic index of IPdR could be better than that of IUdR as a radiosensitizer for primary liver cancers or tumors metastasized to the liver. Based on structure and activity relationship studies, nucleoside analogues which could be activated by this enzyme to compounds capable of inhibiting DNA synthesis could be designed and should be explored as agents against cancer, viruses or parasites in the liver.     

Synthesis and Anti-HIV-1 Activity of New 2-[(2-Phthalimidoethyl)thio]-4(3H)-pyrimidinone Derivatives

A series of new 2-[(2-phthalimidoethyl)thio]-4(3H)-pyrimidinones (I – VI) have been obtained through S-alkylation of the corresponding 2-thiouracils (VII – XII) with 2-(tosyloxyethyl)phthalimide. Some of the synthesized compounds inhibit HIV-1 replication in the micromolar concentration range. Among these, the most active compound is 6-methyl-2-[(2-phthalimidoethyl)]-4(3H)-pyrimidinone. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 9, pp. 16 – 17, September, 2005.     

Nucleosides. 146. 1-Methyl-5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil, the C-nucleoside isostere of the potent antiviral agent 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (FMAU). Studies directed toward the synthesis of 2'-deoxy-2'-substituted arabino nucleosides. 6

The synthesis of 5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1-methyluracil (1, C-FMAU), an isostere of the potent antiviral and antitumor nucleoside 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (2'-fluoro-5-methyl-ara-U or FMAU), was achieved. Pseudouridine (2) was converted into 4,5'-anhydro-3'-O-acetyl-2'-O-triflylpseudouridine (4), which was treated with tris(dimethylamino)sulfur (1+) difluorotrimethylsilicate (TASF) to give 4,5'-anhydro-5-(3-O-acetyl-2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1- methyluracil (5b) in 40% yield. Acid hydrolysis of the 4,5'-anhydro linkage of 5b with Dowex 50 (H+) afforded C-FMAU. The inhibitory activity of C-FMAU against HSV-1 and HSV-2 was about 10-fold less than that of FMAU in tissue culture. This compound, however, did not show significant activity in mice inoculated with HSV-1 or HSV-2.     

(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-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.     

Nucleosides. 136. Synthesis and antiviral effects of several 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-alkyluracils. Some structure-activity relationships

In order to study structure-activity relationships between antiherpetic activity and the size of the C-5 alkyl substituents of 2'-fluoro-ara-U derivatives, six new nucleosides (1c-h) were synthesized. The 5-allyl analogue 1c was prepared by a Pd(II)-catalyzed reaction of 5-(chloromercuri)-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil with allyl chloride. Partial hydrogenation of 1c afforded the 5-n-propyl derivative 1d (FPAU). Nucleosides 1e-h were obtained by condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinosyl bromide with the corresponding 5-substituted uracils. Preliminary in vitro data show that, as the alkyl side chain is increased by one carbon unit, the antiherpetic potency is decreased by approximately 1 log order. The cytotoxicity also diminishes as the size of the 5-substituent is increased. FPAU exerts good activity against HSV-1 and HSV-2. FiPAU still shows good therapeutic indices, whereas the higher alkyl analogues are essentially inactive.     

Nucleosides. 133. Synthesis of 5-alkenyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosines and related pyrimidine nucleosides as potential antiviral agents

The synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosines with a halovinyl or vinyl substituent at C-5 was accomplished from the corresponding 5-iodo (FIAC, 1) and/or 5-chloromercuri nucleoside analogues with use of Li2PdCl4- and Pd(OAc)2-mediated coupling reactions. Thiation of the benzoylated derivative of the 5-ethyluracil nucleoside 3 followed by S-methylation and then ammonolysis provided 5-ethyl-2'-fluoro-ara-C. 5-Ethynyl-2'-fluoro-ara-C (19a) and 5-ethynyl-2'-fluoro-ara-U (19b) were also obtained from the persilylated 5-iodo nucleosides 1 and 16, respectively, by PdII/CuI catalyzed coupling with (trimethylsilyl)acetylene. With use of selective sugar deprotection of the initial coupling products with H2O/Me2SO, the corresponding 5-[2-(trimethylsilyl)ethynyl] derivatives 18a and 18b could be isolated. Most of the new compounds showed activity in vitro against both HSV-1 and HSV-2, as did the known corresponding 5-alkenyluracil nucleosides synthesized earlier. The 5-vinylcytosine and -uracil nucleosides 10 and 24, respectively, were highly effective against HSV-1 (ED90 = 0.40 and 0.043 microM, respectively) and HSV-2 (ED90 = 0.59 and 0.56 microM, respectively). Unlike BVDU, the 2'-fluoroarabinosyl derivatives of 5-(halovinyl)cytosine and -uracil showed activity against both types of herpes simplex virus. The therapeutic indices of these compounds are in some cases superior to those of 2'-fluoro-5-methyl-ara-U (FMAU, 2). Moderate antileukemic activity was observed in vitro for the 5-alkynyl and 5-vinyl compounds. The competition of these compounds with thymidine for viral-induced thymidine kinases was also studied.     

Nucleosides. 110. Synthesis and antiherpes virus activity of some 2'-fluoro-2'-deoxyarabinofuranosylpyrimidine nucleosides.

A series of 5-substituted 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosines 7a-d and their corresponding uracils 9a-d,f were prepared by condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinosyl bromide (5) with appropriately trimethylsilylated pyrimidines followed by saponification of the protected nucleosides 6 or 8. 1-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (7e) was obtained by iodination of 7a. Iodination of 8a followed by removal of the protecting acyl-protecting groups afforded the 5-iodo nucleoside 9e. Several of these 2'-fluoro-substituted nucleosides completely obviated replication of herpes simplex virus type 1 (HSV-1) in monolayers of Vero cells at concentrations of 10-100 microgram/mL. The 5-iodocytosine analogue 7e was the most effective, showing 99.5% suppression of viral replication even at concentrations of 0.1 microgram/mL. The cytotoxicity of 7e to L5178Y or P815 cells in culture was minimal. A comparison of the efficacy of 7e against HSV-1 with other known nucleoside antiviral agents indicates that further in vitro and in vivo evaluation of 7e is warranted.     

Synthesis of pyrimidin-2-one nucleosides as acid-stable inhibitors of cytidine deaminase

One of the problems encountered in the use of tetrahydrouridine (THU, 2) and saturated 2-oxo-1,3-diazepine nucleosides as orally administered cytidine deaminase (CDA) inhibitors is their acid instability. Under acid conditions these compounds are rapidly converted into inactive ribopyranoside forms. A solution this problem was sought by functionalizing the acid-stable but less potent CDA inhibitor 1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (1) with the hope of increasing its potency to the level achieved with THU. The selection of the hydroxymethyl substituent at C-4, which led to the synthesis of 4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (10), 3,4-dihydro-4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (7), and 3,4,5,6-tetrahydro-4-(dihydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-p yrimidinone (28) was based on the transition-state (TS) concept. The key intermediate precursor, 4-[(benzoyloxy)methyl]-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-2(H) -pyrimidinone (24), was obtained via the classical Hilbert-Johnson reaction between 2-methoxy-4-[(benzoyloxy)methyl]pyrimidine (20) and 2,3,5-tri-O-benzoyl-1-D-ribofuranosyl bromide (21). Deprotection of 24 afforded compound 10, while its sodium borohydride reduction products afforded compounds 7 and 28 after removal of the blocking groups. Syntheses of 3,4-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (9) and 3,6-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (8), which lack the hydroxymethyl substituent, was accomplished in a similar fashion. The new compounds bearing the hydroxymethyl substituent were more acid stable than THU, and their CDA inhibitory potency, expressed in terms of Ki values, spanned from 10(-4) to 10(-7) M in a manner consistent with the TS theory. Compound 7, in particular, was superior to its parent 1 and equipotent to THU (Ki = 4 X 10(-7) M) when examined against mouse kidney CDA. The superior acid stability of this compound coupled to its potent inhibitory properties against CDA should provide a means of testing oral combinations of rapidly deaminated drugs, viz. ara-C, without the complications associated with the acid instability of THU.     

IPdR: a novel oral radiosensitizer

IPdR (5-iodo-2-pyrimidinone-2'-deoxyribose) is a novel orally available, halogenated thymidine (TdR) analog and is a potential radiosensitizer for use in human tumors, such as rectal, pancreas, sarcoma and glioma tumors. IPdR is a prodrug that is efficiently converted to IUdR (5-iodo-2'-deoxyuridine), an intravenous radiosensitizer by a hepatic aldehyde oxidase, resulting in high IPdR and IUdR plasma levels in mice for > or = 1 h after oral IPdR. Athymic mice tolerated oral IPdR to doses up to 1500 mg/kg/day t.i.d. for 6 - 14 days without significant systemic toxicities. A number of in vivo preclinical studies have demonstrated that IPdR is a superior radiosensitizer compared with IUdR given as a continuous infusion in terms of safety and efficacy with a significantly lower toxicity profile, including gastrointestinal and hematologic side effects. A preclinical study has shown that IPdR is effective in inducing human colon cancer xenograft radiosensitization in drug-resistant DNA mismatch repair-proficient and -deficient tumor models, as well as in human globlastoma xenograft. In anticipation of performing a clinical Phase I trial in humans, investigators also studied the drug pharmacokinetics and host toxicities in two non-rodent, animal species during a 14-day treatment course. Dose-limiting systemic toxicities (diarrhea, emesis, weight loss and decreased motor activity) were observed in ferrets receiving IPdR at 1500 mg/kg/day on a 14-day schedule that were not found previously in athymic mice. Recently, a once-daily IPdR dosing up to 2000/mg/kg for 28 days in Fischer-344 rats showed reversible mild-to-moderate systemic toxicities without any severe or life-threatening toxicities. However, in all preclinical toxicity studies so far, no significant hematologic, biochemical or histopathologic changes have been found. Hepatic aldehyde oxidase activity was reduced in a dose-dependent fashion in the ferret liver, suggesting partial enzyme inactivation by this IPdwR schedule, but that is not found in Fischer-344 rats. The plasma pharmacokinetic profile in Rhesus monkeys showing biexponential clearance are similar to previously published data in athymic mice. In this paper, the authors review the development, mechanism of action, preclinical data and rationale for clinical studies.     

IPdR: a novel oral radiosensitizer

IPdR (5-iodo-2-pyrimidinone-2′-deoxyribose) is a novel orally available, halogenated thymidine (TdR) analog and is a potential radiosensitizer for use in human tumors, such as rectal, pancreas, sarcoma and glioma tumors. IPdR is a prodrug that is efficiently converted to IUdR (5-iodo-2′-deoxyuridine), an intravenous radiosensitizer by a hepatic aldehyde oxidase, resulting in high IPdR and IUdR plasma levels in mice for ≥ 1 h after oral IPdR. Athymic mice tolerated oral IPdR to doses up to 1500 mg/kg/day t.i.d. for 6 – 14 days without significant systemic toxicities. A number of in vivo preclinical studies have demonstrated that IPdR is a superior radiosensitizer compared with IUdR given as a continuous infusion in terms of safety and efficacy with a significantly lower toxicity profile, including gastrointestinal and hematologic side effects. A preclinical study has shown that IPdR is effective in inducing human colon cancer xenograft radiosensitization in drug-resistant DNA mismatch repair-proficient and -deficient tumor models, as well as in human globlastoma xenograft. In anticipation of performing a clinical Phase I trial in humans, investigators also studied the drug pharmacokinetics and host toxicities in two non-rodent, animal species during a 14-day treatment course. Dose-limiting systemic toxicities (diarrhea, emesis, weight loss and decreased motor activity) were observed in ferrets receiving IPdR at 1500 mg/kg/day on a 14-day schedule that were not found previously in athymic mice. Recently, a once-daily IPdR dosing up to 2000/mg/kg for 28 days in Fischer-344 rats showed reversible mild-to-moderate systemic toxicities without any severe or life-threatening toxicities. However, in all preclinical toxicity studies so far, no significant hematologic, biochemical or histopathologic changes have been found. Hepatic aldehyde oxidase activity was reduced in a dose-dependent fashion in the ferret liver, suggesting partial enzyme inactivation by this IPdwR schedule, but that is not found in Fischer-344 rats. The plasma pharmacokinetic profile in Rhesus monkeys showing biexponential clearance are similar to previously published data in athymic mice. In this paper, the authors review the development, mechanism of action, preclinical data and rationale for clinical studies.     

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.     

2'-Fluorinated arabinonucleosides of 5-(2-haloalkyl)uracil: synthesis and antiviral activity

The synthesis of 5-(2-fluoroethyl)-2'-deoxyuridine (FEDU, 4b), its 2'-fluoro analogue 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-(2-fluoroethyl)-1H,3H- pyrimidine-2,4-dione (FEFAU, 4k), and the 2'-fluoro analogue of the potent antiherpes virus compound 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), 5-(2-chloroethyl)-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1H,3H-pyr imidine - 2,4-dione (CEFAU, 4i), is described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus (HSV) types 1 and 2 and varicella zoster virus (VZV). All compounds were shown to possess significant and selective antiviral activity. FEDU proved less potent than CEDU against VZV replication; however, it was more active against HSV-2. CEFAU showed marked activity against HSV-1, HSV-2, and VZV. The compound containing fluorine at both positions, FEFAU, exhibited the strongest antiviral potency against HSV-1, HSV-2, and VZV. It inhibited HSV-1 at a concentration of 0.03-0.2 microgram/mL, HSV-2 at 0.1-0.3 microgram/mL, and VZV at 0.03 microgram/mL. Neither FEDU nor CEFAU or FEFAU exerted a significant inhibitory effect on cell proliferation at a concentration of 100 micrograms/mL. Thus, the cytotoxicity of these compounds is as low as that of CEDU and compares favorably to that of previously described 2'-fluoroarabinosyl nucleoside analogues.     

Synthesis and antiviral properties of 5-(2-substituted vinyl)-6-aza-2'-deoxyuridines

The following 5-(2-substituted vinyl)-6-aza-2'-deoxyuridines were synthesized: (E)-5-(2-bromovinyl) (2) (6-aza-BVDU), 5-(2-bromo-2-fluorovinyl) (a mixture of E and Z isomers) (3), (E)-5-(2-chlorovinyl) (4), (E)-5-[2-(methylthio)vinyl] (5), 5-(2,2-dibromovinyl) (6), and 5-(3-furyl) (7). The synthesis of 2-6 utilized Wittig-type reactions on 5-formyl-1-(2'-deoxy-3', 5'-di-O-p-toluoyl-beta-D-erythro-pentofuranosyl)-6-azauracil (16). 6-Aza-BVDU (and its alpha-anomer) was also synthesized from (E)-5-(2-bromovinyl)-6-azauracil (12) by using standard deoxyribosidation methodology. Compound 7 was prepared from 5-(3-furyl)-6-azauracil (33) via a ribosidation/deoxygenation sequence. An attempt to prepare the corresponding 5-(2,2-difluorovinyl) analogue afforded instead a mixture of the 5-[(2,2-difluoro-2-methoxy)ethyl] and 5-(2,2,2-trifluoroethyl) derivatives 29 and 30. Compounds 2-7, 29, and 30 were tested for in vitro activity against herpes simplex virus types 1 and 2 (HSV-1, HSV-2). 6-Aza-BVDU (2) exhibited ID50s of 8 micrograms/mL vs. HSV-1 and 190 micrograms/mL vs. HSV-2. BVDU (1) had ID50s of 0.015 and 1.6 micrograms/mL against HSV-1 and HSV-2, respectively. Compound 4 showed a similar profile of activity, but the other analogues were either weakly active or inactive.     

Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.     

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.     

Fluorocarbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluorocarbocyclic 2'-deoxy guanosines.

A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.     

Nucleosides. 129. Synthesis of antiviral nucleosides: 5-alkenyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracils

Synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracils containing a vinyl (4a), 2-halovinyl (4b-d), or ethyl substituent at C-5 was achieved. These nucleosides were found to be about a log order less active than 2'-fluoro-5-iodo-ara-C (FIAC) against HSV-1, but they are much less cytotoxic against normal human lymphocytes than FIAC. Nucleosides 4a and 4e showed good activity against HSV-1 (ED50 = 0.16 and 0.24 microM, respectively) and HSV-2 (ED50 = 0.69 and 0.65 microM) with very little cytotoxicity (ID50 greater than 100 microM).     

Synthesis and biological activity of 5-(2,2-difluorovinyl)-2'-deoxyuridine

5-(2,2-Difluorovinyl)uracil (IV) was synthesized from 2,4-dimethoxy-5-bromopyrimidine by sequential formylation, difluoromethylenation, and removal of the 2- and 4-methyl groups. Condensation of the trimethylsilyl derivative of IV with protected D-erythro-pentofuranosyl chloride followed by separation of anomers and deblocking gave 5-(2,2-difluorovinyl)-2'-deoxyuridine (V). Compound V was active against herpes simplex virus type 1 (HSV-1) infection as well as tumor cells transformed by the HSV-1 thymidine kinase gene.