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A galactan sulfate (GS) was isolated from an aqueous extract of the red seaweed Aghardhiella tenera and partially purified. GS inhibited the cytopathic effect of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in MT-4 cells at concentrations 10-fold higher than those required for the inhibition by dextran sulfate (MW 5000) of the cytopathic effect of HIV-1 and HIV-2 (50% inhibitory concentrations: 0.5 and 0.05 mu gml(-1), respectively). GS suppressed syncytium formation between MOLT-4 cells and persistently HIV-1- or HIV-2-infected HUT-78 cells at concentrations higher than 5 mu gml(-1). Like dextran sulfate (DS) and aurintricarboxylic acid (ATA), GS inhibited the binding of HIV-I to the cells and the binding of anti-gp120 mAb to HIV-1 gp120. Like DS and ATA, GS proved active not only against HIV-I and HIV-2 but also against other enveloped viruses, i.e. herpes-, toga-, arena-, myxo- and rhabdoviruses. GS represents a natural polysaccharide with broad-spectrum activity against a number of important viral pathogens. ispartof: Antivir Chem Chemoth vol:5 issue:5 pages:297-303 status: published

The synthesis and antiviral activity of a new series of acyclic nucleoside analogues containing a (2-hydroxyethoxy)ethyl moiety is discussed.

The thymidine analogue (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd), which is an antiviral agent effective against herpes simplex virus type 1 and varicella zoster virus, has also proved to be a potent inhibitor of dihydrouracil dehydrogenase, the major degrading enzyme of the anticancer drug fluorouracil (FUra). To evaluate the effect of BVdUrd on the pharmacokinetics of FUra in cancer patients, BVdUrd was administered orally at a daily dose of 250 mg (five patients) or 3 x 250 mg (five patients). FUra was infused at doses of 110-400 mg over 10 min. Blood and urine samples were collected regularly during a period of up to 50 h. BVdUrd was rapidly absorbed, peak plasma levels of 0.6-7.1 micrograms/ml being achieved after 1-2 h. Following a rapid decline, plasma BVdUrd levels remained higher than 10 ng/ml for up to 2 days after BVdUrd administration. The total body clearance of FUra was approximately 61/h and t1/2 markedly increased to 4-7 h. The mean urinary excretion of FUra was 50%. No differences in FUra kinetics were observed between patients receiving one or three oral doses of BVdUrd. We conclude that the concomitant use and subsequent interaction of FUra and the antiviral agent BVdUrd resulted in an impressive inhibition of FUra breakdown and marked increase of renal clearance. The findings indicate that the simultaneous use of FUra and drugs resembling this antiviral agent in patients may result in unexpected toxicity. Further experience with BVdUrd and new analogues might enable the development of new FUra treatment schedules and treatment designs e.g. combinations with leucovorin.

A number of 3,5-diarylidene-4-piperidones (1) and some related quaternary ammonium salts (5) as well as closely related analogs were prepared principally as candidate cytotoxic agents in two screens. The first test system used an average of 54 human tumor cell lines from eight neoplastic diseases, namely leukemia, melanoma, colon, non-small-cell lung, small-cell lung, central nervous system, ovarian, and renal cancers. Selective toxicity was demonstrated by some of the compounds, especially toward leukemia. The second screen used L 1210 lymphoid leukemia cells. In general, the compounds were less cytotoxic than the reference drug melphalan in both screens. Linear plots were made between the Hammett (σ), fragment (f), and molar refractivity (MR) constants of the nuclear substituents in series 1 and 5with the IC50 figures of both the human tumor cell lines and L 1210 cells. Evaluation against the human tumor cell lines revealed that increases in the f values were correlated with elevation of cytotoxicity in both series 1 and 5; MR constants were also important in series 5. In the L 1210 screen, σ and MR constants were positively correlated with cytotoxicity. X-ray crystallography was undertaken on 3,5-bis-[[4′-(methylthio)phenyl]methylene]-1-methyl-4-piperidone methiodide (5d), which had significant cytotoxicity, and 3,5-bis(4-pyridylmethylene)-1-methyl-4-piperidone methiodide (6), which was virtually inactive in both screens. A number of structural features were noted, and in particular a smaller interplanar angle made by one of the arylidene groups with the central piperidine ring in the case of 5d than either of the 4-pyridylmethylene moieties in 6 may contribute to the variation in cytotoxicity between these two compounds. Eleven compounds which were examined for activity against a panel of 12 viruses were shown to display only marginal potencies or to be inactive at the highest concentrations utilized. Two compounds with slight activity both had the hydrophilic carboxylate ion in the aryl rings. From this study, various conclusions pertaining to future molecular modification with a view to increasing cytotoxic potency were made.

Novel 5,6-dichlorobenzimidazole nucleoside analogues structurally related to the well-known riboside DRB have been synthesized. The 1′,2′- trans nucleosides were prepared by condensation of peracylated sugars with 5,6-dichlorobenzimidazole, whereas the 1′,2′- cis β-D-arabinofuranosyl and β-D-lyxofuranosyl nucleosides were obtained by inversion of configuration on the sugar moiety. Chiral acyclic derivatives were stereospecifically prepared by ring-opening of furano- or pyrano-nucleosides by means of periodate oxidation, followed by borohydride reduction. The in vitro activities against a range of DNA and RNA viruses, as well as the cytotoxicities in human T-lymphocyte MT-4 cells, have been determined for these novel compounds and for DRB. No truly selective activity (i.e. clearly below the cytotoxic concentration) was observed against any of the viruses used. Some of the compounds, including DRB, were cytotoxic to MT-4 cells at CC50values of less than 10 μg ml−1.

Varicella zoster virus (VZV), a member of the herpesvirus family, is responsible for a primary infection (varicella, chickenpox) as well as a recurrent disease (zoster, shingles). The course of varicella is generally benign in immunocompetent patients. For zoster, post-herpetic neuralgia is the most common complication. In immunocompromised patients, particularly patients suffering from the acquired immune deficiency syndrome (AIDS), transplant recipients and cancer patients, VZV infections can be life-threatening. For these patients and also for immunocompetent patients at risk, such as pregnant women or premature infants, the current treatment of choice is based on acyclovir (ACV) by either intravenous or oral route. The low oral bioavailability of ACV, as well as the emergence of drug-resistant virus strains, have stimulated efforts towards the development of new compounds for the treatment of VZV infections. Among these new compounds, penciclovir (PCV) and its oral prodrug form, famciclovir (FCV), 882C87, BVDU, BVaraU and the oral prodrug form of acyclovir (valacyclovir) rank among the most promising. Like ACV itself, all these drugs are dependent on the virus-encoded thymidine kinase (TK) for their intracellular activation (phosphorylation), and, therefore, cross-resistance to these drugs may be expected for those virus mutants that are TK-deficient and thus resistant to ACV. However, such TK(-) VZV mutants are still sensitive to the acyclic nucleoside phosphonates (i.e. HPMPC), which for their phosphorylation do not depend on the virus-encoded TK. The molecular targets within the viral replicative cycle, with which these different compounds interact, are discussed, as are the in vitro activity and in vivo efficacy of the most promising compounds. Other aspects, such as vaccination and passive immunization, are briefly mentioned.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) grow in vitro in a broad range of human and animal cells. Here we describe the activity of several nucleoside and nucleotide analogues against HSV-1 and HSV-2 in human brain-tumour-derived cells. Of the compounds tested, ( E)-5-(2′-bromovinyl)-2′-deoxyuridine (BVDU) proved to be the most potent inhibitor of the wild-type, thymidine-kinase-positive (TK+) HSV-1. In contrast to BVDU, acyclovir and ganciclovir, which were virtually inactive against TK−HSV-1, the two acyclic nucleoside phosphonates ( S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and ( S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) were equally active against TK+and TK−HSV-1 strains. Depending on the brain tumour cell line used, differences in virus-drug sensitivity varied up to 30-fold. Upon passage of the virus in the brain tumour cell lines in the presence of the compounds, virus resistance developed rapidly to BVDU, acyclovir and foscarnet but not to HPMPC. Furthermore, the HSV-1 mutant that had become resistant to BVDU, acyclovir or foscarnet remained as sensitive to HPMPC and HPMPA as did the wild-type HSV-1 (KOS strain).

Mutant HIV-1 that expresses a Glu138-->Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that expresses a Tyr181-->Cys substitution in its RT [(Y181C)RT] were not protected by nevirapine and TIBO and were only temporarily protected by BHAP. HIV-1 mutant that emerged under the latter conditions contained a Cys181-->Ile substitution in their RT [(LC181I)RT]. This mutant proved highly resistant to all HIV-1-specific RT inhibitors tested, except for several 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives. When recombinant (C181I)RT was evaluated for susceptibility to the HIV-1-specific RT inhibitors, it was resistant to all inhibitors except the HEPT compounds. Since a (Y181F)RT HIV mutant strain was isolated from cells infected with (Y181C)RT HIV-1 and treated with BHAP, we postulate that the Ile codon was derived from a Cys-->Phe transversion mutation (TGT-->TTT), followed by a Phe-->Ile transversion mutation (TTT-->ATT).

As a continuation of previous studies on the synthesis and antitumor activity of geiparvarin analogues bearing a carbamate moiety in the alkyl side chain, a series of N-substituted [(E)-3-(4,5-dihydro-5,5-dimethyl-4-oxo-2-furanyl)-2- butenyl]carbamates (15a-f) were synthesized and tested with the objective to investigate the reason for the marked difference of cytostatic activity found between alkyl and phenyl derivatives. A series of compounds, characterized by different physicochemical properties, were designed in order to study this hypothesis. Moreover, to further investigate the modification of the alkenyl side chain, (E)- and (Z)-[2-(4,5-dihydro-5,5-dimethyl-4-oxo-2-furanyl)propenyl]-7H-furo[3,2- g][1]benzopyran-7-one (11a,b) were synthesized, the latter compounds being the combination of two units, namely, the 3(2H)-furanone ring system endowed with potent alkylating properties and the furocoumarin portion which binds to DNA resulting in potential DNA-targeted alkylating agents. The compounds were tested for their cytostatic activity against proliferation of murine (L1210) and human (Molt/4, CEM, or MT-4) tumor cells. The highest cytostatic activity found within both series of carbamic derivatives (15a-d,k and 15e,g-j) was associated with the highest global lipophilicity. With regard to compounds 11a,b, the cytostatic activity of (Z)-furocoumarin 11b might be related to a specific interaction with DNA (i.e., intercalation).

Modified cyclodextrin sulphate (mCDS) in which lipophilic groups were introduced to cyclodextrin sulphate (CDS) was synthesized and proved more inhibitory to the replication of HIV-1 and HIV-2 than CDS or dextran sulphate (DS). The anti-coagulant activity of mCDS was lower than that of DS. Cyclodextrin phosphate (CDP) showed anti-HIV activity similar to that of CDS, and its anti-coagulant activity was even lower than that of mCDS. Flow cytometric analysis suggested that the mechanism of the anti-HIV-1 action of CDS, mCDS, and CDP is based on inhibition of HIV-1 binding to the cells. The peak blood concentration after oral administration of mCDS11(potassium tris[6-benzylthio-6-deoxy]-β-cyclodextrin hexadecasulphate) to rabbits was about 1000 times higher than the concentration showing anti-HIV activity. The retention time in the blood was also long (blood half-life: 4 h). These results point to the potential usefulness of oral mCDS administration in the prophylaxis and/or therapy of HIV infections.

A series of triterpene compounds characterized by a stringent structure-activity relationship were identified as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication. Currently studied botulinic derivatives have 50% inhibitory concentrations (IC50) against HIV-1 strain IIIB/LAI in the 10 nM range in several cellular infection assays but are inactive against HIV-2. These compounds did not significantly inhibit the in vitro activities of several purified HIV-1 enzymes. Rather, they appeared to block virus infection at a postbinding, envelope-dependent step involved in the fusion of the virus to the cell membrane.

By using an assay system based on a human hepatoblastoma cell line (HB611) that continuously synthesizes hepatitis B virus (HBV) DNA, 56 acyclic nucleoside phosphonate analogues were examined for their inhibitory effects on HBV DNA synthesis. The following compounds were found to inhibit HBV DNA synthesis at concentrations that were significantly lower than their minimum cytotoxic concentrations; 9-(2-phosphonylmethoxyethyl)adenine (PMEA), 9-(2-phosphonylmethoxyethyl) guanine(PMEG), 9-(2-phosphonylmethoxyethyl) guanine ethyl ester (PMEGEE), 9 - (2 - phosphonylmethoxyethyl) - 1 - deazaadenine (PMEC1A), 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), ( S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), 9-(3-isopropoxy-2-phosphonylmethoxypropyl)adenine (IPPMPA), 9-( RS)-(2-phosphonylmethoxypropyl)adenine (PMPA) and 9-(3-hydroxy-2-phosphonylmethoxypropyl)-2, 6-diaminopurine (HPMPDAP). The most selective compounds (with indexes greater than 100) were PMEDAP, PMEA, IPPMPA, and PMPA. Acyclic pyrimidine nucleoside phosphonate analogues did not prove markedly selective as anti-HBV agents. Diphosphoryl derivatives of some acyclic purine nucleoside phos-phonates (i.e. PMEA, PMEDAP, HPMPA) were prepared. They proved inhibitory to HBV DNA polymerase but not cellular DNA polymerase α.

A novel series of hybrid molecules with cholesterol and naphthalenesulfonic acid have been synthesized and evaluated for their inhibitory activity against myxoviruses (respiratory syncytial virus [RSV], influenza A and B virus), Naphthalenesulfonic acids (compounds 1–3) and cholesterols (compounds 4 and 5) did not inhibit myxovirus-induced cytopathic-ity. However, hybrid molecules (compounds 6 and 7) proved active against RSV and influenza A virus but not influenza B virus. The antiviral effects of the hybrid compounds 6 and 7 were comparable to those of dextran sulfate. Mixtures of naphthalenesulfonic acid and cholesteryl chloroformate (compounds 1 and 5 molecules, corresponding to the hybrid molecule 6; and compounds 2 and 5, corresponding to the hybrid molecule 7) did not show antiviral activity. The mode of action of the cholesterol-linked naphthalenesulfonic acids can be attributed to inhibition of virus-cell fusion (influenza A virus) or inhibition of both virus-cell binding and fusion (RSV).

Palladium-catalyzed cross-coupling of 8-bromo-2'-deoxyadenosine with terminal alkynes in the presence of copper(I) iodide in dimethylformamide resulted in a series of 8-(1-alkyn-1-yl)-2'-deoxyadenosines. Hydrogenation of alkynyl derivatives over 10% Pd/C under atmospheric pressure gave 8-n-alkyl analogues in nearly quantitative yields. On partial saturation of heptynyl, pentynyl, and propynyl derivatives over Lindlar catalyst, the corresponding cis-olefins were obtained along with minor amounts of trans isomers. Of the analogues tested, the following showed some activity, i.e. they were found to be active at concentrations that were at least 3-fold lower than the cytotoxic concentrations: the 8-heptynyl derivative against vaccinia virus (VV), vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and respiratory syncytial virus (RSV); the 8-propyl derivative against varicella-zoster virus (VZV) and CMV; the 8-pentyl derivative against CMV; the 8-heptyl derivative against VV, CMV, RSV, and influenza A; and the 8-heptenyl derivative against VV, RSV, and influenza A. The unsubstituted 2'-deoxyadenosine did not show any antiviral effect, except against RSV. Except for 8-propyl-dA, the antivirally active dA analogues were rather inhibitory to the growth of human embryonic lung cells. The most cytotoxic was the 8-ethynyl derivative.

MKC-442 (6-benzyl-1-ethoxymethyl-5-isopropyluracil or I-EBU) has recently been identified as a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Since the compound has favorable pharmacokinetic and toxicity profiles in vivo, we have evaluated MKC-442 for its inhibitory effect on the replication of HIV-1 in various cell cultures, including human peripheral blood lymphocytes and monocyte-macrophages. The 50 and 90% effective concentrations for HIV-1 (HTLV-IIIB strain) replication in MT-4 cells were 15 and 98 nM, respectively. MKC-442 was also inhibitory to HIV-1 replication in peripheral blood lymphocytes and monocyte-macrophages as determined by the production of p24 antigens in the culture supernatant. Fluorescence-activated cell sorter analysis revealed that MKC-442 was equally active against zidovudine-resistant mutants and zidovudine-susceptible strains. Furthermore, combinations of MKC-442 with either 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, or 2',3'-dideoxyinosine synergistically inhibited the replication of HIV-1. Thus, MKC-442 has been considered as a candidate for clinical efficacy studies.

An analysis of the major frequency shifts of the optically pumped cesium beam primary frequency standard of the Laboratoire Primaire du Temps et des Frequences (LPTF) is presented, together with a description of the most relevant characteristics of the device. The short-term frequency stability was measured against a hydrogen maser. The square root of the Allan variance /spl sigma//sub y/(/spl tau/) is, at present, 5.8 /spl times/ 10/sup -13/ /spl tau//sup -1/2/, with a flicker level at 3.3 /spl times/ 10//sup -15/. This extremely good frequency stability enables a highly precise analysis of the most important systematic frequency shifts to be performed in a short time. A first accuracy evaluation resulted in an uncertainty (1/spl sigma/) of 1.1 /spl times/ 10/sup -13/. >

The addition reaction of either 3-bromo-5-lithiopyridine (2a) or 3-cyano-5-lithiopyridine (2b) to 2,3:4,5-di-O-isopropylidene-aldehydo-D-arabinose (1) or 2,4:3,5-di-O-benzylidene-aldehydo-D-lyxose (8) gave respectively a D-gluco/D-manno mixture of 3-bromo- and 3-cyano-5-(2,3:4,5-di-O-isopropylidene-pentitol-1-yl)pyridine (3a,b) or a D-galacto/D-talo mixture of respectively 3-bromo- and 3-cyano-5-(2,4:3,5-di-O-benzylidene-pentitol-1-yl)pyridine (9a,b). Mesylation of C-1' followed by reaction with CF3COOH/H2O resulted in the formation of the corresponding D-arabino- or D-lyxofuranosyl pyridine C-nucleosides. The cyano group of (5b) and (11b) was converted into a carbamoyl group using Amberlite IRA 400 (OH-). 3-Cyano-5-D-arabinofuranosylpyridine (5b) was converted into 3-thiocarbamoyl-5-D-arabinofuranosylpyridine (7) using H2S and triethylamine. None of the test compounds showed a marked cytostatic or antiviral activity in vitro.

The in vivo activity of the Keggin polyoxotungstate PM-19 [K7(PTi2W10O40)·6H2O] was investigated against herpes simplex virus type 2 (HSV-2) in ddY mice. A single dose of PM-19 at 100mg kg−1administered intraperitoneally (i.p.) immediately after i.p. infection of ddY mice with HSV-2 offered 92% protection against an otherwise lethal HSV-2 infection. PM-19 was less or not effective if given by any route other than the i.p. route. When repeated doses of PM-19 were administered i.p. on day 0 (immediately after infection) and day 1 and 2 after infection, it proved protective over a dosage range of 0.1-50 mg kg−1day−1, its ED50 (50% effective dose, based on the number of survivors) being 0.25 mg kg−1day−1. Under these conditions, ACV was not effective even at doses up to 100 mg kg−1day−1.

Several novel fluorinated acyclic nucleoside phosphonates [i.e. 9-(3-fluoro-2-phosphonylmethoxy propyl)adenine (FPMPA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)-2,6-diaminopurine (FPMPDAP)] were evaluated for their inhibitory effect against feline immunodeficiency virus (FIV) replication in Crandell feline kidney (CrFK) cells and feline peripheral blood lymphocytes (PBL) in vitro. Whereas 3-azido-3-deoxythymidine (AZT) was not able to achieve complete suppression of viral antigen expression and reverse transcriptase activity in the FIV-infected cell culture supernatants at 25 μM, FPMPA, FPMPDAP, and the 9-(2-phosphonylmethoxyethyl)purine derivatives PMEA and PMEDAP fully protected FIV-infected cells at μM. Both FPMPA and FPMPDAP were endowed with a higher antiviral potency and/or therapeutic selectivity than PMEA and PMEDAP in inhibiting FIV infection, mainly due to a markedly lower toxicity for the cell cultures.

A series of new N,N ′-disubstituted thioureas were prepared and evaluated in vitro for their activity against myxoviruses. Compound N -[4-(3-propoxy-1 H -1,2,4-triazol-1-yl)phenyl]- N ′-(2-pyrazinyl)thiourea 5d exhibited specific activity against respiratory syncytial virus in HeLa cells. Compounds N -(1-adamantanyl)- N ′-[4-(3-ethoxy-1 H -1,2,4-triazol-l-yl)phenyl]thiourea 5g and N -(1-adamantanyl)- N ′-[4-(3-propoxy-1 H -1,2,4-triazol-1-yl)phenyl]thiourea 5h proved active against influenza A in Madin-Darby canine kidney (MDCK) cells and respiratory syncytial virus in HeLa cells.

A series of new 1 H -1,2,4-triazole derivatives was synthesised and evaluated in vitro for activity against various DNA and RNA viruses. Compounds 1-benzyl-5-(4-methylphenyl)-3-(2-pyrazinyl)-1 H -1,2,4-triazole 2b , 1-benzyl-5-(3-methylphenyl)-3-(2-pyrazinyl)-1 H -1,2,4-triazole 2c and 1-[(2-hydroxyethoxy)methyl]-5-(3-methylphenyl)-3-(2-pyrazinyl)-1 H -1,2,4-triazole 4h proved active against reovirus-1, and compounds 1-[(2-hydroxyethoxy)methyl]-5-phenyl-3-(2-pyrazinyl)-1 H -1,2,4-triazole 4f and 1-[(2-hydroxyethoxy)methyl]-5-(3-methylphenyl)-3-(2-pyrazinyl)-1 H -1,2,4-triazole 4h against parainfluenza-3 virus, in Vero cells at concentrations that were 5- to 40-fold lower than the cytotoxic concentration required to alter normal cell morphology.

For the majority of native species, human-created habitats provide a hostile environment that prevents their colonization. However, if the conditions encountered in this novel environment are part of the fundamental niche of a particular species, these low competitive environments may allow strong population expansion of even rare and stenotopic species. If these species are potentially harmful to humans, such anthropogenic habitat alterations may impose strong risks for human health. Here, we report on a recent and severe outbreak of the viciously biting and day-active mosquito Anopheles plumbeus Stephens, 1828, that is caused by a habitat shift toward human-created habitats. Although historic data indicate that the species was previously reported to be rare in Belgium and confined to natural forest habitats, more recent data indicate a strong population expansion all over Belgium and severe nuisance at a local scale. We show that these outbreaks can be explained by a recent larval habitat shift of this species from tree-holes in forests to large manure collecting pits of abandoned and uncleaned pig stables. Further surveys of the colonization and detection of other potential larval breeding places of this mosquito in this artificial environment are of particular importance for human health because the species is known as a experimental vector of West Nile virus and a potential vector of human malaria.

The study of strategies for the treatment of human cytomegalovirus (HCMV) infection starts with the discovery of compounds that are potent and selective inhibitors of HCMV replication. Selectivity means that the window between the concentrations that block viral replication on the one hand and those that affect host cell functions on the other is sufficiently wide. Because HCMV induces an obvious cytopathic effect (CPE) in human fibroblast cultures, potential antiviral activity can easily be scored microscopically. For this type of assay, a relatively low input of virus should be used, so that the CPE does not appear within the first 2-3 d after infection. If too high a multiplicity of infection (MOI) is used, the input virus will result in a substantial amount of CPE shortly (i.e., within 24 h) after infection. As the CPE induced by this input virus will not be blocked by compounds that inhibibit new progeny virus formation, the use of a high MOI will mask a potential inhibitory effect of a given compound on HCMV replication. Scoring of CPE can be done either by counting the HCMV-induced plaques (although this is a rather labor-intensive method for large-scale screening purposes) or simply by scoring the CPE on a scale of 1-5 (or 10). Each assay should always be validated by using a reference compound with proven anti-HCMV activity (e.g., ganciclovir, cidofovir, foscarnet).

Herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV) and human cytomegalovirus (CMV) cause diseases that are usually self-limiting in the immunocompetent host. However, HSV-1, HSV-2, VZV, and CMV are major causes of morbidity and mortality in the immunocompromised patient. Prolonged antiviral treatment is often required for the clinical management of herpesvirus infections in the immunocompromised patient, and this favors the emergence of drug-resistant strains. The isolation of acyclovir-resistant (ACV(r)) HSV-1, HSV-2, and VZV strains as well as ganciclovir-resistant (GCV(r)) CMV strains has been reported with increasing frequency and is a major concern (1-3). Resistance to foscarnet (phosphonoformic acid [PFA]), the drug of choice when ACV or GCV fails, has also been described in the clinic (4,5). Furthermore, double resistance to both GCV and PFA (for CMV) and to both ACV and PFA (for HSV) has been observed in immuno-compromised patients after sequential and concomitant treatment with either or both drugs.

The high replication rate of HIV, together with the low fidelity of its reverse transcriptase, provides the virus with an unprecedented genomic flexibility. This allows a fast adaptation to selective pressure, including antiviral drugs, resulting in the development of drug-resistant strains. The present improvements in the treatment of AIDS patients are at least partly owing to antiviral therapy. To assess the implications of HIV drug resistance on patient management, drug resistance assays for clinical HIV isolates are widely being used. Ideally, monitoring drug resistance should help clinicians in their treatment decisions. If patients would really benefit clinically from this strategy, then the gain from clinical improvement and from omitting drugs to which the virus is already resistant would outweigh the cost of drug resistance testing. In the next few years, researchers should consolidate the clinical benefit of antiviral drug resistance testing, and for this they need fast, reliable and cheap assays. All present assays are in vitro assays, which can only partly mimic the in vivo situation with confounding factors, such as cellular resistance (1). Efforts are presently made to establish in vivo assays (2; see also Chapter 10 ).

Simulating coherent control with femtosecond pulses on a polyatomic molecule with anharmonic splitting was demonstrated. The simulation mimicked pulse shaping of a Spatial Light Modulator (SLM) and the interaction was described with the Von Neumann equation. A transform limited pulse with a fluence of 600 J/m 2 produced 18% of the population in an arbitrarily chosen upper vibrational state, n =2. Phase only and amplitude only shaped pulse produced optimum values of 60% and 40% respectively, of the population in the vibrational state, n=2, after interaction with the ultra short pulse. The combination of phase and amplitude shaping produced the best results, 80% of the population was in the targeted vibrational state, n=2, after interaction. These simulations were carried out with all the population initially in the ground vibrational level. It was found that even at room temperatures (300 Kelvin) that the population in the selected level is comparable with the case where all population is initially in the ground vibrational state. With a 10% noise added to the amplitude and phase masks, selective excitation of the targeted vibrational state is still possible.

A series of silicon-containing polyoxotungstates belonging to the ‘Keggin-type’ (‘Keggin’, ‘Keggin sandwich’) were evaluated for their antiviral activity against enveloped viruses (myxo-, herpes- and retroviruses). The compounds exhibited antiviral activity against influenza virus type A, respiratory syncytial virus (RSV), herpes simplex virus type-1 (HSV-1), type-2 (HSV-2), thymidine kinase-deficient (TIC) HSV-1, human cytomegalovirus (HCMV), human immunodeficiency virus type-1 (HIV-1) and type-2 (HIV-2) at concentrations that were well below their cytotoxic threshold. The ‘Keggin’ compound JM2815 (K5[Si-(TiCp)W11O39].12H2O) and the ‘Keggin sandwich’ compound JM1590 (K13[Ce(SiW11O39)2].26H2O) resulted in the highest selectivity indices against HIV-1 and HIV-2, and compound JM2820 ([Me3NH]8.[Si2Nb6W18O77]) was the most potent inhibitor of HSV and HCMV replication. These compounds proved active against HCMV and HSV when present during virus adsorption, and against influenza virus A and RSV when present after virus adsorption. Polyoxosilicotungstates inhibited the binding of radiolabeled HCMV particles to the cells at concentrations that were antivirally active, and the compounds were able to displace HCMV particles that were bound to a heparin-Sepharose matrix. Presumably, the polyoxosilicotungstates interact with positively charged domains on the viral envelope site(s) involved in the attachment of the (HCMV) virions to the cell surface receptor heparan sulphate.

Viruses causing hemorrhagic fevers in man belong to the following virus groups: togavirus (Chikungunya), flavivirus (dengue, yellow fever, Kyasanur Forest disease, Omsk hemorrhagic fever), arenavirus (Argentinian hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever), filovirus (Ebola, Marburg), phlebovirus (Rift Valley fever), nairovirus (Crimian-Congo hemorrhagic fever) and hantavirus (hemorrhagic fever with renal syndrome, nephropathic epidemia). Hemorrhagic fever virus infections can be approached by different therapeutic strategies: (i) vaccination; (ii) administration of high-titered antibodies; and (iii) treatment with antiviral drugs. Depending on the molecular target of their interaction, antiviral agents could be classified as follows: IMP dehydrogenase inhibitors (i.e., ribavirin and its derivatives); OMP decarboxylase inhibitors (i.e., pyrazofurin); CTP synthetase inhibitors (i.e., cyclopentylcytosine and cyclopentenylcytosine); SAH hydrolase inhibitors (i.e., neplanocin A); polyanionic substances (i.e., sulfated polymers); interferon and immunomodulators.

9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), a potent inhibitor of human immunodeficiency virus (HIV) replication, was evaluated for its activity against human cytomegalovirus (HCMV) in vitro, and murine cytomegalovirus (MCMV) and rat CMV (RCMV) in vivo. PMEDAP strongly inhibited HCMV-induced cytopathicity in human embryonic lung (HEL) cell cultures (EC50 11 microM) and caused a concentration-dependent suppression of viral DNA synthesis (IC50 20 microM) [corrected]. PMEDAP had no effect on the expression of HCMV-specific immediate early antigens (IEA) as measured on day 1 post-infection, but inhibited the expression of HCMV late antigens as measured on day 6 post-infection (EC50 20 microM) [corrected]. The diphosphate derivative of PMEDAP (PMEDAPpp) selectively inhibited HCMV-induced DNA polymerase (IC50 0.1 microM). PMEDAP proved markedly effective in reducing the mortality rate of NMRI mice that had been infected intraperitoneally or intracerebrally with a lethal dose of MCMV. PMEDAP exhibited greater anti-MCMV activity when administered as a single dose immediately after infection than when this dose was divided over repeated administrations. 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) also prevented MCMV-induced mortality, but only at a dose ten-fold higher than that of PMEDAP. PMEDAP also delayed death in severe combined immune deficiency (SCID) mice that had been infected with MCMV. The effect of PMEDAP on RCMV infections in rats was less pronounced.

The construction of an optically pumped Cs beam primary frequency standard was undertaken in the PTF laboratory. The main features of its design are described. Preliminary results and values of the major frequency shifts are then given. The measured short-term stability is reported. >

The efficacy of HPMPC and DHPG against systemic and intracerebral murine cytomegalovirus (MCMV) infections was examined in immunocompetent NMRI mice and in mice with severe combined immunodeficiency (SCID). HPMPC proved to be far superior to DHPG in preventing mortality and growth retardation in MCMV-infected NMRI mice even when given as a single dose on the day of infection. In intraperitoneally infected SCID mice, HPMPC administered as a single dose of 2, 10, 20 or 50 mg/kg per week increased the survival period of the mice by 22, 49, 77 and 156 days, respectively. In contrast, DHPG at daily doses of 10, 20 or 50 mg/kg for five consecutive days every week did not delay death by more than 13, 17 and 21 days, respectively. About one week before the MCMV-infected SCID mice (treated with either DHPG or HPMPC) died, they developed signs of neurological disease and intranuclear inclusion-bearing cells were found in their brains. The virus that was recovered from the brains of these mice did not prove to be resistant to HPMPC or DHPG. Only the virus recovered from the brains of mice treated with HPMPC at a dosage of 50 mg/kg/week had a slightly decreased susceptibility to HPMPC. When HPMPC (50 mg/kg for 4 consecutive days) was administered to SCID mice at the time when neurological symptoms became apparent, death of the animals could be delayed by another 35 to 40 days.

After they have been transfected with the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) thymidine kinase (TK) gene murine mammary carcinoma (FM3A) cells become highly sensitive to the growth inhibitory properties of the antiherpetic agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 9(-)[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV), 9(-)[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, ganciclovir), and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). BVDU was 100-fold more potent an inhibitor of HSV TK gene-transfected tumor cell growth (50% inhibitory concentration (IC50), 0.0020-0.0047 microM) than FMAU or DHPG (IC50, 0.051-0.277 microM) and 1000-fold more potent than ACV (IC50, 0.42-4.9 microM). As a rule, the test compounds were more cytostatic to HSV-2 TK than HSV-1 TK gene-transfected FM3A cells. This may be ascribed to the higher phosphorylating capacity (Vmax/Km) of HSV-2 TK than HSV-1 TK and/or to the higher TK enzyme levels of the HSV-2 TK gene-transfected FM3A cells than the HSV-1 TK gene-transfected FM3A cells. Thymidylate synthase of the HSV TK gene-transfected FM3A cells appears to be the target enzyme for the cytostatic action of BVDU, but not FMAU, DHPG, or ACV. Instead, the cytostatic activity of DHPG seems to be correlated with its conversion to the triphosphate form and subsequent incorporation into the DNA of HSV TK gene-transfected FM3A cells.

9-(2-Phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) is a broad-spectrum antiviral agent with potent activity against DNA viruses and retroviruses. We now demonstrate that PMEDAP is highly efficacious when given orally to mice infected with either Moloney murine sarcoma virus (MSV), Friend leukemia virus (FLV), or murine cytomegalovirus (MCMV). PMEDAP markedly delayed MSV-induced tumor initiation when administered orally at 50, 100, or 250 mg/kg/day during 5 subsequent days. At the highest dose (250 mg/kg/day), PMEDAP completely prevented tumor formation in the MSV-infected animals. PMEDAP also caused 84-96% inhibition of FLV-induced splenomegaly when given orally to FLV-infected mice at 50-250 mg/kg/day. These PMEDAP treatment regimens were also markedly effective in increasing the survival rate of MCMV-infected mice. Intraperitoneal PMEDAP achieved a comparable antiviral activity at 2- to 5-fold lower doses than oral PMEDAP. However, the therapeutic index (ratio of the toxic dose to the antivirally effective dose) of oral PMEDAP was substantially higher than that of intraperitoneal PMEDAP. Oral PMEDAP at doses of 100, 250, or 500 mg/kg resulted in plasma PMEDAP levels of 0.5-2.5 micrograms/ml, which were sustained for 3 or 6 hours after administration and may account for the high antiviral efficacy achieved.

The (S)- and (R)-enantiomers of acyclic purine nucleoside phosphonate analogs (i.e., 3-hydroxy-2-phosphonomethoxypropyl [HPMP] derivatives, 3-fluoro-2-phosphonomethoxypropyl [FPMP] derivatives, and 2-phosphonomethoxypropyl [PMP] derivatives of adenine [A], 2-aminopurine, 2,6-diaminopurine [DAP], and guanine [G]) have been synthesized and evaluated for antiviral activity. As a rule, the HPMP derivatives proved effective against DNA viruses but not RNA viruses or retroviruses. In particular, (S)-HPMPA, (S)-HPMPDAP, and (R)- and (S)-HPMPG were exquisitely inhibitory to herpes simplex virus type 1 (50% effective concentrations, 0.63, 0.22, 0.10, and 0.66 microM, respectively). The FPMP and PMP derivatives showed marked inhibitory activities against retroviruses but not DNA viruses. The (S)-enantiomer of FPMPA and the (R)-enantiomer of PMPA were approximately 30- to 100-fold more effective against human immunodeficiency virus and Moloney murine sarcoma virus (MSV) than their enantiomeric counterparts. In contrast, both (S)- and (R)-enantiomers of the DAP and G derivatives proved equally effective against retroviruses, except for (R)-PMPDAP, which was 15- to 40-fold more inhibitory than (S)-PMPDAP. (R)-PMPDAP emerged as the most potent and selective inhibitor of MSV-induced transformation of murine C3H/3T3 cells and human immunodeficiency virus-induced cytopathicity in MT-4 and CEM cells (50% effective concentration, approximately 0.1 to 0.6 microM). When administered intraperitoneally at a single dose as low as 2 mg/kg, (R)-PMPDAP efficiently decreased MSV-induced tumor formation in newborn NMRI mice and significantly increased the survival time of MSV-infected mice. In addition, upon oral administration to MSV-infected mice, (R)-PMPDAP showed marked antiretroviral efficacy.

Currently, only two drugs, i.e. ganciclovir, (DHPG, cytovene®)_and foscarent (PFA, Foscavir®), are licenced for the treatment of severe CMV infections. Both compounds may have serious side effects and can lead to the emergence of drug-resistant virus strains. Recent pertain to the efficacy of prophylactic or pre-emptive therapy with ganciclovir to prevent the development of CMV infections in transplant recipients. The search for safe and effective anti-CMV chemotherapeutics recently led to the development of some new promising anti-CMV compounds (HPMPC, cyclobutylguanine, benzimidazoles), of which HPMPC is currently undergoing clinical evaluation. Intravenous hyperimmune and normal gamma-globulin have been used prophylactically or therapeutically in the treatment of CMV disease. Some of the trials indicate that immunoglobulin prophylaxis might reduce the incidence or severity of CMV infections in transplant recipients. Some studies also claim prophylactic efficacy for acyclovir, whereas other conclude that acyclovir has little, if any, prophylactica activity in the prevention of CMV infections. The use of live attenuated or subunit CMV vaccines for the prevention of primary CMV infections in solid organ transplant recipients and preggnant women deserves further consideration. Another promising, recently reported, approach is the adoptive transfer of CMV-specific cytotoxic T-cells to the immuno suppressed host with the goal of conferring an adapted immune response.

Different types of anti-sticking coatings have been applied industrially on injection moulds for various types of plastics. Very often these tests are being done on a trial-and-error basis and results obtained are difficult to interpret. WTCM/CRIF has developed laboratory equipment where the injection moulding process can be simulated and demoulding forces and friction coefficients can be measured. These measurements were compared with surface energy calculations of the coated surfaces and of the plastic materials in order to find a correlation. Using this approach it must be possible to make an easy and cheap selection of promising coatings towards plastic injection moulding. Another important advantage is that the understanding and modelling of the mould–plastic interface becomes possible. This new way of coating selection for plastic injection moulding has been demonstrated for various PVD coatings and verified for different industrial injection moulding applications.

The synthesis of some 9-(2-cyclobutylethyl)guanine derivatives and analogous carbonucleosides from 1S-alpha-pinene is here presented. None of them showed detectable selectivity when assayed in the performed anti-viral tests.

Several cyclobutyladenine and analogous carbonucleosides were synthesized from 1R-alpha-pinene and their anti-viral activity was tested. One of them (3e) showed interesting selectivity against both TK+ and TK- VZV.

A series of 3'-deoxy-3'-fluoro- and 2'-azido-2',3'-dideoxy-3'-fluoro-D-ribofuranosides of natural heterocyclic bases have been synthesized with the use of universal carbohydrate precursors, viz., 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-fluoro-D-ribofuranose and methyl 2-azido-5-O-benzoyl-2,3-dideoxy-3-fluoro-beta-D-ribofuranoside, respectively. The cytostatic and antiviral activity of the compounds was evaluated against a variety of tumor cell lines and DNA/RNA viruses, respectively. As the most active compound, from both a cytostatic and antiviral activity viewpoint, emerged 3'-deoxy-3'-fluoroadenosine. It inhibited the proliferation of some tumor cell lines (i.e. murine leukemia L1210 and human T-lymphocyte MT-4) at a concentration of 0.2-2 micrograms/mL, and proved inhibitory to the replication of positive-stranded RNA viruses (i.e. polio, Coxsackie, Sindbis, Semliki forest), double-stranded RNA viruses (i.e. reo), and some DNA viruses (i.e. vaccinia) at a concentration of 1-4 micrograms/mL, which is well below the cytotoxicity threshold (40 micrograms/mL).

The synthesis of 5-heteroaryl-substituted 2'-deoxyuridines is described. The heteroaromatics were obtained from three different 5-substituted 2'-deoxyuridines. Cycloaddition reaction of nitrile oxides on the 5-ethynyl derivative 1 gave the isoxazoles 4a-e. The thiazole derivatives 14a-c were obtained from the 5-thiocarboxamide 11, while 5-pyrrol-1-yl-2'-deoxyuridine (17) could be synthesized directly from 5-amino-2'-deoxyuridine. The compounds were evaluated for antiviral activity. Selective activity against herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) was noted for 5-(3-bromoisoxazol-5-yl)-2'-deoxyuridine (4c). The compound was inactive against herpes simplex virus type 2, cytomegalovirus, and thymidine kinase (TK)-deficient mutants of HSV-1 and VZV, which indicates that, most likely, its antiviral activity depends on phosphorylation by the virus-specified TK.

2'-Deoxyuridines with a five-membered heterocyclic substituent in the 5-position were synthesized by palladium-catalyzed coupling reactions of 5-iodo-2'-deoxyuridine with the activated heteroaromatics. Further modification of the compound with the 5-thien-2-yl substituent gave 5-(5-bromothien-2-yl)-2'-deoxyuridine and 5-(5-chlorothienyl-2-yl)-2'-deoxyuridine. Both compounds show potent and selective activity against herpes simplex virus type 1 and varicella-zoster virus.