key: cord-333675-vkk2frnf
authors: Hamada, Manabu; Roy, Vincent; McBrayer, Tamara R.; Whitaker, Tony; Urbina-Blanco, Cesar; Nolan, Steven P.; Balzarini, Jan; Snoeck, Robert; Andrei, Graciela; Schinazi, Raymond F.; Agrofoglio, Luigi A.
title: Synthesis and broad spectrum antiviral evaluation of bis(POM) prodrugs of novel acyclic nucleosides
date: 2013-07-04
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2013.06.053
sha: 
doc_id: 333675
cord_uid: vkk2frnf

A series of seventeen hitherto unknown ANP analogs bearing the (E)-but-2-enyl aliphatic side chain and modified heterocyclic base such as cytosine and 5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxamide or 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as key synthetic step. All novel compounds were evaluated for their antiviral activities against a large number of DNA and RNA viruses including herpes simplex virus type 1 and 2, varicella zoster virus, feline herpes virus, human cytomegalovirus, hepatitis C virus (HCV), HIV-1 and HIV-2. Among these molecules, only compound 31 showed activity against human cytomegalovirus in HEL cell cultures with at EC(50) of ∼10 μM. Compounds 8a, 13, 14, and 24 demonstrated pronounced anti-HCV activity without significant cytotoxicity at 100 μM.

The discovery by A. Holý and E. De Clercq in 1986 of broadspectrum antiviral activity of (S)-HPMPA [9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine] and PMEA [9-[2-(phosphonome thoxy)ethyl]adenine] led to a new family of nucleotides designated as acyclic nucleoside phosphonates (ANP) [1e4],. ANPs are nucleotide analogs that are characterized by the presence of a phosphonate group linked to a pyrimidine or purine base through an aliphatic linker. Three of these are approved drugs for the treatment of severe/fatal infectious diseases and represent three different types of ANPs: (i) HPMP derivatives such as (S)-1-[3hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir (Vistide Ò )] which is approved for the treatment of cytomegalovirus (CMV) retinitis in AIDS patients [5] ; (ii) PME derivatives such as PMEA [adefovir (in its oral prodrug form, adefovir dipivoxil (Hepsera Ò )] for the treatment of hepatitis B virus infections [6] , and (iii) PMP derivatives such as PMPA [tenofovir (in its oral prodrug form, tenofovir disoproxil fumarate (Viread Ò )] is used for the treatment of HIV infections (AIDS) and hepatitis B virus [7] . From these data, it appears that small chemical alterations in the acyclic side-chain lead to marked differences in antiviral activity and the spectrum of activity of acyclic nucleoside phosphonates against various classes of viral agents [1] .

Thus, the synthesis and biological evaluation of a large panel of ANPs were systematically investigated as potential antiviral compounds [1] . In our search for antiviral compounds, we synthesized a new class of acyclic nucleoside phosphonates based on a 4-phosphono-but-2-en-1-yl base motif in which the oxygen heteroatom has been replaced with a double bond having trans stereochemistry [8] . We have shown that this modification allows mimicry of the three-dimensional geometry provided by the backbone of PMEA, PMPA, and CDV while maintaining an electronic contribution similar to that brought by the oxygen atom [8] . Several new derivatives are efficiently activated by human thymidylate kinase (hTMPK), and the best substrates were converted to bis-(pivaloyloxymethyl)ester phosphonate prodrugs and found to be active against several herpes viruses in cell culture.

On the basis of these findings, it was interesting to design and synthesize hitherto unknown ANP analogs bearing the biolabile phosphonate (E)-but-2-enyl aliphatic side-chain and a series of modified heterobases selected from the literature as lead nucleobases with antiviral properties, such as cytosine and 5-fluoro cytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxamide or 4-substituted-1,2,3-triazoles (Fig. 1) . From a chemical synthesis point of view, the strategy based on olefin cross metathesis we have developed to obtain a large library of (E)-4-phosphono-but-2 0 -en-1 0 -yl pyrimidine nucleosides [9e11].

First, we turned our attention to the synthesis of cytosine derivatives as cytosine modified nucleosides form a prolific family of antitumor and antiviral agents [12] . As it could be excepted, even if the ruthenium carbene complex 3 (GrubbseNolan catalyst 2nd generation) is less affected by free amine and nitrogen-containing groups than the Grubbs's 1st generation catalyst [13] , the crossmetathesis reaction between unprotected N 1 -crotylated cytosine 2 and bis-(POM) allylphosphonate 1 failed (Scheme 1).

The successful cross-metathesis occurred with protected N 1crotylated cytosines 6a, b. Thus, cytosine 4a and 5-fluorocytosine 4b were converted to their N 4 -bis-Boc cytosine derivatives 5a, b, respectively, through a N-peracylation followed by subsequent and regioselective N 1 deprotection by a saturated solution of NaHCO 3 in methanol [14, 15] . Crotylation of the N 1 position of 5a, b using Cs 2 CO 3 and crotyl bromide afforded the desired compounds 6a (85%) and 6b (81%). Compound 6a, b were then engaged in the olefin cross metathesis reaction with bis(POM)-allylphosphonate 1 using 5 mol% of the (NHC)Ru]CHR Nolan's catalyst, Cl 2 (PCy 3 ) (IMes)Ru (CHPh) (3), in dry CH 2 Cl 2 (0.1 M) at reflux to afford (E)-N 1 -(4 0bis(POM)-phosphinyl-2 0 -butenyl)-bis-Boc-cytosine 7a, b in moderate yields (43% (for R ¼ H) and 26% (for R ¼ F)). The removal of the Boc group requires oftentimes harsh conditions (e.g. trifluoroacetic acid, trimethylsilyl iodide, hydrochloric acid in ethyl acetate, potassium carbonate, etc.) that are not compatible with the POM moiety. However, Hwu et al. [16] have reported an efficient and milder selective Boc deprotection under neutral conditions using ceric ammonium nitrate (CAN) that is consistent with the stability of the phosphonate biolabile group. Thus, protected ANP 7a, b were reacted with a catalytic amount of ceric ammonium nitrate (CAN) (20 mmol%) in CH 3 CNeMeOH (1:1) to give the expected compounds 8a, b, respectively, in moderate yield, with no observed removal of the POM moiety (Scheme 2).

We turned then our attention to the synthesis of the pyrazinecarboxamide derivative 14 since, among the modified nucleobases, a series of pyrazinecarboxamide derivatives (including T-705, favipiravir) developed by Furuta et al. [17, 18] have demonstrated good activity in various RNA viral infections. In a first attempt, we struggled to introduce the pyrazine moiety through direct N-alkylation of 10 with the corresponding (E)-4-phosphonobut-2 0 -en-1 0 -yl bromide (9) , in the presence of K 2 CO 3 in anhydrous DMF, (Pathway A). Unfortunately only the bis(POM)-but-1,3-dienyl phosphonate 9 0 resulting from undesired bromine elimination was obtained, (Scheme 3). Thus, we decided to reach the pyrazine phosphonate analogs 13, 14 following the same strategy developed for the cytosine derivatives, (Pathway B). Starting from the 3hydroxypyrazine-2-carboxamide 10, the N 1 -Crotyl-3-oxo-pyrazine-N 3 -bis-Boc-carboxamide 11 was obtained in 49% yield, via the two step crotylation/N-Boc-protection. Next, N 1 -crotyl-3-oxo-pyrazine-N 3 -bis-(Boc)-carboxamide 11 in CH 2 Cl 2 was treated in the presence of bis(POM)-allylphosphonate 1 and (NHC)Ru]CHR Nolan's catalyst 3 to give the desired product 12 in 22% yield. To obtain the carboxamide 14, we first applied our previous described methods, using ceric ammonium nitrate and CH 3 CNeMeOH (1:1) . Surprisingly, the methylester 13 was isolated as the major compound in 47% yield. Thinking that the presence of the undesired product 13 was due to the use of methanol as co-solvent, the preparation of the desired amide 14, was achieved in CH 3 CN using ceric ammonium nitrate in 25% yield.

Based on the above results, we extended this approach to the formation of a ribavirin analog bearing the 1,2,4-triazole derivative [19, 20] . Starting from 15, the protection of both nitrogens provided compound 16, in quantitative yield, which is directly used in the next step. An attempt to selectively Boc deprotect at the N 1 position Alternatively, we decided to successively alkylate the N 1 position of the 1,2,4-triazole-3-carboxamide in the presence of crotyl bromide and Cs 2 CO 3 , followed by the protection of the free nitrogen in dry THF in the presence of Boc 2 O and DMAP at room temperature to give the desired triazole 19 in 34% in two steps (Scheme 4). N 1crotyl-1,2,4-triazole-3-bis-Boc-carboxamide 19 was then subjected to the olefin cross metathesis reaction, with bis(POM)-allylphosphonate in CH 2 Cl 2 to obtain the desired compound 20 in 16% yield. The bis-Boc groups were cleaved according to the previous procedure, by treatment with CAN in a mixture of CH 3 CNeMeOH (1:1) to give the free ANP analog 21 in 42% yield (Scheme 4).

To complete our investigation, we elaborated a small library of ANPs in their prodrug form bearing the substituted 1,2,3-triazolyl moiety 24e33. The triazole derivatives were obtained using Cu(I)catalyzed azideealkyne cycloaddition (CuAAC) using selected alkynes and phosphonate azide 22 [21e24] ,. The introduction of the azide group could be easily performed from the key intermediate previously described. The olefin cross metathesis of bis(POM)allylphosphonate with (E)-1,4-dibromobut-2-ene catalyzed by catalyst 3 afforded (E)-4-bromo-bis(POM)-allylphosphonate (9) in 88% yield. Then, the introduction of azido on 9 with sodium azide in DMSOeTHFeH 2 O (5:2:1) afforded (E)-4-azide-bis(POM)-allylphosphonate 22 in 69% yield (Scheme 5). Among the modifications on the base moiety, carboxylic acid is an unavoidable function. In a first attempt, following our previously described method C, in the presence of propiolic acid under microwave irradiation, we observed the formation and isolation of the unexpected decarboxylated product 23 in 16% yield [25, 26] . To circumvent this decarboxylation, Hall et al. reported boronic acid catalysis (BAC) for the activation of carboxylic acids, to lead to a classic dipolar [3 þ 2] cycloaddition with several azides [27] . To obtain the desired (E)-4-(4-carboxylic acid-[1,2,3]-triazol-1-yl)methyl-bis(POM)-but-2enylphosphonate derivative 24, we selected the BAC of the azideealkyne cycloaddition, which was converted in 58% yield in the presence of 2-nitrophenylboronic acid at room temperature.

Finally, the copper-catalyzed azide-alkynes 1,3-dipolar cycloaddition (CuAAC) affording chemo-selectively and complete regioselectively the (1,4) substituted-1,2,3-triazoles [22] , that permitted the synthesis of a number of ANPs analogs, (Table 1) . A first series of bis(POM)-(1,4-disubstituted-1,2,3-triazol)-but-2enyl-phosphonate congeners bearing a substituted 1,2,3-triazole by phenylacetylene moieties (chosen from apolar to bulky substituents) was obtained by CuAAC reaction with Cu(0)/CuSO 4 $5H 2 O as catalyst in the presence of substituted phenylacetylenes, ethynylthiophene and non-aromatic alkynes, in moderate to good yields ranging from 35 to 93% (Table 1 , entries 1e6) at room temperature (method A. The reaction between our synthon 22 and 2ethynylthiophene or prop-2-ynol at room temperature only led to trace amounts of the expected products 31 and 32. These were, however, isolated. The cycloaddition was then carried out at 60 C Table 1 , entries 7 and 8, method B) to afford compounds 31 and 32 in 45 and 95% yield, respectively. However, the alkyne listed in Table 1 , entry 9 did not allow the formation of the triazole product 33 under thermal conditions. Microwave heating is known as a powerful tool that can produce a variety of nucleoside products [23] . Following our previous work [24] , the microwave irradiation allowed to obtain in moderate yields the desired ANPs 33 and 34 in 44% and 34% yield respectively (Table 1, entry 9 and 10, method C).

The title bis(POM) (E)-4-phosphono-but-2-en-1-yl acyclic nucleosides were subjected to an in vitro antiviral screening using a wide spectrum of viruses, in MDKC cell cultures for anti-influenza virus activity, in Vero cell cultures for an antiviral activity against Para-influenza- 3 shown) only compound 31 showed activity at an EC 50 of w10 mM (AD-169 strain) with no observed cytotoxicity at 100 mM ( Table 2) .

The compounds were also evaluated for inhibition of hepatitis C virus (HCV) in the subgenomic HCV replicon system in Huh 7 cells [28] , and cytotoxicity testing was performed in PBMC, human lymphoblastoid CEM and Vero cells [29, 30] . Data for anti-HCV activity and anti-HIV activity, and the cytoxicity are shown in Table 3 . Many of the compounds were found to be moderately cytotoxic, which must be considered when interpreting the anti-HCV data. Compounds 8a, 13, 14, and 24 demonstrated anti-HCV activity without significant cytotoxicity at 100 mM. The other molecules showed pronounced inhibition at 10 mM, but at compound concentrations that were close to their cytostatic activity.

We have efficiently synthesized a series of seventeen hitherto unknown ANP analogs bearing the E-but-2-enyl aliphatic side chain and a series of modified heterocyclic bases such as cytosine, 5fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxam ide and 4-substituted-1,2,3-triazoles and evaluated their antiviral activities. Among the tested molecules only compound 31 showed activity against human cytomegalovirus at an EC 50 of w10 mM (AD-169 strain) at subtoxic concentrations with no observed cytotoxicity up to 100 mM. Compounds 8a, 13, 14, and 24 demonstrated pronounced anti-HCV activity at 10 mM without significant cytotoxicity at 100 mM. Further structural optimization of both the (E)-but-2-enyl aliphatic side chain and the heterocycle is well under way, alongside more detailed biological testing of the most active compounds, with the aim of improving their antiviral potency. 

Commercially available chemicals were of reagent grade and used as received. Solvents were dried following standard procedures. The reactions were monitored by thin layer chromatography (TLC) analysis using silica gel plates (Kieselgel 60F 254 , E. Merck). Column chromatography was performed on Silica Gel 60M (0.040e0.063 mm, E. Merck). The 1 H, 31 P and 13 C NMR spectra were recorded on a Varian Inova Unity 400 spectrometer (400 MHz) in (d 4 ) methanol, CDCl 3 , shift values in parts per million relative to SiMe 4 as internal reference. High Resolution Mass spectra (HRMS) were performed on a Bruker maXis mass spectrometer by the "Fédération de Recherche ICOA/CBM (FR 2708) platform".

Physico-chemical data are in agreement with reported information [9] . CAS number: 1258789-63-7. 

To the stirred suspension of cytosine in an argon atmosphere (444.0 mg, 4.0 mmol, 1.0 equiv.) in dry THF (13 mL), DMAP (44.0 mg, 0.4 mmol, 0.1 equiv.) Boc 2 O (3.60 g, 16 mmol, 4.0 equiv.) were added. After 20 h stirring at room temperature, the mixture was diluted with EtOAc and then extracted with (2 Â 30 mL) EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The resulting tri-Boc-cytosine was used for the next step without further purification. To a solution of tri-Boccytosine in methanol (40 mL) was added saturated NaHCO 3 aq. solution (18 mL) at room temperature. After stirring 2 h at 60 C, confirming the complete consumption of substrate by examining a TLC developed with petroleum ethereEtOAc (1:1). After removal of methanol in vacuo, the mixture was diluted with EtOAc (30 mL), quenched with water (20 mL) and finally extracted with EtOAc (2 Â 20 mL). The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was purified by silica gel column chromatography with petroleum ethere EtOAc (1:1 to 1:20) to give 5a (465 mg, 37%) as a white solid. 1 

In an analogous manner to the preparation of 5a, 5b was prepared from 5-fluoro-cytosine (158.0 mg, 24%) as a white solid. 1 To a solution of 5a (90.0 mg, 0.29 mmol, 1.0 equiv.) in dry DMF (1 mL) was added Cs 2 CO 3 (104.3 mg, 0.32 mmol, 1.1 equiv.) and crotyl bromide (43.0 mg, 0.32 mmol, 1.1 equiv.) at room temperature and stirred under an argon atmosphere for 2 h. The resulting mixture was then diluted with EtOAc (2 Â 20 mL), quenched with water and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was purified by silica gel column chromatography with petroleum ethereEtOAc (1:1) to give a mixture of Z (minor)/E (major) N 4 ,N 4 -bis-Boc-N 1 -crotyl-cytosine 6a (90.0 mg, 85%) as a yellow oil. 1 In a similar manner as described for 8a, a solution of 7b (35.5 mg, 0.05 mmol) in 0.8 mL of CH 3 CNeMeOH (1:1) was treated with ceric ammonium nitrate (5.5 mg, 0.01 mmol), to give (E)-N 1 -(4 0 -bis(POM)phosphinyl-2 0 -butenyl)-5-fluoro-cytosine 8b (8.2 mg, 35%) as a colorless oil. 1 

To a solution of bis(POM)-allylphosphonate 1 (560 mg,1.60 mmol, 1.0 equiv.) in 24 mL of dry CH 2 Cl 2 was added (E)-1,4-dibromobut-2ene (1.37 g, 6.40 mmol, 4.0 equiv.) and (NHC)Ru]CHR Nolan's catalyst 3 (68.6 mg, 0.08 mmol, 0.05 equiv.). After 16 h of stirring at 45 C under an argon atmosphere, all volatiles were evaporated and the residue was purified by silica gel chromatography eluting with petroleum ethereEtOAc (4:1) to give (E)-4-bromo-bis(POM)-but-2enylphosphonate 9 (623 mg, 88%) as a brown oil. 1 

In a similar manner as described for 6a, a solution of 3hydroxypyrazine-2-carboxamide 10 (139.1 mg, 1.0 mmol) in dry DMF (3 mL) was treated with Cs 2 CO 3 (358.4 mg, 1.1 mmol) and crotyl bromide (148.5 mg, 1.1 mmol) for 4 h at 70 C, to give a crude mixture of (E/Z) N 1 -crotyl-3-oxo-pyrazine-2-carboxamide. After evaporation of all volatiles and rapid silica gel chromatography, the residue (134.0 mg) was treated with DMAP (7.7 mg, 0.07 mmol) and Boc 2 O (602.4 mg, 2.76 mmol) in dry THF (3 mL), to give N 1 -crotyl-3oxo-pyrazine-2-bis-Boc-carboxamide 11 (134.0 mg, 49%) as a colorless oil. 1 In a similar manner as described for 7a, a solution of bis(POM)allylphosphonate 1 (273.5 mg, 0.78 mmol) and N 1 -crotyl-3-oxopyrazine-N,N-bis-Boc-carboxamide 11 (338.2 mg, 0.86 mmol) in dry CH 2 Cl 2 (8 mL) were treated with (NHC)Ru]CHR Nolan's catalyst 3 (66.2 mg, 0.078 mmol), to give 12 (122.8 mg, 22%) as a yellow oil. 1 155.4, 135.1, 130.4, 130.3, 126.1, 126.0, 124.7, 83.4, 83.3, 53.3 

To a solution of 1,2,4-triazole-3-carboxamide 15 (224.5 mg, 2.0 mmol, 1.0 equiv.) in dry DMF (6 mL) was added Cs 2 CO 3 (716.8 mg, 2.2 mmol, 1.1 equiv.) and crotyl bromide (270.0 mg, 2.2 mmol, 1.1 equiv.) and the reaction solution was stirred at room temperature under an argon atmosphere for 2 h and then warmed at 70 C for 12 h. After concentration to dryness in vacuo, the residue was subjected to silica gel chromatography with CH 2 Cl 2 e MeOH (5:1) and employed in the next step without further purification. The residue (330 mg) was suspended in THF (10 mL), DMAP (44 mg, 0.2 mmol, 0.1 equiv.) and Boc 2 O (1.31 g, 6.0 mmol, 3.0 equiv.) were added under an argon atmosphere. The solution was stirred for 20 h at room temperature and then the mixture was diluted with EtOAc and then extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ethereEtOAc (4:1) to give a mixture of Z (minor)/E (major) N 1 -crotyl-1,2,4-triazole-3-bis-Boccarboxamide 19 (248.0 mg, 34%) as a colorless oil. 1 In a similar manner as described for 8a, a solution of (E)-N 1 -(4 0bis(POM)-phosphinyl-2 0 -butenyl)-1,2,4-triazole-3-bis-Boc-carboxamide 20 (44.3 mg, 0.065 mmol) in 1 mL of CH 3 CNeMeOH (1:1) was treated with ceric ammonium nitrate (7.2 mg, 0.013 mmol), to give (E)-N 1 -(4 0 -bis(POM)phosphinyl-2 0 -butenyl)-1,2,4-triazole-3carboxamide 21 (14.0 mg, 42%) as a colorless oil. 

To a solution of (E)-4-bromo-bis(POM)-but-2-enylphosphonate 9 (222.0 mg, 0.50 mmol, 1.0 equiv.) in mixture of DMSO (5 mL), THF (2 mL) and H 2 O (1 mL) was added sodium azide (163.0 mg, 2.50 mmol, 5.0 equiv.). After 24 h stirring at room temperature, the mixture was diluted with EtOAc and water and then extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with petroleum ethere EtOAc (4:1) to give (E)-4-azide-bis(POM)-but-2-enylphosphonate 

Procedure A: To a solution of alkyne (1.3 equiv.) and (E)-4-azidobis(POM)-but-2-enylphosphonate 22 (0.11 mmol, 1.0 equiv.) in t-BuOH/H 2 O (1:1 ratio, 400 mL) were added Cu powder (11.6 mg, 0.40 mmol, 5.0 equiv.) and CuSO 4 (5.0 mg, 0.020 mmol, 0.25 equiv.). The resulting suspension was stirred 8 h at room temperature, then the crude mixture was diluted in EtOAc (1 mL), and directly transferred on a preparative thin layer silica plate to give (E)-4 0 -(1,2,3-triazol-1-yl)-bis(POM)-but-2 0 -enylphosphonate.

Procedure B: Following the procedure described above, the resulting suspension was stirred 16 h at 60 C.

Procedure C: In a similar procedure, the mixture was stirred 1 h under microwave conditions at 125 C. The title compound was prepared from 22 with procedure C to give 23 (16%) as a colorless oil. 1 The title compound was prepared from 22 with procedure A to

give 25 (70%) as a yellow oil. 1 The title compound was prepared from 22 with typical procedure C to give 33 (44%) as a colorless oil. 1 The title compound was prepared from 22 with typical procedure C to give 34 (34%) as a yellow oil. 1 Viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC 50 or compound concentration required reducing virus-induced cytopathogenicity or viral plaque (VZV) formation by 50%. The minimal cytotoxic concentration (MCC) of the compounds was defined as the compound concentration that caused a microscopically visible alteration of cell morphology. Alternatively, the cytostatic activity of the test compounds was measured based on inhibition of cell growth. HEL cells were seeded at a rate of 5 Â 10 3 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37 C, the cell number was determined with a Coulter counter. The cytostatic concentration was calculated as the CC 50 , or the compound concentration required to reduce cell proliferation by 50% relative to the number of cells in the untreated controls. The methodology of the anti-HIV assays was as follows: human CEM (w3 Â 10 5 cells/ cm 3 ) cells were infected with 100 CCID 50 of HIV(III B ) or HIV-2(ROD)/ml and seeded in 200 mL wells of a microtiter plate containing appropriate dilutions of the test compounds. After 4 days of incubation at 37 C, HIV-induced CEM giant cell formation was examined microscopically.

Hepatitis C antiviral activity was evaluated as previously described [28] . Huh 7 Clone B cells containing HCV Replicon RNA were seeded in a 96-well plate at 3000 cells/well, and the compounds were tested at 10 mM in triplicate immediately after seeding. Following five days incubation (37 C, 5% CO 2 ), total cellular RNA was isolated using the RNeasy96 well extraction kit from Qiagen. Replicon RNA and an internal control (TaqMan rRNA control reagents, Applied Biosystems) were amplified in a single step multiplex Real Time RT-PCR Assay. The antiviral effectiveness of the compounds was calculated by subtracting the threshold RT-PCR cycle of the test compound from the threshold RT-PCR cycle of the no-drug control (DCtHCV). A DCt of 3.3 equals a 1-log reduction (equal to 90% less starting material) in Replicon RNA levels. The cytotoxicity of the compounds was also calculated by using the DCt rRNA values. RS-446 (2 0 -C-Me-C) was used as the control and tested at 10 mM. To determine EC50 and EC90 values [31] , DCt values were first converted into fraction of starting material and then were used to calculate the % inhibition.

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