Guy Tourigny

Anti-Herpes Virus Activity of 5-Methoxymethyl-2′-Deoxycytidine in Combination with Deaminase Inhibitors

5-Methoxymethyl-2′-deoxycytidine (MMdCyd) is an anti-metabolite with selective anti-herpes activity and low cytotoxicity. MMdCyd is dependent upon initial activation by the viral-induced deoxythymidine-deoxycytidine (dThd/dCyd) kinase for its activity against herpes simplex virus (HSV). Antiviral activity of MMdCyd is cell-dependent and is influenced by the deaminase content of the cell line used for assays. The antiviral potency against HSV-1 in this study was higher in RK-13 cells (ED50 3–5 μm) than in Vero and HEP-2 cells (ED50 14–26 μm). The potency of MMdCyd increased approximately 20-fold against HSV-1 and twofold against HSV-2 in the presence of tetrahydrodeoxyuridine (H4dUrd; which inhibits both dCyd deaminase and dCMP deaminase) in Vero cells. MdCyd in combination with H4dUrd was effective in preventing the cytopathogenic effect of HSV-1 and decreasing the production of infectious virus particles. The IC99 (concentration required to reduce the yield of infectious virus obtained 72 h after infection by 99% relative to control cultures) was 1.6 μm. In combination with tetrahydrouridine (H4Urd; an inhibitor of Cyd/dCyd deaminase) the potency of MMdCyd was only slightly enhanced (ED50 7–8 μm). Dihydrodeoxyuridine and deoxyuridine reversed the antiviral activity of MMdCyd. The minimum cytotoxic concentration for rapidly dividing cells (RK-13, HEP-2 and Vero) for MMdCyd was greater than 3 mm. H4Urd and H4dUrd were devoid of cytotoxicity and antiviral activity up to 2.12 mm (the highest concentration tested). Diacetyl-MMdCyd (pro-drug form) was approximatewly 20 times less potent than MMdCyd.

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g+ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.

Mechanism of selective inhibition of herpes simplex virus replication by deoxycytidine analogues : interaction of 5-methoxymethyl-2'-deoxycytidine-5'-triphosphate with DNA polymerases

5-Methoxymethyl-2′-deoxycytidine (MMdCyd) is a selective anti-herpes agent that is dependent upon initial activation by herpes simplex virus (HSV)-induced deoxythymidine/deoxycytidine kinase. 5-Methoxymethyl-2′-deoxycytidine triphosphate (MMdCTP) was synthesized. The nature of the interaction of MMdCTP and dCTP with the DNA polymerase of Escherichia coli, HSV-1 and human DNA polymerase α was determined using specific and optimized assay conditions for each enzyme. MMdCTP was a better substrate for HSV-1 DNA polymerase compared to dCTP. At a nucleotide concentration of 10 μm MMdCTP utilization was 130% that of an equimolar concentration of dCTP. Under similar conditions, human DNA polymerase α utilized MMdCTP about as efficiently as dCTP. E. coli DNA polymerase I preferentially utilized dCTP. The IC50 values of MMdCTP were 8 × 10−7 m and 29 × 10−7 m for HSV-1 and human α DNA polymerase, respectively. MMdCTP is a competitive inhibitor of HSV-1 DNA polymerase with respect to dCTP incorporation (Ki = 3.8 × 10−7 m). Preferential utilization of MMdCTP and its eventual incorporation into HSV DNA seems to account for the antiviral action of MMdCyd.

Structure and Conformation of the Antiviral Agent 5-Methoxymethyl-2′-deoxyuridine

Abstract The three dimensional structure of the activiral agent, 5-methoxymethyl-2′-deoxyuridine (MmdUrd) was determined by x-ray diffraction methods. MMdUrd crystallized in space group P212121 of the orthorhombic system with a = 9.166(1)A, b, = 25.348(1)Amm c = 5.270(1)A and Z = 4. The conformation of the glycosyl bond is anti (χ = 233.30), the deoxyribose ring has the C(2′)-endo envelope conformation (2E), the CH2OH side chain has the g+ conformation and the methoxy group at the C(5) position is on the same side of pyrimidine plane as the 0(4′) oxygen. NMR spectroscopy was used to determine the conformation in solution. The spectra indicate that the sugar ring exists in a 60:40 equilibrium of the S- and N-states. The population of the three rotamers about the exocyclic c(4′)–C(5′) bond were estimated to be g+:t:g::61%:31%:8%. The correlaiton of molecular conforation with antiviral activity is discussed.

Relationship between conformation and antiviral activity-II. 5-Methoxymethyl-2'-deoxycytidine and 5-methoxymethyl-N4-methyl-2'-deoxycytidine

5-methoxymethyl-N4-methyl-2′-deoxycytidine (N4-Me-MMdCyd) and 5-methoxymethyl-N4-methyl-2′-deoxycytidine-5′-monophosphate (N4-Me-MMdCMP) were synthesized to confer resistance to deamination by deaminating enzymes. N4-Me-MMdCyd and N4-Me-MMdCMP were inactive against Herpes simplex virus type 1 (HSV-1) and also nontoxic to VERO cells up to 1796 μM (highest concentration tested). 5-methoxymethyl-2′-deoxycytidine-5′-monophosphate (MMdCMP) was more potent than the nucleoside against HSV-1 in VERO cells. In HSV-infected VERO cells (10 PFU/cell), N4-Me-MMdCyd caused only slight perturbations of deoxyribonucleoside triphosphate pools. 5-methoxymethyl-N4-methyl-2′-deoxycytidine-5′-triphosphate (N4-Me-MMdCTP) was synthesized and the nature of interaction of N4-Me-MMdCTP and dCTP with DNA polymerase of Escherichia coli, HSV-1 and human α was investigated. N4-Me-MMdCTP was neither an effective substrate nor a strong inhibitor of Escherichia coli, HSV-1 or human α DNA polymerase. The relationship between molecular conformation and antiviral activity for MMdCyd and N4-Me-MMdCyd is discussed. The conformation of the deoxyribofuranose ring in MMdCyd and N4-Me-MMdCyd are different. In N4-Me-MMdCyd, the exocyciic C(5′) side chain has the t conformation whereas MMdCyd has the g+ rotomer conformation. The orientation of the N4-methyl group may also impede binding to the HSV-induced kinase by steric hindrance and/or by hindering hydrogen bonding between the enzyme and the lone pair of electrons at N(3). The results suggest that attempts to render resistance to deamination by alkylation at the N(4) position of the cytosine moiety is not likely to yield compounds with activity against HSV.

Synthesis, molecular conformation and activity against herpes simplex virus of (E)-5-(2-bromovinyl)-2'-deoxycytidine analogs.

Analogs of (E)-5-(2-bromovinyl)-2 ′-deoxycytidine (BrVdCyd) (1) by substitution at N4 were synthesized to impart resistance against deamination. The anti-HSV-1 activity and solution conformation of these analogs were determined. N4-Acetyl-BrVdCyd (2) was a potent inhibitor of HSV-1 replication whereas N4-propanoyl-BrVdCyd (3) had good activity and N4-Butanoyl-BrVdCyd (4) had only low activity against HSV-1 replication. N4-Methyl-BrVdCyd (5) was devoid of activity against HSV-1.