Synthesis of new (+-)-3,5-dihydroxypentyl nucleoside analogues from 1-amino-5-(benzyloxy)pentan-3-ol and their antiviral evaluation

The synthesis and antiviral evaluation of a series of (+-)-3,5- dihydroxypentyl nucleoside analogues related to acyclic nucleoside antiviral agents are reported. All purine and pyrimidine nucleoside analogues described in this paper have been obtained from 1-amino-5-(benzyloxy)pentan-3-ol. A synthesis of this amine is reported from 1-(benzyloxy)but-3-ene after epoxidation and regiospecific diethylaluminum chloride catalyzed opening of the epoxide by trimethylsilyl cyanide. The compounds were tested in vitro in infected MRC5 and CEM cells. None of the compounds exhibited antiviral activity against HSV-1, HCMV, and HIV-1 with the exception of the guanine derivative 7, which inhibited the cytopathic effect of HSV-1 by 50% at 12.5 micrograms/mL.     

in vitro metabolism of 1-β-d-arabinofuranosylcytosine and 1-β-2'-fluoro-arabino-5-iodocytosine in normal and herpes simplex type 1 virus-infected cells

Phosphorylation of 1-β-D-2'-F-arabino-5-iodocytosine (FIAC), a newly synthesized pyrimidine nucleoside with potent antiherpesvirus activity, was compared with that of its parent compound, 1-β-D-arabinofuranosylcytosine (ara-C). While ara-C was phosphorylated extensively by homogenates of normal, rapidly proliferating mouse tissues, FIAC was a poor substrate for the nucleoside kinase occurring in such normal tissues. With cell homogenates of noninfected Vero cells, thymidine (TdR) was phosphorylated about fifty and twenty times more efficiently than FIAC and ara-C, while infection of Vero cells with Herpes Simplex Virus Type 1 (HSV-1) resulted in a 23-fold increase of TdR- and a 1270-fold increase of FIAC phosphorylation. In contrast, phosphorylation of ara-C was increased only by a factor of 2.6. While the reaction products obtained with homogenates of normal mouse tissues were 5'-mono-, di- and triphosphates of ara-C and FIAC, the reaction products with noninfected and infected Vero cell homogenates were predominantly monophosphates. In contrast, TdR was efficiently phosphorylated to its 5'-mono-, di- and triphosphates by such homogenates. In intact HSV-1-infected Vero cells, FIAC was rapidly taken up and phosphorylated to FIACMP and to an as yet unidentified metabolite. In contrast, TdR was taken up and phosphorylated to 5'-mono-, di- and triphosphates and ara-C was taken up moderately but metabolized poorly to its 5'-mono-, di- and triphosphates. Thus, in normal tissues, FIAC was a poorer substrate than ara-C for nucleoside kinases, but in intact HSV-1-infected Vero cells FIAC was efficiently phosphorylated and thus behaved like a TdR analog, except that it was phosphorylated only to the 5'-monophosphate and a hitherto unidentified metabolite. The greatly increased phosphorylation of FIAC by HSV-1-infected Vero cells probably accounts, at least in part, for its great selectivity of action.     

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).     

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)-cyclobutyl] guanine [(+-)-BHCG or SQ 33,054]: a potent and selective inhibitor of herpesviruses

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine [(+-)-BHCG or SQ 33,054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (+-)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (+-)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (+-)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.     

Carbocyclic analogues of 5-halocytosine nucleosides

Carbocyclic analogues of 5-halocytosine nucleosides were prepared by direct halogenation of the carbocyclic analogues of cytidine, 2'-deoxycytidine, 3'-deoxycytidine, or ara-C. The 5-chloro and 5-bromo derivatives of the cytidine (carbodine) and of the 2'-deoxycytidine analogues and the 5-iodo derivatives of all four of the cytosine nucleoside analogues were prepared. All of the C-5-halocytosine nucleosides, as well as the parent C-cytosine nucleosides, were tested against a strain of herpes simplex virus type 1 (HSV-1) that induces thymidine kinase in host cells. Carbodine, 5-bromocarbodine, C-2'-deoxycytidine, C-5-bromo-2'-deoxycytidine, the four C-5-iodocytosine nucleosides, and C-ara-C inhibited replication of this strain of HSV-1 in cultured cells. Most of these compounds were tested also against the type 2 virus (HSV-2) in vitro and were active. The greatest activity observed was exerted by C-5-iodo-2'-deoxycytidine in inhibiting replication of HSV-1 in L929 cells. In tests against these DNA viruses, carbodine, a ribofuranoside analogue that had been shown previously to be highly active against human influenza A virus in vitro, was the most active compound against HSV-2 and one of the most active compounds against HSV-1 in Vero cells. 5-Bromocarbodine was active against influenza virus, but it was less active than carbodine.     

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.     

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.     

Comparison of the antiviral effects of 5-methoxy-methyldeoxyuridine-5′-monophosphate with adenine arabinoside-5′-monophosphate

Methoxymethyldeoxyuridine-5′-monophosphate (MMUdR-MP) and arabinofuranosylade-nine-5′-monophosphate (Ara-AMP) had significant antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in RK-13 and Vero cells. MMUdR-MP and Ara-AMP were more potent than methoxymethyldeoxyuridine (MMUdR) and arabinofuranosyladenine (Ara-A) against the MS strain of HSV-2. MMUdR-MP inhibited replication of HSV-1^r (mutant resistant to MMUdR). MMUdR in combination with Ara-AMP showed additive activity; whereas the MMUdR-MP and Ara-AMP combination was antagonistic against herpes viruses. MMUdR in combination with Ara-A was synergistic in reducing the log virus yield. Cytotoxicity (microscopic lesions) was observed on exposure to MMUdR-MP and Ara-AMP at 450 and 90 μM, respectively. Rapidly proliferating RK-13 cells exposed to Ara-AMP (64 μM) were killed. In the same system, the cells surviving after incubation with MMUdR-MP (650 μM), multiplied at an almost normal rate.     

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.     

Inhibitory effect of cycloSaligenyl-nucleoside monophosphates (cycloSal-NMP) of acyclic nucleoside analogues on HSV-1 and EBV

The in vitro antiviral activity of a new series of cycloSal-pro-nucleotides derived from the acyclic nucleoside analogues aciclovir and penciclovir against herpes simplex virus type 1 (HSV-1), thymidine kinase deficient (TK-) HSV-1, and Epstein-Barr virus (EBV) was evaluated. Using the XTT-based tetrazolium reduction assay EZ4U, the cycloSal derivatives were examined for their antiviral and cytotoxic effects in HSV-1 as well as HSV-1-TK--infected Vero cells. The anti-EBV activity was assessed by means of an EBV DNA hybridization assay using a digoxigenin-labeled probe specific for the Bam H1-W-fragment of the EBV genome and by measuring viral capsid antigen (VCA) expression in P3HR-1 cells by indirect immunofluorescence. Among the new cycloSal-phosphotriesters the three aciclovir monophosphates proved to be potent and selective inhibitors of HSV-1 replication, EBV DNA synthesis and EB-VCA expression. Of interest is the retention of activity of the aciclovir monophosphates in HSV-1-TK--infected cells. Particularly 3-methyl-cycloSal-aciclovir monophosphate retained the same effectiveness, as compared to the wild type virus strain. In contrast to the aciclovir pro-nucleotides the penciclovir cycloSal-phosphotriesters exhibited at best only a marginal antiviral effect on HSV and EBV replication.     

The relationship between the antiviral activity of 5'-amino-5'-deoxythymidine and its incorporation into herpes simplex virus type 1 DNA

As part of our studies on the molecular basis for the antiherpes activity of 5'-AdThd (5'-amino-5'-deoxythymidine), a study of the HSV-1 DNA synthesized in infected Vero cells exposed to 5'-AdThd was undertaken. Unlike many other antiviral nucleoside analogs, 5'-AdThd did not inhibit HSV-1 DNA synthesis. Analysis of the DNA synthesized in the presence of [14C]5'-AdThd revealed that the analog was incorporated into the viral DNA in a dose-dependent manner and that the degree of incorporation correlated with the antiviral activity as measured by yield reduction assays. Analysis of the 5'-AdThd substituted DNA by centrifugation in neutral and alkaline sucrose gradients revealed no double-stranded breaks but an increase in single-stranded breaks, at very high concentrations of the analog. Analysis of HSV-1-specific RNAs revealed a shift from poly(A+) to poly(A-) RNA. The degree of this shift paralleled the substitution of 5'-AdThd for thymidine in the HSV-1 DNA.     

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.     

Antiviral action of 5-amino-2-(2-dimethyl-aminoethyl)benzo-[de]-isoquinolin-1,3-dion e

A newly synthesized imide derivative of 3-nitro-1,8-naphthalic acid, 5-amino-2-(2-dimethylaminoethyl)benzo-[de]-isoquinolin-1,3-dione (designated M-FA-142), was tested on chick embryo cells against herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV), and on Vero cells against African swine fever virus (ASFV). At a concentration of 4 micrograms/ml the drug inhibited VV replication by about one order of magnitude, and that of HSV-1 by about three orders of magnitude. A minor effect was shown against ASFV. Virus inhibition was found to depend on the amount of drug and multiplicity of infection. No virucidal effect was observed on the viruses tested, except for a slight effect on HSV-1. Inhibition of virus growth could be reversed when the drug was removed from the cell culture medium. Serial passages of HSV-1 and VV in the presence of the drug caused the appearance of drug-resistant viruses.     

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.     

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.     

Phosphonoformate and phosphonoacetate derivatives of 5-substituted 2'-deoxyuridines: synthesis and antiviral activity

The synthesis of potential "combined prodrugs" wherein phosphonoformate or phosphonoacetate was attached to the 5'-position of 2'-deoxyuridine, 2'-deoxythymidine, 5-iodo-2'-deoxyuridine (IDU), 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), or 5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or to the 3'-position of CEDU is described. The antiviral activities of these derivatives and of reference compounds were compared in Vero, HEp-2, and primary rabbit kidney cells against herpes simplex virus types 1 and 2 (HSV-1 and -2). The CEDU and BVDU analogues were also evaluated against systemic and intracutaneous HSV-1 infection in mice. The nature of the 5-substituent proved critical for antiviral activity, since only the 5-iodo-, 5-(2-bromovinyl)-, and 5-(2-chloroethyl)-substituted derivatives were inhibitory to the herpesviruses. Furthermore, the type specificity is determined by the nature of the 5-substituent: the IDU analogues were similarly inhibitory to HSV-1 and -2 whereas the CEDU and BVDU analogues inhibited HSV-2 replication only at considerably higher concentrations than HSV-1. In vivo, several derivatives were shown to possess significant antiviral activity; however, none surpassed its respective parent compound, CEDU or BVDU, in potency. It seems improbable, therefore, that a synergistic effect between PFA or PAA and the nucleoside analogue occurred. The extent of in vitro and in vivo activity of the CEDU and BVDU 5'-phosphonoformates and 5'-phosphonoacetates is most plausibly explained by the ease by which the "combined prodrugs" are hydrolyzed and the parent compound, CEDU and BVDU, respectively, is released.     

The anti-herpesvirus activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)-cycloprop-1'-yl]methyl]guanine is markedly potentiated by the immunosuppressive agent mycophenolate mofetil

Mycophenolic acid (MPA), the active form of the immunosuppressive agent mycophenolate mofetil (MMF), was found to markedly potentiate the anti-herpesvirus activity of the novel anti-herpesvirus agent A-5021, (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine. For example, at a concentration of 1 microg/ml MPA, the activity of A-5021 against HSV-1, HSV-2 and TK(-) HSV-1 increased by a factor of 130, 14 and > or = 189, respectively. Exogenously added guanosine reversed this potentiating effect, suggesting that a depletion of the endogenous dGTP pools enhanced the inhibitory effect of the 5'-triphosphate metabolite of A-5021 on the viral DNA polymerase. The combined effect of A-5021 and MPA on the growth of uninfected Vero cells was additive rather than synergistic. The combination of topically applied MMF (5%) with 0.05% A-5021 (a subactive concentration) completely protected against HSV-1-induced cutaneous lesions in hairless mice, whereas therapy with either compound used alone had no protective effect. These findings may have implications for those transplant recipients that receive MMF as (part of) their immunosuppressive therapy and that develop intercurrent herpesvirus infections for which they need treatment.     

Synthesis, antiviral and cytotoxic activity of 2'-deoxyuridines, 2'-fluoro-2'-deoxyuridines and 2'-arabinouridines containing 5-(1-hydroxy-2-halo-2-ethoxycarbonylethyl)-, 5-(1-hydroxy-2-iodo-2- carboxyethyl)- and 5-[1-hydroxy (or methoxy)-2-iodoethyl] substituents.

The 5-[1-hydroxy-2-chloro-2-(ethoxycarbonyl)ethyl]-2'-deoxyuridine (7) and 5-[1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl]-2'- fluoro-2'-deoxyuridine/uridine nucleosides (8, 9) were synthesized by the regiospecific addition of HOX (X = Br or Cl) to the vinyl substituent of the respective (E)-5-[2-(ethoxycarbonyl)-vinyl]-2'-deoxyuridines (6a-b) and uridine (6c). A related reaction of (E)-5-(2-carboxyvinyl)-2'-deoxyuridines (10a-b) and uridine (10c) with iodine and potassium iodate afforded the 5-(1-hydroxy-2-iodo-2-carboxyethyl) derivatives (11-13). 5-(1-Hydroxy-2-iodoethyl)-arabinouridine (18) was obtained by the reaction of (17) with iodine in the presence of the oxidizing agent iodic acid. Treatment of (18) with methanolic sulfuric acid afforded 5-(1-methoxy-2-iodoethyl)-arabinouridine (19) in 65% yield. Of the newly synthesized compounds, 7, 11 and 12 showed activity in vitro against HSV-1. The most active compound (12, ID50 = 0.1 microgram/ml) was 10 times less active than acyclovir (ID50 = 0.01 microgram/ml) against HSV-1. Compounds 7 and 11 were cytotoxic to L1210 cells in culture, exhibiting an ED50 of 7.2 and 4.7 micrograms/ml respectively, relative to melphalan (ED50 = 0.15 microgram/ml), but were inactive against the KB cell line.     

Design, synthesis and antiviral activity of novel 4,5-disubstituted 7-(beta-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazines and the novel 3-amino-5-methyl-1-(beta-D-ribofuranosyl)- and 3-amino-5-methyl-1-(2-deoxy-beta-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-hexaazaacenaphthylene as analogues of triciribine

The synthesis of several heterocyclic analogues of the biologically important nucleoside antibiotic toyocamycin and the tricyclic nucleoside triciribine (TCN) were prepared along with their 2'-deoxy counterparts. Coupling of 2-nitropyrrole-3,4-dicarboxamide (15) under a variety of conditions with alpha-chloro-2-deoxy-3,4-di-O-toluoyl-D-ribofuranose (16a) gave mixtures of the alpha and beta anomers. A coupling of 15 with 1-chloro-2,3,5-tri-O-benzoyl-D-ribofuranose (18) gave exclusively the beta anomer. Individually, the two pyrrole nucleosides were treated with Pd/C, H2 to reduce the nitro groups and cyclized with nitrous acid, and the corresponding 4-position was functionalized as a triazoyl derivative. Nucleophillic displacement was carried out with ammonia to give a mixture of 4-amino-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (26) and 2-amino-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyrrole-3,4-dicarbonitrile (27), the latter being formed via a retro-Diels-Alder reaction. The subsequent addition of hydrogen sulfide, water, methanol, hydroxylamine, cyanamide, hydrazine and methylhydrazine to the 5-cyano group was carried out to give the corresponding analogues. In the case of methyl hydrazine, subsequent treatment with NaOMe in methanol gave the title hexaazaacenaphthylenes. Biological evaluation of the compounds established that the pyrrole (17beta, 19-21) and most of the pyrrolotriazine (22, 24, 28, 32-34) nucleosides were inactive or weakly active against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1). In contrast 29 and 31 were active against one or both of these viruses but activity was poorly separated from cytotoxicity. In contrast, the 2-aza analogue of sangivamycin (30) was active against HCMV and HSV-1 but this apparent activity was most likely due to its high cytotoxicity. The tricyclic nucleoside 12, was active against its target virus, human immunodeficiency virus type 1 (HIV-1), but this activity was not well separated from cytotoxicity.     

Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.