Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

Synthesis and Antiviral Activity Assay of Novel (E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine

Abstract

(E)-3′,5′-diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Different Mechanisms of Inhibition of DNA Synthesis by (E)-5-(2-Bromovinyl)-2′-deoxyuridine in Cells Transfected with Gene for Thymidine Kinase of Herpes Simplex Virus Type 1 and in Cells Infected with the Virus

Abstract

The effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) on deoxyribonucleoside 5′-triphosphate pools was studied in cells transfected with gene for thymidine kinase of herpes simplex virus type 1 and cells infected with the virus. When infected cells were treated with BVDU, the triphosphate form of the nucleoside analog was detected. When transfected cells were treated with BVDU, the triphosphate form was not detected and the pattern of changes in the pools was the same as after 5-fluoro-2′-deoxyuridine treatment. BVDU seems to inhibit DNA synthesis differently in the two cell lines and nucleotide metabolism in the transfected cells was not the same as in the infected cells.     

Evaluation of Antiherpetic Compounds Using a Gastric Cancer Cell Line: Pronounced Activity of BVDU against Herpes Simplex Virus Replication

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV-induced cytopathogenicity in gastric adenocarcinoma MKN-28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV-1 and HSV-2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC₅₀) values of the antiherpes agents in MKN-28 cells were compared with those in human embryo lung MRC-5 cells, all compounds, except for BVDU, showed higher EC₅₀ values in MKN-28 cells. For BVDU the EC₅₀ values in MKN-28 cells were 0.8 (HSV-1) and 0.036 (HSV-2) times the EC₅₀ values in MRC-5 cells. Thus BVDU was 27.5 times more active against HSV-2 in MKN-28 cells than in MRC-5 cells. The MKN-28 gastric cancer cells may be useful for the rapid screening of anti-HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.     

Effect of 2-Amino Substitution on the Antiviral Effects of 5-Ethyl-2′-Deoxyuridine and (E)-5-(2-bromovinyl)-2′-Deoxyuridine and Their Incorporation into DNA

Abstract

The 2-amino derivatives of 5-ethyl-2′-deoxyuridine (EDU) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) have been synthesized and evaluated for anti-herpesvirus activity. They were at least 1000-fold less effective against herpes simplex virus replication than the parent compounds EDU and BVDU. The 5′-triphosphates of the 2-amino substituted EDU, BVDU and thymidine derivatives were also synthesized and examined on their substrate/inhibitor properties against different DNA polymerases. None of the compounds proved markedly inhibitory to HSV-1 DNA polymerase or cellular DNA polymerase a. Nor were they incorporated into the growing DNA chain.     

Synthesis,Protonation Behavior,Conformational Analysis,and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU)

Abstract

(E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through ¹H NMR spectroscopy. In addition, a convenient synthesis of N-3′ and N-5′ acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5′-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3′-amino group, thus affording derivatives 9.     

Synthesis and Biological Properties of (+) and (-)-(E)-5-(2-bronovinyl)-21 -deoxy-11 a-Carbauridine

Abstract

Carbocyclic (+)- and (-)-(E)-5- (2-bromovinyl)-2′-deoxyuridlne have been prepared from (+)- and (-)-endo-norborn-5-en-2-y1 butyrate. In cell cultures both (+)- and (-)-C-BVDU showed activity against herpes simplex virus types 1 and 2, (+)-C-BVDU being only slightly less active than BVDU itself. (-)-C-BVDU gave a smaller but still significant antiviral effect. A nomenclature for carbocyclic nucleosides is proposed.     

Combination of Azidothymidine (AZT) and (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU) Inhibits the Replication of Herpes Simplex Virus Type 1 (HSV-1) and Type 2 (HSV-2) and Varicella Zoster Virus (VZV) Strains That Are Deficient in the Expression of the Viral Thymidine Kinase (tk)

Abstract

Combinations of high concentrations of AZT with BVDU, acyclovir (ACV) or ganciclovir (GCV) show antagonism against TK⁺ HSV-1, but not TK⁺ VZV strains, in cell cultures. When BVDU and AZT were used in combination against TK^? HSV-1, TK^? HSV-2 and TK^? VZV strains, a pronounced inhibition of viral replication was observed. This potentiating effect was not seen if AZT was combined with ACV or GCV.     

Synthesis of (Z)-(1-Fluoro-2-hydroxymethyl-cyclopropylmethyl)purines

Abstract

(Z)-(1-fluoro-2-hydroxymethylcyclopropylmethyl)purines were designed, synthesized and evaluated their antiviral activity against poliovirus, HSV, and HIV.     

Synthesis of [1′-Fluoro-2′,2′-bis-(hydroxymethyl)-cyclopropylmethyl]purines as Antiviral Agents

Abstract

[1′-fluoro-2′,2′-bis-(hydroxymethyl)cyclopropylmethyl]purines were designed, synthesized and their antiviral activity against poliovirus, HSV and HIV was evaluated.     

Synthesis of Hydantoin Nucleosides with Naphthylmethylene Substituents in the 5-Position

Abstract

(Z)-5-(Naphthylmethylene)-2-thiohydantoin derivatives (3a,b,12a-d) were prepared directly fiom condensations of 2-thiohydantoin derivatives (1,l la,b) with naphthaldehydes. Bisglycosylation took place on reaction of (Z)-5-(naphthylmethylene)- 2-thiohydantoin derivatives (3a,b) with glycosyl halides (4a,b) under alkaline conditions. The bisglycosilated hydantoins produced N₃ glycosylated hydantoins on treatment with ammonia in methanol. (Z)-5-(2-Naphthylmethylene)-2-(benzylidene E-hydrazono)hydantoin (9a) and (Z)-5-(2-naphthylmethylene)-2-(polyhydroxyalkylidene E-hydrazono)hydantoins (9b,c) were prepared fiom the reaction of (Z)-5-(2-naphthyylmethylene)-2- methylmercaptohydantoin (7) with benzylidene E-hydrazone (8a) and monosaccharide E-hydrazones (8b,c). S-Glycosylation also took place when N₃ substituted hydantoins were reacted. The hydantoin nucleosides were tested for their potential activity against HTV and HSV.     

3′-(N-Hydroxyimino)-2′,3-dideoxynucleosides and Their Derivatives: Synthesis,Broad Spectrum Antiviral Properties and Synthetical Application for the Preparation of Other Nucleoside Analogues

Abstract

A series of 3′-(N-hydroxyimino)-2′,3′-dideoxynucleosides bearing different nucleic bases has been prepared. In vitro antiviral activity studies showed that among these compounds the thymine derivative possesses significant activity against HIV, HSV, EBV and HBV. Conveniently 5′-protected 3′-(N-hydroxyimino)-2′,3′-dideoxythymidine was further used as a synthon for the preparation of other nucleoside analogues.     

Synthesis and Molecular Modeling of Novel HSV1 Uracil-DNA Glycosylase Inhibitors

Abstract

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported ¹. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved². Starting with the optimized model of binding between 6-(4′-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.     

Relationship Between Conformation and Antiviral Activity -IV. 5-Ethyl-2′-Deoxyurldine and 5-Ethyl-2′-Deoxycytidine +

Abstract

5-Ethyl-2′-deoxyuridine(EtdUrd), though a potent inhibitor of herpes simplex virus (HSV) replication, is rapidly catabolized to produce an inactive pyrimidine base by thymidine and/or uridine phosphorylases. 5-Ethyl-2′-deoxycytidine (EtdCyd) was synthesized to confer metabolic stability and thus improve efficacy against systemic HSV infections. EtdCyd was inactive against HSV in the presence or absence of deaminase inhibitors in VERO cells up to 2 mM. The relationship between molecular conformation and antiherpes activity for EtdUrd and EtdCyd is discussed.

     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Synthesis and Antiviral Activities of New Pyrazolo[4,3‐c]quinolin‐3‐ones and Their Ribonucleoside Derivatives

Several new pyrazolo[4,3‐c]quinolin‐3‐one ribonucleosides (5a–g) and their corresponding heterocycle moieties (3a–g) were synthesized and evaluated against vaccinia virus (VV) and herpes simplex virus type 1 (HSV‐1). The derivatives 3c and 3d showed modest inhibitory activity against vaccinia virus reaching 70% at a concentration of 100 µM. All heterocyclic compounds (3a–f) showed a modest inhibition against HSV‐1, reaching the maximal inhibitory effect around 20–30%. The antiviral effects of most of the pyrazolo[4,3‐c]quinolin‐3‐one ribonucleosides (5a–f) on VV and HSV were not impressive.     

Synthesis, 18F‐Radiolabelling and Biological Evaluations of C‐6 Alkylated Pyrimidine Nucleoside Analogues

Synthesis of pyrimidine derivatives with a side‐chain attached to the C‐6 of pyrimidine ring (6–14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV‐1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 µM) exhibited better binding affinity for HSV‐1 TK than acyclovir (ACV, 170 µM) and ganciclovir (GCV, 48 µM). Catalytic turnover constant (k _cat) of 12 (0.08 s^? 1) was close to the k _cat values of ACV (0.10 s^? 1) and GCV (0.10 s^? 1). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV‐1 TK‐transduced and non‐transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV‐1 TK could be developed as non‐toxic PET‐tracer molecule. Thus, ¹⁸F labelling of the precursor 14 was performed by nucleophilic substitution using [¹⁸F] tetrabutylammonium fluoride as the fluorinating reagent.     

Chemical and Enzymatic Synthesis and Antiviral Properties of 2′-Deoxy-2′-fluoroguanosine

Abstract

Chemical and enzymatic methods were employed for the synthesis of the title compound, 2′F-Guo 7. High antiviral activity of 2′F-Guo was established in chick embryo cells infected with influenza virus FPV/Rostock/34 (H7N1) and herpes simplex virus (HSV) type I (1C strain).     

Synthesis and Biological Evaluation of 3-Deazacytidine and 3-Deazauridine Derivatives

Abstract

The synthesis of new 5-and 6- subsituted 3-deazapyrimidine ribonucleosides has been performed by reacting the silylated bases and 1-0-acetyl-2, 3, 5-tri-O-benzoyl-β-D-ribofuranose in dichloroethane in the presence of stannic chloride.

None of these compounds exhibited any antiviral activity in vitro against HSV1 and RV31.     

Mechanism of Antiviral Activity of 5-Ethyl-2′-Deoxyuridine

Abstract

5-Ethyl-2′-deoxyurldine (EDU) is phosphorylated to a much greater extent by herpes simplex virus (HSV)-infected Vero cells than by mock-infected cells. Within the infected cells, EDU is preferentially incorporated into viral DNA and more inhibitory to viral than cellular DNA synthesis