JAC Advance Access published online on October 22, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm376
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Preclinical development of bicyclic nucleoside analogues as potent and selective inhibitors of varicella zoster virus
1 Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK 2 Instituto de Quimica Medica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain 3 FermaVir Pharmaceuticals, Inc., 420 Lexington Avenue, Suite 445, New York, NY 10170, USA 4 Morphology and Molecular Pathology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium 5 Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
* Corresponding author. Tel/Fax: +44-029-2087-4537; E-mail: mcguigan{at}cardiff.ac.uk
Received 20 June 2007; returned 27 July 2007; revised 3 September 2007; accepted 4 September 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Objectives: To progress the anti-varicella-zoster-virus (VZV) aryl bicyclic nucleoside analogues (BCNAs) to the point of Phase 1 clinical trial for herpes zoster.
Methods: A new chromatography-free synthetic access to the lead anti-VZV aryl BCNAs is reported. The anti-VZV activity of lead Cf1743 was evaluated in monolayer cell cultures and organotypic epithelial raft cultures of primary human keratinocytes. Oral dosing in rodents and preliminary pharmacokinetics assessment was made, followed by an exploration of alternative formulations and the preparation of pro-drugs. We also studied uptake into cells of both parent drug and pro-drug using fluorescent microscopy and biological assays.
Results: Cf1743 proved to be significantly more potent than all reference anti-VZV compounds as measured either by inhibition of infectious virus particles and/or by viral DNA load. However, the very low water solubility of this compound gave poor oral bioavailability (
14%). A Captisol® admixture and the 5'-monophosphate pro-drug of Cf1743 greatly boosted water solubility but did not significantly improve oral bioavailability. The most promising pro-drug to emerge was the HCl salt of the 5'-valyl ester, designated as FV-100. Its uptake into cells studied using fluorescent microscopy and biological assays indicated that the compound is taken up by the cells after a short period of incubation and limited exposure to drug in vivo may have beneficial effects.
Conclusions: On the basis of its favourable antiviral and pharmacokinetic properties, FV-100 is now being pursued as the clinical BCNA candidate for the treatment of VZV shingles.
Key Words: antiviral , VZV , BCNAs , herpes
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
In 1999 we observed a significant and selective anti-varicella-zoster-virus (VZV) action by some unusual bicyclic nucleoside analogues (BCNAs) 1 (Figure 1).1 The early drug leads had a long alkyl side chain on the bicyclic base part, with an optimum length of
C8–C10. The in vitro potencies of these compounds against VZV were 300-fold more potent than the clinically established antiherpetic agent aciclovir (2).1 We subsequently noted that replacement of the alkyl chain by a p-alkylphenyl unit produced a significant boost in potency against VZV.2 The most potent analogue was the p-pentylphenyl BCNA analogue Cf1743 (3), which exhibited activity against a broad range of VZV isolates at subnanomolar concentrations, with cytotoxic concentrations exceeding 200 µM. Thus, 3 is exquisitely more potent than aciclovir against VZV and exhibits an extremely high therapeutic index, measured as the ratio of cell growth inhibition over antiviral activity. Moreover, unlike aciclovir, which is a broad-spectrum antiherpetic agent, the BCNAs 1 and 3 are highly specific for VZV with no significant activity against any other virus,1,2 including other members of the herpes virus family and the closely related simian varicella virus.3 A broad variety of analogues of 1 and 3 have been synthesized and we have investigated the structure–activity relationships and pharmacology of the BCNAs.4–6 At the conclusion of these studies the pentylphenyl analogue (3) was identified as a potential clinical candidate and we embarked on a development programme in support of this. We herein report on scale-up studies for the synthesis of 3, in particular, attempts to achieve a chromatography-free purification method. However, preliminary pharmacokinetics (PK) on 3 indicated a need for further enhancement in terms of AUC. Formulation-based approaches were successful in improving the solubility of 3 but did not have a significant impact on oral PK. Thus, we turned to pro-drug methods and now report the success of a valine ester approach leading to the clinical candidate FV-100. Salt formation from this candidate and solid phase stability studies are also reported, besides fluorescent cell microscopy studies on the pro-drug and the parent compound.
|
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Thin-layer chromatography
Pre-coated, aluminium backed plates (60 F-54, 0.2 mm thickness; supplied by E. Merck AG, Darmstad, Germany) were used and were developed by ascending method. After solvent evaporation, compounds were detecting by quenching of the fluorescence, at 254 or 366 nm, upon irradiation with a UV lamp.
Glass columns were slurry packed in the appropriate eluent under pressure, with 40–60 µm silica gel, 60A (Phase Sep, UK). Samples were applied as a concentrated solution in the same eluent, or pre-adsorbed on silica gel. Fractions containing the product were identified by TLC, pooled and the solvent removed in vacuo.
1H, 13C, 19F, 31P NMR spectra were recorded on a Bruker Avance 500 spectrometer (500 MHz for proton) and autocalibrated to the deuterated solvent reference peak. All 13C NMR were proton decoupled. The following abbreviations are used in the assignment of NMR signals: s (singlet), d (doublet), t (triplet), 
t (pseudotriplet), q (quartet), qt (quintet), sept (septet) m (multiplet), bs (broad signal), dd (double doublet), dt (double triplet) and dm (double multiplet).
Low-resolution mass spectra were run on a VG Platform II Fisons instrument (Fisons, Altrincham, UK) (atmospheric pressure ionization, electrospray mass spectrometry) in either negative or positive mode. High-resolution mass spectra were performed by the service at the University of Birmingham, UK.
CHN microanalysis was performed as a service by The School of Pharmacy at the University of London.
Infrared spectra were recorded on a Perkin Elmer 1600 series FTIR spectrometer as solids, using a dry potassium bromide matrix.
Ultraviolet Spectrometry was carried out on a Cary 100 version 9.00 spectrometer.
The solvents used were anhydrous and used as purchased from Aldrich. All glassware was oven dried at 130°C for several hours and allowed to cool under a stream of dry nitrogen.
3-(2-Deoxy-ß-D-ribofuranosyl)-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (3)
5-Iodo-2-deoxyuridine (15 g, 42.4 mmol) was dissolved in dry DMF (230 mL) with stirring at room temperature. 1-Ethynyl-4-pentylbenzene (24.7 mL, 21.8 g, 127 mmol, 3 equiv.) was added, followed by tetrakis triphenyl phosphine palladium (0) (4.9 g, 4.24 mmol, 0.1 equiv.) and the mixture stirred until it become clear.
Then, copper (I) iodide (1.61 g, 8.47 mmol, 0.2 equiv.) was added, followed by dry disopropylethylamine (14.8 mL, 10.95 g, 84.7 mmol, 2 equiv.), and the mixture was stirred at room temperature under a dry nitrogen atmosphere for 15 h. Copper (I) iodide (1.61 g, 8.47 mmol, 0.2 equiv.) was added with stirring followed by triethylamine (230 mL, excess), and the mixture heated with stirring at 80°C for 8 h. The solvent was removed under high vacuum and the residue washed with dichloromethane (1.5 L) using a Buchner funnel. The remaining solid was recrystallized from methanol to yield an off-white solid (7.12 g, 42%). 1H NMR (DMSO) 
: 8.84 (1H, s), 7.72 (2H, d, J 7.8 Hz), 7.30 (2H, d, J 7.8 Hz), 7.19 (1H, s), 6.20 (1H, t, J 6.0 Hz), 5.29 (1H, d, J 3.8 Hz), 5.17 (1H, t, J 3.6 Hz), 4.27 (1H, m), 3.95 (1H, m), 3.72 (1H, m), 3.66 (1H, m), 2.60 (2H, t, J 7.4 Hz), 2.43 (1H, m), 2.11 (1H, m), 1.58 (2H, m), 1.30 (4H, m), 0.85 (3H, t, J 6.7 Hz). 13C NMR (DMSO) : 171.0, 153.9, 153.7, 144.0, 137.8, 128.9, 125.8, 124.5, 106.9, 98.6, 88.2, 87.6, 69.5, 60.7, 41.3, 34.9, 30.8, 30.3, 21.9, 13.8. IR: 3250, 2940, 2460, 1680, 1580. HPLC: H2O/CH3CN gradient 0–100 in 10 min: Rt 8.93 min (99.8%). Found MH+ 399.196593 C22H27N2O5 requires 399.191998.
3-(2-Deoxy-ß-D-ribofuranosyl)-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one-5'-monophosphate, ammonium salt (9)
Under an argon atmosphere, to a suspension of 3 (0.64 g, 1.6 mmol) in triethylphosphate (5 mL), POCl3 (0.22 mL, 2.4 mmol) was added at 0°C, and the reaction stirred at the same temperature for 2 h. After this time TLC (EtOAc/MeOH = 9:1 and iPrOH/NH3/H2O = 7:1:2) showed the disappearance of starting material, and a 0.4 M NH4HCO3 solution was then added until the effervescence stopped. After 10 min the mixture was concentrated under reduced pressure, the residue was dissolved in water (10 mL) and filtered off. The filtrate was subsequently purified by column chromatography (iPrOH/NH3 = 9:1 and iPrOH/NH3/H2O = 9:1:2), the appropriate fractions were combined and the solvent was removed in vacuo to yield the product as a white solid (363.2 mg, 44%). Finally, the compound underwent a C18 reverse phase extraction using as eluent an aqueous solution of NH4HCO3 (4 mM), followed by a gradient of the same solution and MeCN 10% and 20%. 1H NMR (DMSO) : 9.05 (1H, s, H-4), 7.64 (2H, H-A)–7.24 (2H, H-B) (AB system, J 7.6 Hz), 7.22 (1H, s, H-5), 6.18 (1H, t, J 6.1 Hz, H-1'), 4.33–4.32 (1H, m, H-3'), 4.01 (2H, m, H-5'), 3.96–3.90 (1H, m, H-4'), 2.56 (2H, t, J 7.7 Hz, 
-CH2), 2.42–2.34 (1H, m, H-2'), 2.13–2.05 (1H, m, H-2'), 1.60–1.51 (2H, m, ß-CH2), 1.33–1.24 (4H, m, CH2), 0.86 (3H, t, J 6.8 Hz, CH3). 13C NMR (DMSO) : 16.7 (CH3), 24.8, 33.2, 33.7, 37.8 (CH2), 43.9 (C-2'), 66.2 (C-5'), 72.2 (C-3'), 89.6, 90.3 (C-1', C-4'), 102.2 (C-5), 110.0 (C-4a), 127.2, 131.8 (C-Ph), 128.8 (C-ipso), 141.4 (C-4), 146.6 (C-para), 156.3 (C-6), 156.7 (C-2), 173.8 (C-7a). 31P NMR (DMSO) : 1.14. MS (ES–) m/e 477.2 (M–, 100%). Accurate mass: C22H26N2O8P requires 477.1427; found 477.1422.
3-(2-Deoxy-ß-D-ribofuranosyl)-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one, 5'-valyl ester (10)
Compound 3 (200 mg, 0.5 mmol) was dissolved in dry DMF (5 mL), followed by the addition of polymer-bound triphenylphosphine [370 mg, 1.1 mmol (3 mmol p/g resin)] and di-tert-butylazodicarboxylate (DBAD) (231 mg, 1.0 mmol) to the mixture, and it was stirred for 20 min. A solution of FMOC-Val-OH (340 mg, 1.0 mmol) in DMF (5 mL) was added dropwise over a period of 30 min. The reaction mixture was stirred at room temperature under an argon atmosphere until a complete disappearance of the starting material (overnight). The resin was filtered off and washed with ethyl acetate. Piperidine (1 mL, 10 mmol) was added to the solution and stirred for 10 min. The solvent was removed under reduced pressure without warming over 35°C and the residue was dissolved in ethyl acetate (20 mL), washed with 10% NaHCO3 (3 x 20 mL) and brine (2 x 20 mL). The final residue was purified by column chromatography (gradient CH2Cl2/MeOH, 0% to 10%) to give 137 mg of valyl ester (55% yield) as a yellow solid. 1H NMR (CDCl3) : 8.3 (1H, s), 7.55 (2H, d), 7.15 (2H, d), 6.6 (1H, s), 6.25 (1H, t), 4.45–4.30 (4H, m), 3.23 (1H, d), 2.80 (1H, m), 2.53 (2H, t), 2.12 (1H, m), 1.97 (1H, m), 1.60 (2H, m), 1.24 (4H, m), 0.90–0.78 (9H, m). 13C NMR (CDCl3) : 175.16, 171.62, 156.26, 154.89, 145.19, 135.29, 129.02, 125.69, 124.95, 108.60, 96.82, 88.73, 85.08, 70.90, 64.19, 60.19, 41.91, 35.82, 32.32, 31.44, 30.89, 22.50, 19.30, 17.24, 13.99. HPLC: H2O/CH3CN gradient 0–100 in 15 min: Rt 12.47 min (99.1%). Found MH+ 498.21986 C27H35N3O6 requires 498.25259.
3-(2-Deoxy-ß-D-ribofuranosyl)-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one, 5'-valyl ester, HCl salt (10.HCl)
Compound 10 (300 mg) was dissolved in THF (3 mL). Under vigorous stirring 1 M HCl (2 mL) was added dropwise at 0°C and the mixture was stirred for 10 min. The solvents were removed under reduced pressure to obtain 322 mg (100%) of yellow oil that solidified with the addition of ether. 1H NMR (dDMSO) : 8.6 (4H, bs), 7.70 (2H, d), 7.30 (2H, d), 7.20 (1H, s), 6.22 (1H, t), 5.60 (1H, bs), 4.48 (2H, m), 4.30 (1H, m), 4.16 (1H, m), 3.98 (1H, m), 2.61 (2H, t), 2.44 (1H, m), 2.25 (1H, m), 2.18 (1H, m), 1.57 (2H, m), 1.32 (4H, m), 1.00–0.83 (9H, m). 13C NMR (DMSO) : 171.13, 168.88, 153.97, 153.70, 144.18, 137.94, 129.04, 125.77, 124.58, 107.22, 98.75, 87.71, 84.13, 69.73, 65.26, 57.35, 40.18, 34.88, 30.88, 30.39, 29.38, 21.91, 18.26, 17.55, 13.90. HPLC: H2O/CH3CN gradient 0–100 in 15 min: Rt 12.47 min (98%). MS (ES+): 498.2.
Determination of molar absorptivity (
)
An accurate solution of a known concentration was prepared by transferring 1 mg of the compound to be determined into a 100 mL volumetric flask and dissolving in ethanol with swirling and sonication before being diluted to volume. The absorbance at the 
max was measured by UV and used to calculate .
Determination of solubility of test compounds
One milligram of the compound to be determined (or the required amount to form a saturated solution) was weighed into a vial and diluted with 5 mL of water. The vial was shaken for 24 h before being filtered through 0.22 mm of Millipore filters and the absorbance determined at the max by UV.
Preparation of Captisol® and cyclodextrin solutions
Solutions of Captisol®/cyclodextrins were prepared according to the following dilutions: 10% w/v, 100 mg of Captisol® in 1 mL; 20% w/v, 200 mg of Captisol® in 1 mL; 40% w/v, 400 mg of Captisol® or the cyclodextrin in 1 mL. The required amount of solubilizing agent was dissolved in water, sonicated for 5 min and diluted to volume.
Ten milligrams of Cf1743 was then added to the respective Captisol®/cyclodextrin solutions to produce a saturated solution, which was allowed to stir for 3 days.
The suspensions were then filtered using 0.22 µm Millipore filters, diluted as required and a UV spectrum obtained. The absorbance at the max was used to calculate the concentration of the test solution as a measure of solubility.
Human embryonic lung (HEL) fibroblasts (ATCC CCL-137) were maintained in the minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine and 0.3% sodium bicarbonate. The VZV strain Oka (ATCC VR-795) and YS (gift from Dr Sakuma, Department of Microbiology, Asahikawa Medical College, Japan) were used as reference strains. Seven wild-type clinical VZV isolates were also tested. The VZV clinical isolates were freshly isolated from the skin lesions of patients with either varicella or zoster.6a VZV virus stocks were prepared in HEL cells. When a 70% cytopathic effect was obtained, the cells were trypsinized and resuspended in a medium containing 10% DMSO and stored as frozen aliquots at –80°C. Virus stocks were titrated on HEL cell monolayers, and plaque-forming units (PFU)/mL titres were measured. Primary human keratinocytes (PHKs) were isolated from neonatal foreskins. Tissue fragments were incubated with trypsin–EDTA for 1 h at 37°C. The epithelial cells were detached and cultured with Serum-Free Keratinocyte Medium (Keratinocyte-SFM), Gibco, Invitrogen Corporation, UK. These PHKs were used for both the antiviral assays in monolayers and for the organotypic cultures.
The source of the compounds used in the present experiment was as follows: aciclovir [9-(2-hydroxyethoxymethyl)guanine], GlaxoSmithKline, Research Triangle Park, NC, USA; brivudin [BVDU; (E)-5-(2-bromovinyl)-1-(ß-D-deoxyribofuranos-1-yl-uracil)] Searle, High Wycombe, UK; penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine], Novartis, Basel, Switzerland; cidofovir [HPMPC; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine], Gilead Sciences, Foster City, CA, USA; and foscarnet [PFA; phosphonoformate sodium salt], Sigma Chemicals, St Louis, MO, USA. The dilutions of the different compounds were made in MEM containing 2% FCS, 2 mM L-glutamine and 0.3% sodium bicarbonate.
VZV drug susceptibility assays were performed on confluent HEL cells in 96-well microtitre plates. Monolayers were infected with 20 PFU of cell-associated virus per well. For each assay, virus controls (infected-untreated cells) were included. After a 2 h incubation period, the virus inoculum was removed and the medium was replaced by the different dilutions (in duplicate) of the tested molecules. Serial dilutions of test compounds were incubated with the infected monolayers for 5 days. After the 5 day incubation period, the cells were fixed and stained with Giemsa, and the level of virus-induced cytopathic effect was determined by counting the number of plaques for each dilution. Activity was expressed as EC50 (effective compound concentration required to reduce virus-plaque formation by 50%) compared with the untreated control. The activity of CF-1743 and FV-100 on the reduction of viral plaque formation was compared with the reference compounds BVDU and aciclovir.
Confluent PHKs grown in 24-well microplates were infected with VZV and incubated for a period of 2 h. Virus inoculum was removed and the medium containing different concentrations of the test compounds were added. The cell cultures were incubated at 37°C for 3 days and 5–6 days. Cells were then recovered from each well by trypsinization and resuspended in medium, and titres of VZV-infected cells (PFU/mL) were determined. An aliquot of 200 µL of each sample was frozen and used later to determine the VZV-DNA copy numbers by real-time PCR.
Evaluation of antiviral compounds against VZV in organotypic epithelial raft cultures
The organotypic epithelial raft cultures were prepared as previously described.19 Two series of rafts were done to evaluate the antiviral effects of the compounds against VZV; one was processed for histology and the other one for quantification of virus DNA by real-time PCR. Rafts were infected with the OKA VZV strain at 4 or 5 days post-lifting and the medium was replaced by a medium containing serial dilutions of the compounds. The medium was changed every other day till the cultures were fixed in 10% buffered formalin at day 12 for histological examination or processed for viral quantification by real-time PCR. To prepare the samples for the quantitative PCR (Q-PCR), the formed epithelium in the rafts was separated from the dermal equivalent and DNA was extracted using the tissue QIAamp DNA Blood Minikit (Qiagen, Basel, Switzerland) according to the manufacturer's instructions. Aliquots of 5 µL of each sample were used in the real-time PCRs.
Q-PCR for VZV-DNA by the TaqMan method
Q-PCR was carried out by real-time PCR using the ABI Prism 7000 Sequence Detector (Applied Biosystems, Foster City, CA, USA).
PCR primers for the VZV ORF29 gene (single-stranded DNA-binding protein) ORF29-1239F (forward primer: 5'-CCAGTTTGCCGGACCTCAT-3', nucleotides 1239–1257), VZVORF29-1297R (reverse primer: 5'-AGATCGAGATGGCCACGTTC-3', nucleotides 1278–1297) and the Taqman probe (5'-CTG CGA ATC CC-3', nucleotides 1262–1272) dual-labelled at the 5'-end with the reporter dye molecule, 6-carboxyfluorescein (FAM) and the 3'-end with minor groove binder, were designed by the Primer Express software (Applied Biosystems, Foster City, CA, USA). Q-PCR amplification reactions were set up in a reaction volume of 25 µL using the TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ, USA), containing 5 µL of purified DNA, 900 nM of forward primer and reverse primer and 200 nM TaqMan probe. Thermal cycling conditions were initiated with a 2 min incubation at 50°C, followed by a first denaturation step of 10 min at 95°C and then 55 cycles of 95°C for 15 s (denaturation) and 60°C for 1 min (reannealing and extension). Real-time PCR amplification data were collected continuously and analysed with the Sequence Detection System (Applied Biosystems, Foster City, CA, USA). For the quantification of VZV-DNA, standard curves were constructed by plotting the cycle thresholds (CTs) against the logarithm of the starting amount of serial dilutions of the plasmid standard (pVZV-ORF29) containing the amplified insert. For the cloning of the pVZV-ORF29 plasmid, a segment of the ORF29 gene was amplified from VZV-purified DNA by PCR using the primers described above, and the PCR product was cloned into the pCR4-TOPO (TOPO TA Cloning kit, Invitrogen, Groningen, Netherlands). The sequence of the cloned VZV ORF29 fragment was confirmed by DNA sequencing in both orientations on a capillary DNA sequencing system (Amersham Biosciences). All samples were tested in triplicate, their respective CTs were determined and the initial starting sequence amount was calculated from standard curves as a mean of the three measurements.
Monolayers of HEL cells grown in 96-well microtitre plates were infected with 20 PFU of cell-associated virus per well. After 2 h of incubation, the virus inoculum was removed and the medium replaced by serial fold dilutions (in duplicate) of the tested molecules. At several times following addition of the test molecules (2, 4, 8, 22, 29, 46, 70 and 118 h), infected cells (one set of microtitre plates per time point) were washed twice or five times with saline to remove the test molecules and then incubated with the medium. After 5 days of incubation the cells were fixed and stained with Giemsa. The activity was determined by counting the number of plaques and calculating the EC50 values for each drug at different time points.
Confluent HEL cell cultures were infected with 20 PFU of cell-associated virus (Oka strain) per well. A variety of concentrations of aciclovir, BVDU and CF-1743 were added to the infected cell cultures at the time of infection (0 h) or at several time points post-infection (one set of microtitre plates per time point). Five days after the initial infection of the cells, the different microtitre plates were fixed with ethanol and stained with Giemsa. The antiviral effects of the compounds were measured by counting the virus-induced plaque number in the cell cultures and the EC50 values were calculated.
HeLa cells were cultured as previously described,18 and all cell culture reagents were from Invitrogen. For microscopy experiments, 1.2 x 105 cells were plated on to 35 mm glass-bottomed culture dishes (MatTek Corporation, Ashland, OR, USA) and incubated under tissue culture conditions for 16–20 h. The cells were then washed two times with D-MEM phenol red-free medium (clear medium) and incubated for 2–60 min at 37°C with a clear medium containing 8 µM of the designated drug. The medium was removed and the cells washed two times in clear medium prior to adding 1 mL of clear medium containing 20 mM HEPES buffer, pH 7.4. The fluorescence of the compound in the live cells was then immediately analysed on a Leica DMIRB inverted fluorescent microscope equipped with a 340–380 nm band pass filter and a 40x oil objective. Images were captured with a QIMAGING RETIGA 1300 camera (Burnaby, BC, Canada) and processed using Improvision Openlab 5.0.2 software (Coventry, UK), either as greyscale or as blue images. For experiments with nigericin (Sigma, Poole, UK), the cells were treated for 60 min with 8 µM FV-100 as above, washed and then incubated for 30 min under tissue culture conditions in a complete medium containing 10 µM nigericin. The cells were then analysed by fluorescence microscopy as described.
All animal experiments were carried out after approval of the protocols by the animal Ethics Committee at the K.U. Leuven, and conform with the rules of working with animals for experimental use. The Cf1743 (3) and the 5'-valine derivative of Cf1743 (10) were administered to NMRI mice (15–20 g) per oral gavage. At different time points, mice were bled by heart puncture to harvest the blood and to determine the levels of 3 and 10 in the plasma of the mice. Blood clotting was prevented with sodium citrate 0.38%. Plasma was collected after centrifugation (2000 rpm, 10 min, 4°C), and cold methanol (1 plasma: 3 methanol) was added, followed by centrifugation of the extracts (15 000 rpm, 15 min, 4°C). These extracts were subjected to HPLC analysis. The samples were injected into a Merck LiChroCART 125-4 RP select B (5 µm), and the following gradient (flow 1 mL/min) was used: 2 min at 98% NaH2PO4 (Acros, NJ, USA) 50 mM + heptanesulfonic acid 5 mM, pH 3.2 (buffer) (Sigma, St Louis, MO, USA) and 2% acetonitrile (ACN) (Rathburn, Walkerburn, Scotland), 6 min linear gradient to 80% buffer and 20% ACN, 2 min linear gradient to 75% buffer and 25% ACN, 2 min linear gradient to 65% buffer and 35% ACN, 8 min linear gradient to 50% buffer and 50% ACN, 10 min isocratic flow, 50 min linear gradient to 98% buffer and 2% ACN, 5 min equilibration at the same conditions. The presence of Cf1743 and BCNA-derived metabolites was visualized by fluorescence detection (excitation at 340 nm and emission at 415 nm). The area under the curve was plotted against time post-drug administration.
Pharmacokinetic parameters were calculated using the Siphar/Win software; values for the AUC were fitted to a one- or two-compartment model and used to calculate the terminal plasma half-life (t1/2), distribution volume (V) and total body clearance (CLt) of the BCNA.
Cf1743 was administered by the intraperitoneal route at 100 mg/kg/day for 10 consecutive days or by the peroral route at 50 mg, 25 mg and 12.5 mg/kg administered twice a day for 19 days. In the intraperitoneal experiment, the mice were anaesthetized with diethyl ether and perfused with buffered formalin (4%). Subsequently, the organs were removed and immersed in the same buffered formalin for 4 h prior to transfer to 80% methanol. In the peroral experiment, mice were weighed every 2–3 days and observed daily for potential visible side effects. Two weeks after the last drug administration (day 5), mice were killed and a necropsy was performed. Gross inspection of pleural, pericardial and peritoneal cavities was followed by removal of heart, lungs, thymus, liver, kidneys, spleen, brain, muscle and testes or ovaries. Organs were fixed in a 4% formalin solution. Four micron paraffin sections of each tissue were prepared in a standard way, stained with haematoxylin for 10 min and examined by light microscopy.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Chemistry
Our previously reported synthetic route to the BCNA family 1 and 3 is entirely based on the pioneering work of Robins and Barr.7,8 This involves the Pd-catalysed coupling of 5-iodo-2'-deoxyuridine (4) (Figure 2) with terminal alkynes to give intermediate 5-alkynyl-2'-deoxyuridines (5), which are converted to the desired bicyclic systems (6) under Cu-catalysis in the presence of base and heating.1,2 In the parent alkyl series, the lead octyl compound (1) was prepared via the 5-decynyl intermediate (5), the yields of the two steps being 60% and 55% (overall yield 33%).1 In the case of the p-pentylphenyl system (3) the intermediate (5) was not isolated, but was reacted in situ to generate 3. The yield after column chromatography was 15%.2 Agrofoglio et al.9 has reviewed various Pd-assisted routes to nucleosides such as these and several synthetic enhancements have been recently reported, such as the use of microwave-catalysed coupling methods,10 and silver nitrate-based cyclizations.11 Although each of these methods offers the potential for improvement over our earlier reported yields of 1 and 3, in each case there remains the need for chromatographic purification. Given the impending need for scale-up synthesis of our lead compounds, we were keen to develop a chromatography-free isolation procedure, and we chose in the first instance to base this on our established synthetic routes.1,2 Thus, treatment of 4 with 3 equivalents of p-pentylphenyl acetylene in DMF in the presence of diisopropylethylamine and tetrakis Pd(0) and CuI gave clean conversion to the 5-alkynyl intermediate on TLC with traces of the fluorescent-cyclized material (3). The addition of further copper iodide, Et3N and heating for 8 h gave a clean conversion almost exclusively to yield 3 on TLC. The removal of solvent and volatiles under high vacuum and thorough washing of the residue with dichloromethane gave a crude product, which was recrystallized from methanol. On a scale of 15 g, the yield of 3 was 42%; an almost 3-fold improvement on our earlier report, and notably now without recourse to chromatography. 1H and 13C NMR data on 3 match entirely those reported previously2 and indicate high purity. HPLC shows a single peak for 3 with a purity assessment of 99%. Microanalytical data also confirmed the purity of 3 prepared by this route. This has formed the synthetic basis of our preclinical development of 3.
|
Antiviral activity of anti-VZV drugs in PHK cell cultures
PHK monolayer cell cultures were infected with VZV (OKA) in the presence of a variety of aciclovir (2), BVDU (7) and Cf1743 (3) concentrations, and titres of VZV-infected cells (PFU/mL) were determined at day 3 post-infection. The drugs dose-dependently inhibited virus-induced plaque formation. Although the EC50s were around 3, 0.3 and 0.03 µg/mL, respectively, complete suppression was afforded by aciclovir (2), BVDU (7) and Cf1743 (3) at 20, 0.5 and 0.05 µg/mL, respectively. Thus, 3 was 10-fold more potent in its anti-VZV activity in PHK cultures than BVDU (7) and 
100-fold superior to aciclovir (2) (Figure 3). When viral DNA was quantified in the cell cultures, at day 3 (Figure 4a–c) and day 5 (Figure 4d–f) post-infection, a dose-dependent inhibition was noticed for all three drugs, 3 being clearly superior to 7 and 2. The effect of the drugs against viral DNA production correlated well with the VZV infectivity data obtained in the same experiment.
|
|
Effect of anti-VZV drugs on the morphological changes in VZV-infected organotypic epithelial raft cultures
The ability of 2 and 3 to reduce the replication and spread of VZV in epithelial raft cultures of human keratinocytes was determined. Organotypic epithelial raft cultures were infected after 4–5 days of differentiation and treated with several concentrations of the drugs. At 12 days post-lifting, the rafts were processed for histology (Figure 5) and viral quantification. Under these experimental conditions, viral infection and spread in non-treated cultures occurred all along the epithelium. Morphological analysis of the organotypic cultures revealed that 2 and 3 fully protected the epithelium against the VZV-induced destruction at 0.4 (or higher concentrations) and at 0.04 µg/mL (or higher), respectively. BVDU (7) showed similar protection as 2 (data not shown). At lower drug concentrations, the drugs afforded partial protection, with areas of the rafts seriously compromised at some places due to virus replication.
|
VZV-DNA quantification in organotypic epithelial raft cultures infected with VZV/OKA and treated with different anti-VZV drugs
A variety of concentrations of compounds 2, 3, 7, penciclovir and foscarnet (PFA) and the acyclic nucleoside phosphonate cidofovir (8) were administered to VZV-infected organotypic epithelial raft cultures. At day 7 post-infection, VZV-DNA was quantified in the infected cell cultures. As evident from Figure 6, compound 3 (Cf1743) gave the most pronounced drop of viral DNA within a broad concentration range (0.00004–4 µg/mL) (up to 3 orders of magnitude at the highest concentrations). The other reference drugs 2 (aciclovir), 7 (BVDU) and 8 (cidofovir) and penciclovir showed comparable activities to each other, but were invariably inferior to 3 (1–1.5 orders of magnitude decreased viral DNA levels at the highest concentrations). PFA was the least active drug among the reference compounds tested. Thus, the histological findings (Figure 5) for the drug-treated VZV-infected raft cultures correlated well with the VZV-DNA quantification (Figure 6) in the epithelial raft cultures, and confirm the significantly enhanced potency of 3 versus all other agents.
|
Effect of a delayed administration of test compounds to VZV-infected cell cultures (time-of-drug addition studies)
Confluent HEL cell cultures were exposed to VZV at a high multiplicity of infection. A variety of concentrations of aciclovir (2), BVDU (7) and Cf1743 (3) were added to the infected cell cultures at the time of infection (0 h) or at several time points post-infection (Figure 7). The antiviral effects of the compounds were measured 120 h post-infection by counting the virus-induced plaque number in the cell cultures. A delay of administration of 2, 7 and 3 for up to 49 h post-infection did not affect the antiviral efficacy of the drugs. At longer delayed time periods, all three drugs started to lose their activity. The most dramatic effect was observed for 3, which lost its antiviral activity by 5 orders of magnitude when added at 55 h post-infection. The other drugs lost 1 or 2 orders of magnitude under similar experimental conditions. However, the antiviral activity of 3 was still comparable to the antiviral activity of 7 when the drugs were added at time points later than 55 h (Figure 7).
|
Effect of a limited exposure of test compounds to VZV-infected cell cultures
VZV-infected HEL cells were incubated with 2, 3, BVDU (7) and cidofovir (8) for 120 h (5 days) (Figure 8), at a variety of concentrations. In parallel experiments the drugs were removed from the virus-infected cell cultures at various time points post-infection. The reduction of plaque numbers by various drug concentrations that were removed at a range of different time points post-virus infection was determined. Data for the standard anti-VZV agent aciclovir (2) are shown in Figure 8(a). When the drug 2 is present for the full 120 h time period, the EC50 is 0.35 µg/mL (1 µM). This is consistent with previously reported data.1,2 However, if the drug is only present for 31 h the EC50 increases to somewhat over 10 µg/mL (30 µM). For incubations shorter than 31 h the efficacy is so reduced that no EC50 could be reached even at the highest concentrations tested (50 µg/mL, 150 µM). This implies that prolonged exposure to aciclovir (>24 h) is necessary to achieve an effective antiviral outcome.
|
BVDU (7) is known to be considerably more potent against VZV in vitro than aciclovir and the data in Figure 8(b) confirm this. At 120 h of exposure of 7 to the virus-infected cells the EC50 is
0.002 µg/mL (6 nM). In this assay BVDU proved 200 times more potent an anti-VZV agent than aciclovir. Moreover, the washout data indicate that diminished drug exposure time has a lesser effect on the loss of antiviral potency of BVDU than noted above for aciclovir. Thus, reducing exposure time from 120 to 31 h, a procedure that markedly reduced the efficacy of aciclovir, causes only an 2-fold reduction in the potency of BVDU. An incubation as short as 24 h resulted in a 20-fold reduction, and in the time scale of 4–10 h of exposure of BVDU to the virus-infected cells no antiviral effect is seen. Thus, at least 10 h of exposure to BVDU is necessary to display any antiviral effect, and 24 h is needed for an effect at low drug concentrations.
The acyclic nucleoside phosphonate cidofovir (8) shows an EC50 of 0.1 µg/mL at 120 h of exposure (Figure 8c), thus being intermediate in activity between aciclovir and BVDU. Reduced exposure times give a stepwise reduction in potency, with an 10-fold reduction at 24 h of exposure and 50-fold reduction at 4 h. Thus, of all the agents examined cidofovir is the most effective at short exposure times.
Lead BCNA 3 showed a somewhat similar profile to 8 (Figure 8d) but with several differences. At the 120 h of exposure, an EC50 of 0.0005 µg/mL (2 nM) is noted. This is twice as active as BVDU and 700 times more active than aciclovir. While the efficacy of 3 is reduced upon shorter exposure, it is still effective at 0.01 µg/mL even at the shortest (4 h) exposure time. At this time point both aciclovir and BVDU have no antiviral effect, and cidofovir is only effective at 5 µg/mL. These data strongly indicate that a limited exposure time of the BCNA is still highly antivirally effective, and it is effective at exposure times where other agents are not effective.
The potential of the BCNA derivative 3 to retain a strikingly more pronounced antiviral activity than aciclovir, BVDU and cidofovir upon short exposure times to the virus-infected cell cultures is a remarkable observation and of paramount importance in the clinical setting. It may be that the BCNAs are taken up more rapidly, or are retained longer intracellularly, once taken up by the cells, than the other anti-VZV counterparts. This may be due to the characteristic properties inherent to the nature of the molecule once trapped (as a 5'-phosphate derivative) into the cells. Metabolic studies are underway to support the cell culture observations.
In vivo pharmacokinetics and toxicology
With multigram quantities of 3 now available, we performed initial pharmacokinetic and toxicology studies. The high lipophilicity of these agents most likely aids their membrane permeation, but also does present challenges associated with their low water solubility. The solubility of 3 in distilled water at ambient temperature is estimated from UV studies to be 0.9 mg/L.12 From our initial in vivo studies we were able to formulate 3 at 2 mg/mL in a mixture of 11% DMSO, 22% cremaphore and 67% PBS. Following a single oral dose of 20 mg/kg to mice, parent drug was detected in the plasma up to 8 h (Figure 9).
|
Plasma drug concentrations versus time were fitted to calculate the elimination rate constant and plasma half-life of the drugs. Values for the AUC (data not shown) were fitted to both a one- and two-compartment model, and used to calculate the VD and CLt of the BCNAs. At an intravenous bolus of 2.5 mg/kg, Cf1743 was cleared at a CLt of 4 L/h/kg, the VD was
4 L/kg while the t1/2 was 41 min. In contrast, at an oral dose of 20 mg/kg, Cf1743 showed a remarkably long plasma t1/2 of 281 min, a CLt of 28 L/h/kg and a VD of 108 L/kg (Table 1). The Fpo was 14%. Interestingly, at 8 h after the oral administration of 20 mg/kg Cf1743, plasma drug levels of 0.02 µg/mL were observed, which represent a concentration of Cf1743 that still exceeded by 200-fold its EC50 value in cell culture.
|
Since the BCNAs are intrinsically endowed with fluorescent properties, the parent drug and its metabolites were separated and quantified in plasma with a fluorescence detector-equipped HPLC. The HPLC of the plasma extracts of mice injected with Cf1743 revealed metabolic conversion of the parent compound. For Cf1743, five different metabolites were detected in the plasma [Figure 10a (intravenous administration) and Figure 10b (peroral administration)]. The most abundant of these metabolites reached 40% of the total BCNA-related fluorescence again. Despite drug metabolism, the parental Cf1743 BCNA always remained the predominant BCNA derivative in plasma.
|
A preliminary mouse toxicology drug exposure study was performed as follows: Cf1743 was administered by the intraperitoneal route at 100 mg/kg/day for 10 consecutive days or by the peroral route at 50 mg/kg (twice a day) for 5 consecutive days. No abnormalities were found that are suggestive of toxicity. No secondary abnormalities such as infections or other accidental abnormalities were found. There were no side effects observed upon visual observation of the drug-treated mice and no indications of weight loss when examined up to 20 days after the start of the treatment (Figure 11) or change in behaviour of the mice after the completed drug administration period could be noticed. None of the drug-treated mice showed any microscopically visible histological abnormalities in the organs examined (i.e. heart, lung, liver, kidney, spleen, thymus, brain, testes and ovaries).
|
Formulation studies
In view of the need to enhance the oral bioavailability of 3, alternative formulations were examined. Our attention was drawn to promising data noted for the proprietary substituted cyclodextrin Captisol® which has been used in clinical settings.13 Therefore, a series of Captisol® admixtures with 3 were prepared, saturated aqueous solutions were made and concentrations of 3 estimated by UV, after appropriate dilutions. As shown in Table 2 the presence of Captisol® very significantly boosted the water solubility of the highly lipophilic drug. Solubility enhancements are linear with Captisol® content up to the highest concentration used (40%), with a 1000-fold boost, from 1 mg/L to 1 g/L, at this concentration. Notably, unmodified ß- and 
-cyclodextrin have a far less pronounced effect (Table 2) indicating the particular efficacy of the side chain modifications present in Captisol®. However, when a 40% Captisol® 0.5% CMC mixture of 3 was evaluated for oral bioavailability in mice we found only limited (2-fold) enhancements in AUC and Cmax, which are markedly lower than the dramatic enhancement in water solubility would have predicted (Table 3 and Figure 12). The lack of significant improvement of oral bioavailability upon formulation with Captisol® led us to design pro-drugs of 3.
|
|
|
Pro-drug synthesis
Based on the clinical success of the 5'-monophosphate of the antileukaemic agent fludarabine as a water-soluble pro-drug,14 we initially synthesized and examined the 5'-monophosphate of 3. Reaction of 3 with phosphoryl chloride in triethyl phosphate gave 9 in moderate yield (Figure 2). The monophosphate of 3 was noted to be considerably (>100-fold) more water-soluble than 3 but PK assays in mice showed only limited enhancements in AUC and Cmax (data not shown).
This led us to consider alternative pro-drugs that might impact on oral bioavailability. Given the well-established efficacy of 5'-valyl pro-drugs of nucleosides such as aciclovir and ganciclovir,15,16 we considered the synthesis of 5'-valyl-pro-drugs of 3.
Compound 3 was reacted with various N-protected valine derivatives under a variety of condensation conditions. The most satisfactory, in terms of yield and 5'-selectivity, was a Mitsunobu reaction between FMOC-Val-OH and 3 under catalysis by resin-bound triphenylphosphine and the stable and commercially available DBAD (Figure 2).17 On a small scale the intermediate FMOC-protected valyl nucleoside could be isolated and purified by preparative centrifugal circular thin-layer chromatography (CCTLC, ‘Chromatotron’). However, on a large scale this method was unsatisfactory, and the intermediate was used crude without any purification, after the removal of the excess phosphine and phosphine oxide by filtration. Brief treatment of this compound preparation with piperidine at room temperature gave the 5'-valyl pro-drug (10) (Figure 2). The structure and purity of 10 were confirmed by 1H and 13C NMR, mass spectrometry, HPLC and microanalysis.
Given the use of valyl aciclovir as its HCl salt and possible stability concerns over the free amino acid in 10 we prepared two salt derivatives of 10. Simple treatment of 10 with HCl in THF or with succinic acid gave the appropriate salt forms in quantitative yield (Figure 2).
From a quantitative UV study we were able to establish the water solubility of 10 and its salts, relative to 3 with data shown in Table 4. Thus, conversion of lead (3) to its free valine ester (10) gives a 22-fold boost in water solubility. A further 24-fold boost is seen for the HCl salt of 10 giving a solubility of 0.5 mg/mL and being >500-fold more soluble than 3. The succinate salt was even more soluble. Indeed, it was difficult to obtain a consistently saturated solution. A solubility range of 17–50 mg/mL was observed. On this basis, both the HCl and succinate salts of 10 were considered satisfactory for onward development.
|
Pro-drug stability
One key issue for any drug substance is its solid-state stability. Hence, 10 and its HCl and succinate salts were examined by quantitative HPLC for the degradation of the parent drug substance over a period of 3–7 days at temperatures of 40–60°C and under anhydrous or humid (75% relative humidity) stress conditions. The data are shown in Table 5. Increasing decomposition of 10 with time and temperature was observed. However, even at 60°C for 7 days, more than 50% of parent pro-drug still remained intact for each sample. There were significant increases in decomposition in the humid stress test, particularly for the free amine (10) and the succinate salt of 10. However, the HCl salt emerged as particularly stable. Even after 7 days at 60°C in moisture, only 1.5% decomposition of the drug substance was noted relative to control compound. Given this promising stability profile and the pronounced water solubility of (10.HCl) this salt derivative of 10 was chosen as the clinical candidate drug (designated FV-100).
|
Oral bioavailability studies
We proceeded to examine the oral bioavailability and pharmacokinetics of 10.HCl as we had done for parent drug (3). The data (Table 6 and Figure 13) show a significant, 10-fold, boost in both AUC and Cmax for 10.HCl over 3, being far more notable than any enhancement noted for 9 or 3 in the presence of Captisol®.
|
|
Antiviral activity of the 5'-valine derivative of 3 (10.HCl)
The activity of the pro-drug 10.HCl as measured by plaque reduction assay was compared with that of the parent compound Cf1743 against two VZV reference strains. EC50 values for the pro-drug 10.HCl were 2.5-fold and 4-fold higher, respectively, for Oka and YS strains, than those of the parent compound (Oka strain: EC50 = 0.0033 ± 0.0027 µM for 3 and 0.0081 ± 0.0048 µM for 10.HCl; YS strain: EC50 = 0.0031 ± 0.0027 µM for 3 and 0.012 ± 0.005 µM for 10.HCl). When tested against seven different clinical isolates 10.HCl was able to inhibit virus plaque formation with an EC50 of 0.0026 ± 0.0030 µM, which was 3-fold higher than that of 3 (EC50 = 0.0009 ± 0.0009 µM).
Entry of the 5'-valine derivative of 3 (10.HCl) in intact cells
Finally, given the natural and inherent UV fluorescence of the BCNA compounds, we wondered whether fluorescent microscopy could be used to track the entry of the clinical candidate (10.HCl) in living cells. For this purpose, human HeLa (cervical cancer) cells were incubated with 10.HCl at 8 µM for 2 min at 37°C prior to washing and direct microscopic analysis using a 340–380 nm band pass filter. Autofluorescence of these cells at 340–380 nm was negligible compared with the very evident blue fluorescence in cells treated with 10.HCl for only 2 min (Figure 14a). This was prominent in reticular-like structures but increasing the incubation period to 1 h showed prominent sequestration of 10 in punctate structures that resembled endocytic organelles such as endosomes and lysosomes18 (Figure 14b). To test this we dissipated the pH gradient across intracellular organelles using the ionophore nigericin and cells treated with this drug showed very little evidence of 10 sequestration (Figure 14c). Since nigericin neutralizes pH gradients across the cell, it would indicate that the trapping of 10 within these vesicular structures is pH dependent. Similarly, such trapping was not evident when cells were incubated with the parent drug (3) for 1 h in the absence of nigericin (Figure 14d). This is to be expected given the absence of the basic (valine) nitrogen in 3. We were unable to detect significant labelling of cellular membranes, the cytosol or the nucleus in any of the microscopy experiments. It should be noted that the fluorescence of these compounds was rapidly diminished by exposure to UV light, and this necessitated a higher concentration of compound than was seen to be necessary in the antiviral assays. This preliminary study is taken to support the notion that 10.HCl is rapidly internalized, in support of its promising bioavailability.
|
We believe that the microscopy and the drug wash-out data, taken together, indicate the possibility that earlier inhibition of viral replication may ensue with these agents as compared with aciclovir and related compounds. These observations, coupled with the extreme potency and selectivity of the current agents such as our lead (10.HCl) indicate the promising clinical potential of these compounds in the treatment of VZV infections.5 Phase 1 clinical trials of this agent are now being planned to investigate its antiviral effect on VZV-associated shingles and post-herpetic neuralgia.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Funding for this work was received in part from FermaVir Pharmaceuticals NYC. Core funding from Cardiff University, Katholieke Universiteit Leuven and EU are noted.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
This work was funded in part by FermaVir Pharmaceuticals; G. H., a co-author of the work, is employed by FermaVir and C. McG. is a founding stockholder in the company. A research and license agreement is in place between FermaVir and Cardiff University and the Rega Institute.
|
Acknowledgements |
|---|
We thank Anita Camps, Lies Van den Heurck, Steven Carmans and Ria Van Berwaer for excellent technical assistance and Christiane Callebaut for fine editorial help. We also thank Helen Murphy for excellent secretarial assistance.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
1 . McGuigan C, Yarnold CJ, Jones G, et al. Potent and selective inhibition of varicella-zoster virus (VZV) by nucleoside analogues with an unusual bicyclic base. J Med Chem (1999) 42:4479–84.[CrossRef][Web of Science][Medline]
2 . McGuigan C, Barucki H, Blewett S, et al. Highly potent and selective inhibition of varicella-zoster virus by bicyclic furopyrimidine nucleosides bearing an aryl side chain. J Med Chem (2000) 43:4993–7.[CrossRef][Web of Science][Medline]
3 . Sienaert R, Andrei G, Snoeck R, et al. Inactivity of the bicyclic pyrimidine nucleoside analogues against simian varicella virus (SVV) does not correlate with their substrate activity for SVV-encoded thymidine kinase. Biochem Biophys Res Commun (2004) 315:877–83.[CrossRef][Web of Science][Medline]
4 . McGuigan C, Brancale A, Barucki H, et al. Furano pyrimidines as novel potent and selective anti-VZV agents. Antivir Chem Chemother (2001) 12:77–89.[Web of Science][Medline]
5 . McGuigan C, Barucki H, Brancale A, et al. Fluorescent bicyclic furo pyrimidine deoxynucleoside analogues as potent and selective inhibitors of VZV and potential future drugs for the treatment of chickenpox and shingles. Drugs Future (2000) 25:1151–61.[Web of Science]
6 . McGuigan C, Balzarini J. Aryl furano pyrimidines: the most potent and selective anti-VZV agents reported to date. Antiviral Res (2006) 71:149–53.[CrossRef][Web of Science][Medline]
6 . Andrei G, Snoeck R, Reymen D, et al. Comparative activity of selected antiviral compounds against clinical isolates of varicella-zoster virus. Eur J Clin Microbiol Infect Dis (1995) 14:318–28.[CrossRef][Web of Science][Medline]
7 . Robins MJ, Barr PJ. Nucleic-acid related-compounds. 39. Efficient conversion of 5-iodo to 5-alkynyl and derived 5-substituted uracil bases and nucleosides. J Org Chem (1983) 48:1854–62.[CrossRef][Web of Science]
8 . Robins MJ, Barr PJ. Nucleic-acid related-compounds. 31. Smooth and efficient palladium copper catalyzed coupling of terminal alkynes with 5-iodouracil nucleosides. Tetrahedron Lett (1981) 22:421–4.[CrossRef][Web of Science]
9 . Agrofoglio LA, Gillaizeau I, Saito Y. Palladium-assisted routes to nucleosides. Chem Rev (2003) 103:1875–916.[CrossRef][Web of Science][Medline]
10 . Petricci E, Radi M, Corelli F, et al. Microwave-enhanced sonogashira coupling reaction of substituted pyrimidines and pyrimidine nucleosides. Tetrahedron Lett (2003) 44:9181–4.[CrossRef][Web of Science]
11 . Aucagne V, Amblard F, Agrofoglio L. Highly efficient AgNO3-catalyzed preparation of substituted furanopyrimidine nucleosides. Synlett (2004) 2406–8.
12 . Adak R, Mollwitz B, Migliore M, et al. SAR of alkyloxyphenyl furano pyrimidines: potent and selective anti-VZV agents. Antiviral Res (2006) 70:A66.[CrossRef]
13 . Couvreur P, Duchene D, Kalles I. Formulation of poorly available drugs for oral administration. Minutes Eur Symp (2006) 264–9.
14
.
Foon KA, Rai KR, Gale RP. Chronic lymphocytic leukemia: new insights into biology and therapy. Ann Intern Med (1990) 113:525–39.
15 . Weller S, Blum MR, Doucette M, et al. Pharmacokinetics of the acyclovir pro-drug valaciclovir after escalating single- and multiple-dose administration to normal volunteers. Clin Pharmacol Ther (1993) 54:595–605.[Web of Science][Medline]
16
.
Pescovitz MD, Rabkin J, Merion RM, et al. Valganciclovir results in improved oral absorption of ganciclovir in liver transplant recipients. Antimicrob Agents Chemother (2000) 44:2811–5.
17 . Pelletier JC, Kincaid S. Mitsunobu reaction modifications allowing product isolation without chromatography: application to a small parallel library. Tetrahedron Lett (2000) 41:797–800.[CrossRef][Web of Science]
18
.
Simpson JC, Griffiths G, Wessling-Resnick M, et al. A role for the small GTPase Rab21 in the early endocytic pathway. J Cell Sci (2004) 117:6297–311.
19
.
Andrei G, van den Oord J, Fiten P, et al. Organotypic epithelial raft cultures as a model for evaluating compounds against alphaherpesviruses. Antimicrob Agents Chemother (2005) 49:4671–80.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. McGuigan and J. Balzarini FV100 as a new approach for the possible treatment of varicella-zoster virus infection J. Antimicrob. Chemother., October 1, 2009; 64(4): 671 - 673. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||