Antiviral acyclonucleoside analogues for use in treating a viral infection

Abstract

The present invention concerns a compound having the following formula (I): ##STR00001##
as well as its use as a medicament, especially for its use for treating viral infections.

Claims

1. A compound having the following formula (I′): ##STR00074## wherein: n is 0, 1 or 2; X.sub.1, X.sub.2, and X.sub.3 are, independently from each other, CH or N; R″ is selected from the group consisting of: H, NH.sub.2, and halogen atoms; R is selected from the group consisting of: —NR.sub.aR.sub.b groups, R.sub.a and R.sub.b being independently from each other H, a (C.sub.1-C.sub.6)alkyl group or a (C.sub.3-C.sub.6)cycloalkyl group; halogen atoms; and (C.sub.1-C.sub.6)alkoxy groups; R′ is a group of formula (1) ##STR00075## wherein R.sub.1 and R.sub.2 are independently from each other selected from the group consisting of: OH; (C.sub.1-C.sub.6)alkoxy groups; —O-A.sub.1-O-A.sub.2 groups; wherein A.sub.1 is an alkylene radical comprising from 1 to 6 carbon atoms, and A.sub.2 is a (C.sub.1-C.sub.20)alkyl group; —O-A.sub.3-O—C(═O)-A.sub.4, wherein A.sub.3 is an alkylene radical comprising from 1 to 6 carbon atoms, and A.sub.4 is a (C.sub.1-C.sub.6)alkyl group; and —O-A.sub.5-O—C(═O)—O—A.sub.6, wherein A.sub.5 is an alkylene radical comprising from 1 to 6 carbon atoms, and A.sub.6 is a (C.sub.1-C.sub.6)alkyl group; or R′ is a group of formula (2): ##STR00076## wherein: R.sub.4 is a (C.sub.6-C.sub.10)aryl group; R.sub.5 is a (C.sub.1-C.sub.6)alkyl group, R.sub.6 is selected from the group consisting of: (C.sub.1-C.sub.6)alkyl groups, and (C.sub.6-C.sub.10)aryl groups, R.sub.3 is selected from the group consisting of: H; (C.sub.1-C.sub.6)alkyl groups; -A.sub.7-OH, wherein A.sub.7 is an alkylene radical comprising from 1 to 6 carbon atoms; and -A.sub.8-O—C(═O)-A.sub.9, wherein A.sub.8 is an alkylene radical comprising from 1 to 6 carbon atoms, and A.sub.9 is a (C.sub.1-C.sub.6)alkyl group; or a pharmaceutically acceptable salt, racemate, diastereoisomer or enantiomer thereof.

2. The compound of claim 1, having one of the following formulae (II) or (II′): ##STR00077## or a pharmaceutically acceptable salt, racemate, diastereoisomer or enantiomer thereof.

3. The compound of claim 1, having one of the following formulae (III) or (III′): ##STR00078## or a pharmaceutically acceptable salt, racemate, diastereoisomer or enantiomer thereof.

4. The compound of claim 1, having the following formula (IV): ##STR00079## or a pharmaceutically acceptable salt, racemate, diastereoisomer or enantiomer thereof.

5. A medicament comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein R.sub.1 and R.sub.2, identical or different, are selected from the group consisting of: —O-A.sub.1-O-A.sub.2, —O-A.sub.3-O—C(═O)-A.sub.4, and —O-A.sub.5-O—C(═O)—O—A.sub.6 groups.

7. The compound of claim 1, wherein R.sub.1 and R.sub.2, identical or different, are selected from the group consisting of: —O—(CH.sub.2).sub.3—O—(CH.sub.2).sub.15—CH.sub.3, —O—CH.sub.2—O—C(═O)-tBu, and —O—CH.sub.2—O—C(═O)—O—iPr.

8. The compound of claim 1, wherein R is NH.sub.2.

9. The compound of claim 1, wherein R.sub.3 is H or —CH.sub.2OCOCH.sub.3.

10. The compound of claim 4, wherein R.sub.3 is H or —CH.sub.2OCOCH.sub.3.

11. The compound of claim 4, wherein R.sub.4 is a phenyl or naphthyl group.

12. A method for treating a viral infection, comprising administering a pharmaceutically acceptable amount of at least one compound of claim 1 to a patient in need thereof.

13. The method of claim 12, wherein the viral infection is an infection due to a DNA virus or an RNA virus.

14. The method of claim 12, wherein the viral infection is an infection due to a virus selected from the group consisting of Hepatitis B virus, Varicella-zoster virus, Cytomegalovirus, Adenovirus, Herpes virus, Poxvirus, Feline corona virus, Filovirus, Papovarirus, Parvovirus, Myxoma virus, and Hepadnavirus.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

(1) FIG. 1 is a graphic representation of the average of triplicate values compared to the average of the untreated samples, reported as virus yield percent.

(2) FIG. 2 is a graphic representation of the potency values for compounds in accordance with the claimed invention.

(3) FIG. 3 provides graphic representations of additional testing of compounds in accordance with the claimed invention.

(4) FIG. 4 is a graphic representation of weight change in mice undergoing treatment in accordance with the claimed invention.

(5) FIG. 5A is a graphic representations of the in vivo evaluation of compounds in accordance with the claimed invention.

(6) FIG. 5B is a graphic representations of the in vivo evaluation of compounds in accordance with the claimed invention.

(7) FIG. 6 provides graphic representations of the testing showing that the compounds in accordance with the claimed invention did not cause overt toxicity in test assays of mice.

(8) FIG. 7 is a graphic representation of additional testing of compounds in accordance with the claimed invention.

(9) FIG. 8 is a graphic representation of additional testing of compounds in accordance with the claimed invention.

(10) FIG. 9 is a graphic representation of additional testing of compounds in accordance with the claimed invention.

(11) FIG. 10 is a graphic representation of additional testing of compounds in accordance with the claimed invention.

(12) The in vivo evaluation of compound (5) was repeated to confirm its antiviral activity against VZV. In addition, a dose-ranging and delayed treatment assay was included to determine the lowest effective dose (FIG. 5Figures 5A and 5B). As before, 5 26 mg/kg was given once daily, by the subcutaneous route, from Days 0-6. This group was compared to Vehicle (Cremophor-DMSO-saline). In the dose ranging assay, 5 high dose (26 mg/kg), medium dose (13 mg/kg) or low dose (6.5 mg/kg) was given once daily, by the subcutaneous route, from Days 3-9. The treatment phase was delayed to Day 3 because this is more clinically relevant. Again, 5 was effective at 26 mg/kg given at the time of infection, and it prevented VZV spread in skin. Virus yield on Day 7, the day after treatment ended, was significantly less than the Vehicle group (p =0.008, Student's t test). The 26 mg/kg dose was also effective when treatment was delayed to Day 3. The medium and low doses of 5 were equally effective. Virus yield on Day 10, the day after treatment ended for these groups, was significantly less than the Vehicle group (p =0.029, 1-way ANOVA, Dunnett's post hoc test). These results indicate that 5 is highly potent against VZV in this mouse model, effectively preventing VZV spread at a low dose of 6.5 mg/kg.

(13) II—Results

(14) TABLE-US-00001 EC.sub.50 (μM).sup.a VZV HCMV TK+ TK− Cytotoxicity (μM) (AD-169) (Davis) (OKA) (07/1) MCC.sup.b CC.sub.50.sup.c Acyclovir 0.4 250 2.7 15.0 ± 1.5 >100 134 ± 10 Brivudine 0.08 250 0.01 117 >100 339 Cidofovir 1.2 1 nd nd nd nd Compound (1) 44.7 44.7 2.04 2.32 >100 5.1 Compound (2) 7.08 7.26 0.17 0.24 >100 14.49 Compound (3) 44.72 36.57 0.86 1.25 >100 99 Compound (4) 8.56 4.06 0.14 0.12 100 2.02 Compound (5) 1.79 0.70 0.0035 0.018 60 0.66 Compound (6) >20 49.70 0.089 0.22 >100 15.42 Compound (7) >20 8.94 >20 >20 100 1.29 Compound (8) >20 13.11 48.9 >20 100 5.01 Compound (9) >100 >100 76.5 >100 >100 ND Compound (10) >100 >100 34.2 24.46 100 ND Compound (11) >100 >100 51.5 49.3 >100 ND Compound (12) 44.72 >20 11.30 11.04 >100 54.93 .sup.aEffective concentration required to reduce virus plaque formation by 50%. .sup.bMinimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. .sup.cCytotoxic concentration required to reduce cell growth by 50%.

(15) Compounds were evaluated for anti-HCMV and anti-VZV activity. Among the synthesized compounds, products 1 to 6 exhibit EC.sub.50 in micromolar and nanomolar concentration activity. Compound 5 with the mixed prodrug (HDP/POC) is active against HCMV (Davis) with an EC.sub.50 of 0.7 μM and VZV (TK−) with an EC.sub.50 of 0.0035 μM and selective Index of 188.

(16) TABLE-US-00002 EC.sub.50 (μM).sup.a HSV-1 Feline Cytotoxicity HSV-1 HSV-2 (KOS ACV) Vaccinia Herpes (μM) (KOS) (G) TK− Virus Virus MCC.sup.b CC.sub.50.sup.c Brivudin 0.04 50 50 22 — >100 >100 Cidofovir 4.5 1.0 2.8 112 — >100 >100 Acyclovir 0.2 0.2 85 >100 — >100 >100 Ganciclovir 0.032 0.06 8.9 >100 1.4 >100 >100 Compound (1) 13.4 13.4 13.4 32.5 >100 >100 5.1 Compound (2) >100 >100 >100 >100 >100 >100 14.49 Compound (3) 3.75 1.85 5.75 >100 >100 >100 99 Compound (4) 0.6 1.1 0.6 2.9 >100 100 2.02 Compound (5) 0.043 ± 0.037 0.015 ± 0.05 0.008 ± 0.002 0.115 ± 0.11  0.35 60 0.66 Compound (6) 0.6 ± 0.2  0.35 ± 0.05 0.4 4.1 ± 2.4 >100 >100 15.42 Compound (7) >100 >100 >100 >100 >100 100 1.29 Compound (8) >100 >100 >100 >100 >100 100 5.01 Compound (9) >100 >100 >100 >100 >100 >100 ND Compound (10) >100 >100 >100 >100 >100 100 ND Compound (11) >100 >100 51.5 49.3 >100 >100 ND Compound (12) 39.5 39.5 42 >100 >100 >100 54.93 .sup.aEffective concentration required to reduce virus plaque formation by 50%. .sup.bMinimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. .sup.cCytotoxic concentration required to reduce cell growth by 50%.

(17) Synthesized compounds were evaluated for anti HSV-1, anti-HSV-2, anti-HSV-1 (TK.sup.−), anti-VV and anti-FHV. Among them numerous compounds (compounds 1, 3, 4, 5 and 6) possess micromolar and sub-micromolar activities against these viruses. Especially compound 5 which showed nanomolar activities (EC.sub.50<0.35 μM) against both tested viruses.

(18) TABLE-US-00003 EC.sub.50 (μM).sup.a Equine Equine Vaccinia Myxomatosis Herpes Herpes Virus Adeno Virus V 1 V 4 TK.sup.− virus (MYXV) Compound (5) <0.125 <0.125 <0.03 <0.037 0.051 .sup.aEffective concentration required to reduce virus plaque formation by 50%.

(19) Compound 5 is active against different families of veterinary viruses such as Equine herpes and Myxomatosis virus with an EC.sub.50 in nanomolar concentration. Against vaccicnia virus TK- and Adeno virus, compound 5 is also active with EC.sub.50 concentrations <30 Nm and <37 Nm respectively.

(20) In Vivo Evaluation

(21) Varicella zoster virus (VZV) causes the childhood disease varicella (chicken pox) and establishes lifelong latency in neurons. The virus may reactivate years later and manifest as herpes zoster (shingles). These infections are characterized by vesicular skin lesions. Antiviral therapies should prevent VZV spread in the skin and reduce virus shedding, so we developed human skin models to evaluate compounds for activity against VZV. We evaluated the compound 5, in three assays to evaluate its antiviral activity against VZV. We used a cell-based assay to determine that 5 is effective against a VZV strain that is resistant to acyclovir in the nanomolar range. 5 was effective, although less potent, in a human skin organ culture assay. The major question we addressed was whether 5 was effective in vivo. In fact, it is highly potent and prevents VZV spread in the mouse model.

(22) Methods

(23) Cells: Human retinal pigment epithelium cells were used to cultivate VZV. ARPE-19 cells (ATCC CRL 2302) were grown in Eagle minimum essential medium with Earle's salts and L-glutamine (HyClone Laboratories), supplemented with 10% heat-inactivated fetal bovine serum (Benchmark FBS; Gemini Bio Products), penicillin-streptomycin (5000 IU/mL), amphotericin B (250 Ig/mL), and nonessential amino acids (all Mediatech). Cells were incubated at 37° C. in humidified 5% CO.sub.2.

(24) Viruses: VZV-BAC-Luc (Zhang Z, Rowe J, Wang W, Sommer M, Arvin A, Moffat J, Zhu H. 2007. Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system. Journal of virology 81:9024-9033), derived from the Parental Oka (POka, Accession number: AB097933) strain was propagated in ARPE-19 cells. The VZV-BAC-Luc TK− strain (ACV.sup.R) was isolated by selection in increasing concentrations of acyclovir. Virus was passaged by diluting infected cells 1:100, transferring to uninfected cells, then incubating for 3-5 days. Stocks of VZV-infected cells were frozen in tissue culture medium with 10% DMSO and stored at −80° C. or in liquid nitrogen. Bioluminescence is measured in the IVIS-50 or IVIS-200 instruments, and it is proportional to viral load and pfu. To prepare the virus for inoculation into human skin in culture or in mice, VZV-infected ARPE-19 cells were trypsinized, then washed and resuspended in tissue culture medium, and used immediately for direct injection.

(25) Evaluation of 5 in ARPE-19 cells. ARPE-19 cells were seeded in 24-well plates and incubated overnight. The medium was removed and VZV-infected cells, either the wild type VZV-BAC-Luc or the ACV.sup.R isogenic mutant, were added in 0.5 mL and incubated for 2 h. 0.5 mL of medium containing either vehicle or 2-fold dilutions of 5, acyclovir, or cidofovir at concentrations between 0.00125 and 40.0 μM were added to triplicate samples, and then the plates were incubated for 3 days. VZV spread was measured by bioluminescence imaging and expressed as Total Flux (photons/sec/cm.sup.2/steradian). The triplicate values were averaged and compared to the untreated group to determine the extent of inhibition (in percent). The EC.sub.50 was estimated by interpolation.

(26) Evaluation of 5 in skin organ culture: The skin organ culture model is described in detail elsewhere (Taylor, S. L. and J. F. Moffat (2005). “Replication of varicella-zoster virus in human skin organ culture.” J Virol 79(17): 11501-115). Human fetal skin was purchased from ABR and delivered by overnight courier on wet ice. The tissue was cleaned, disinfected in betadine and 70% ethanol, then cut into pieces approximately 1-cm.sup.2. Each tissue was injected twice with 30 μL of the VZV-infected cell suspension (˜10.sup.3 pfu) using a 1 cc syringe fitted with a 27-gauge needle attached to a volumetric stepper (Tridak). The needle was lightly dragged across the tissue approximately 5 times to scarify the surface to increase infection. Tissues were incubated at 37° C. for 3 h to allow the virus to adhere, and then placed individually on NetWells (Corning) that had contact with 1.0 mL of tissue culture medium. The next day (Day 1) each piece of skin was soaked in D-luciferin for 45 min, and then scanned in the IVIS-50 instrument to measure Total Flux. The drug treatment began on Day 1 by placing the skin, in NetWells, over medium containing positive control drug cidofovir (5 μM), or 5 at concentrations ranging from 1 nM to 5 μM. Each group contained 6 pieces of skin. The medium and drug were refreshed daily until the final IVIS scan on Day 11. The antiviral effects of 5 were determined by assessing VZV yield compared to the vehicle group.

(27) SCID-Hu mouse model of VZV replication. CB.17 scid/beige male mice (Charles River) were implanted subcutaneously with human fetal skin (the same type of specimens used for the skin organ culture assay). After 3-4 weeks engraftment, the skin implants were inoculated with VZV-BAC-Luc or VZV-ORF57-Luc. The mice were divided into groups (N=10-13) for treatment or controls. The positive control antiviral drug was cidofovir, 10 mg/kg/day given by intraperitoneal injection. The negative control was the vehicle Cremophor-DMSO-saline (1:1:8) given by subcutaneous injection. The treatment groups were 5 in Cremophor-DMSO-saline, 26, 13, or 6.5 mg/kg/day, given by subcutaneous injection. Treatment began 2 hours or 3 days after the inoculation surgery and continued daily or alternating days for up to 9 days. The mice were scanned in the IVIS-200 instrument daily from Day 1-10. The mice were weighed before the start of the study and daily during the treatment phase. The antiviral effects of compound (5) were determined by assessing the VZV yield measured by Total Flux (photons/sec/cm.sup.2/steradian).

(28) Statistical analysis: The results were analyzed for statistical significance by Student's t test or by ANOVA and Dunnett's post hoc test of multiple comparisons (GraphPad Prism).

(29) Results

(30) The antiviral activity of compound (5) was evaluated in a cell-based assay using ARPE-19 cells, which are highly permissive for VZV replication, and the VZV-BAC-Luc strain (wild type). It was not known whether compound (5) would be active against a strain that was resistant to acyclovir due to a mutation in the thymidine kinase gene (ORF36). Thus we also included the ACV-resistant variant of VZV-BAC-Luc in this assay (ACV.sup.R). Two control antiviral compounds, acyclovir (ACV) and cidofovir (CDV), were tested in the assay because the ACV.sup.R strain is sensitive to CDV due to the monophosphate on the cytidine analog. The ARPE-19 cells were inoculated with wild type or ACV.sup.R virus, and then triplicate samples were treated with compound (5), ACV, or CDV in the appropriate concentration range. After 3 days, bioluminescence was measured in the IVIS-50 instrument and the Total Flux was recorded. The average of triplicate values was compared to the average of the untreated samples, and then reported as the virus yield in percent (FIG. 1). The results with the ACV.sup.R strain are open symbols and the results with the wild type strain are solid symbols. As expected, the ACV.sup.R virus was resistant to ACV and the wild type virus was sensitive to ACV, with an approximate EC.sub.50 of 2.5-5 μM. Cidofovir was effective against both virus strains, with an approximate EC.sub.50 of 2.5 μM. Compound (5) was also effective against both wild type and ACV.sup.R viruses with an approximate EC50 of 0.01 μM (10 nM). Notably, compound (5) was nearly 100-times more potent than these approved antiviral drugs.

(31) The highly potent activity of compound (5) warranted further evaluation in the skin organ culture model of VZV replication. This model employs full-thickness human skin that is maintained at the air-liquid interface above tissue culture medium. Small pieces of skin were inoculated with VZV-BAC-Luc by scarification, approximately 6000 pfu/piece, and then incubated overnight. On Day 1, the bioluminescence was measured in the IVIS-50 instrument to determine the initial level of VZV infection. Compound (5) was added to the medium in a range of concentrations from 1 nM to 5 μM. The positive control drug was cidofovir 5 μM. The Vehicle was 0.05% DMSO in tissue culture medium. Each group contained 4-6 pieces. The medium was changed each day to refresh the drugs, and the bioluminescence was measured by IVIS on Days 5, 7, 8, and 11. The values for each piece of skin are shown and the bar is the mean (FIG. 2). As expected, CDV 5 μM was effective after 11 days. 5 was effective at 1 and 5 μM, and this was significant compared to the vehicle (*, p<0.0001, 1-way ANOVA, Dunnett's post hoc test). The differences between CDV and the two highest concentrations of compound (5) were not significant. The skin organ culture assay provided valuable information about the potency of 5 in skin, which is a relevant tissue for VZV disease. Higher concentrations of 5 were required in skin than in cultured cells. We used this information to design the first evaluation of 5 in vivo.

(32) Compound (5) was evaluated in the SCID-Hu mouse model of VZV replication (Rowe, J., R. J. Greenblatt, D. Liu and J. F. Moffat (2010). “Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice.” Antiviral Res 86(3): 276-285). CB.17 scid/beige male mice were implanted subcutaneously with human fetal skin (the same type of specimens used for the skin organ culture assay). After 3-5 weeks engraftment period, the skin implants were inoculated with VZV-BAC-Luc. The mice were divided into 3 groups of 10-13 mice: vehicle (Cremophor-DMSO-saline 1:1:8), cidofovir 10 mg/kg in saline, and 5 26 mg/kg in Cremophor-DMSO-saline. 5 at 26 mg/kg is the molar equivalent dose to cidofovir 10 mg/kg. Cidofovir was given by the intraperitoneal route, once daily, from Days 0-8. Vehicle and 5 were given by the subcutaneous route, once daily, from Days 0-8. The mice were scanned in the IVIS-200 instrument daily from Day 1-9 and the Total Flux values indicated VZV-infected cells in the skin xenografts (FIG. 3). The left panel of FIG. 3 shows the average Total Flux values for each group, with error bars omitted for clarity. VZV grew normally in the Vehicle group. Both cidofovir and 5 prevented VZV spread during the treatment phase. Virus yield was compared on Day 9, and the absolute Total Flux values were significantly different between the Vehicle and drug treatment groups (* p=0.0051, 1-way ANOVA, Dunnett's post hoc test).

(33) This was the first evaluation of 5 in vivo and it was not known whether the compound was overtly toxic to mice. The mice were weighed before the start of the study and daily during the treatment phase. Their change in weight during the treatment phase was compared to the initial weight and the average for the group is shown in FIG. 4 (error bars omitted for clarity). As expected, all mice lost weight after the inoculation surgery. Cidofovir caused moderate weight loss, which is typical of this nephrotoxic drug. Compound (5) did not cause weight loss and was indistinguishable from the Vehicle group. The mice appeared healthy and there were no signs of distress, such as ruffled fur, diarrhea, or hunched posture.

(34) The in vivo evaluation of compound (5) was repeated to confirm its antiviral activity against VZV. In addition, a dose-ranging and delayed treatment assay was included to determine the lowest effective dose (FIGS. 5A and 5B). As before, 5 26 mg/kg was given once daily, by the subcutaneous route, from Days 0-6. This group was compared to Vehicle (Cremophor-DMSO-saline). In the dose ranging assay, 5 high dose (26 mg/kg), medium dose (13 mg/kg) or low dose (6.5 mg/kg) was given once daily, by the subcutaneous route, from Days 3-9. The treatment phase was delayed to Day 3 because this is more clinically relevant. Again, 5 was effective at 26 mg/kg given at the time of infection, and it prevented VZV spread in skin. Virus yield on Day 7, the day after treatment ended, was significantly less than the Vehicle group (p=0.008, Student's t test). The 26 mg/kg dose was also effective when treatment was delayed to Day 3. The medium and low doses of 5 were equally effective. Virus yield on Day 10, the day after treatment ended for these groups, was significantly less than the Vehicle group (p=0.029, 1-way ANOVA, Dunnett's post hoc test). These results indicate that 5 is highly potent against VZV in this mouse model, effectively preventing VZV spread at a low dose of 6.5 mg/kg.

(35) The weight and condition of the mice was monitored in this assay, and compound (5) did not cause overt toxicity (FIG. 6). As before, the mice did not lose weight and they did not show signs of distress.

(36) Compound (5) is highly active against VZV. It is approximately 100 times more potent than the commercially available drugs acyclovir and cidofovir in cultured cells. This may be due increased penetration through the cell membrane mediated by the hydrophobic moiety on the molecule. It may also be due to the uracil nucleotide analog, which is also highly potent in the drug brivudin that lacks a hydrophobic extension. Notably, Compound (5) is active against a VZV strain that lacks thymidine kinase activity, rendering it effective against viruses that acquire resistance to acyclovir and its derivatives by mutation in the TK gene.

(37) Compound (5) is effective against VZV in the skin organ culture model, but less potent than in cultured cells. Compound (5) prevents VZV spread in skin at 1 μM and higher, which is approximately 10 times greater than the amount needed to fully inhibit VZV in cells. A possible explanation is that the number of cells is greater in the skin explants, which are at least 200 cubic millimeters, than in the cell cultures. The skin also contains adipose cells that may absorb 5 based on its hydrophobic properties. Thus more molecules of 5 may be needed to reach the VZV-infected dermal fibroblasts and the epidermal keratinocytes.

(38) Compound (5) is effective against VZV in the SCID-Hu mouse model of VZV replication. The highest dose of 5 tested, 26 mg/kg/day, the molar equivalent of cidofovir 10 mg/kg/day, prevented VZV spread when given at the time of virus infection, Day 0, and three days after infection. Lower doses, 13 mg/kg/day and 6.5 mg/kg/day, were also effective given three days after infection. Compound (5) is superior to cidofovir because it is more potent and it does not cause weight loss.

(39) In summary, Compound (5) is effective in vivo and is and well-tolerated.

(40) III—Anti-HBV Activity

(41) Protocol

(42) Phenotypic drug resistance testing was performed in hepatocarcinoma cell line Huh7 in presence of a gradient of concentration of drugs. Drugs stock solution were prepared at 4.Math.10.sup.7 nM in DMSO, then aliquoted and stored at −20° C. Huh7 cell line was grown in DMEM medium (ThermoFischer Scientific) supplemented with 10% foetal calf serum (ThermoFischer Scientific), at 37° C. with 5% CO2. At day 0, Huh7 cells were seeded at density of 10 500 cells per well of a 96 wells plate. 100 ng/well of pHBV1.1× were transfected in these cells using Fugene 6 (Promega), according to the manufacturer's protocol. Drugs were added to the cells at a final concentration ranging from 32 to 100 000 nM. All experiments included two negative controls (pCIHBVΔRT and untransfected cells) and one positive control (pGFP encoding green fluorescent protein). All drug concentrations were tested in triplicates. Further steps were performed in level 3 biosafety laboratory, until cell lysis. At day 1 and 4, cell culture medium was removed and replaced with fresh medium and drugs. At day 7, medium was removed and cells were washed twice with PBS. Cellular and viral membranes were lysed with one freeze-thaw cycle at −80° C. and 37° C., followed by a 5 min incubation in IGEPAL® CA-630 1% (Sigma Aldrich). Lysate was clarified by centrifugation at 600 g for 5 min. 10 μL of supernatant were treated with 2U of RQ1 DNase (Promega) for 3 h at 37° C. Intra-capsid viral DNA was extracted with 30 μL of Quickextract (Epibio), according to manufacter's protocol. HBV and AMP DNA were quantified on this extract by duplex real-time PCR using 900 nM primers and 450 nM probes, 5 μL of DNA extract and 12.5 μL TaqMan Universal PCR mastermix II without UNG (ThermoFischer Scientific). Cycling reactions were performed on LightCycler 480 (Roche) with the following cycle parameters: 10 min at 95° C., followed by 45 cycles of 15 s at 95° C., 30 s at 60° C., 30 s at 72° C. Inhibitory concentrations 50% (IC50) were determined by non-linear regression using GraphPad Prism v5.0. Best fit values were obtained by least square regression with two constraints, bottom and top of the curve equal to 0 and 100%, respectively.

(43) Results

(44) TABLE-US-00004 IC50 (nM) Compound 5   400 Compound 13 1 600

(45) The results are also shown in FIGS. 7 to 10.

(46) FIGS. 7 and 8 concern compound 5 and compound 13, respectively.

(47) FIG. 9 concerns the HBV replication of compounds 5 and 13.

(48) FIG. 10 concerns the cytotoxicity of compounds 5 and 13.