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Bioorganic & Medicinal Chemistry 16 (2008) 9610–9615

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Bioorganic & Medicinal Chemistry
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3-Amino-2(5H)furanones as inhibitors of subgenomic hepatitis C virus
RNA replication
Daniela Iannazzo a,*, Anna Piperno a, Giovanni Romeo a, Roberto Romeo a, Ugo Chiacchio b,
Antonio Rescifina b, Emanuela Balestrieri c, Beatrice Macchi c, Antonio Mastino d, Riccardo Cortese e
Dipartimento Farmaco-Chimico, Università di Messina, Via SS. Annunziata, Messina 98168, Italy
Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, Catania 95125, Italy
Dipartimento di Neuroscienze, Università di Roma ‘‘Tor Vergata”, Via Montpellier 1 and IRCSS S. Lucia, Roma 00133, Italy
Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, Università di Messina, Salita Sperone 31, Messina 98168, Italy
CeInge, Via Comunale Margherita 482, Napoli, Italy

a r t i c l e i n f o a b s t r a c t

Article history: A new class of compounds able to block the replication of subgenomic HCV RNA in liver cells is described.
Received 8 May 2008 3-Amino-2(5H)furanones 4 may be regarded as diketoacid analogues and were obtained by basic rear-
Revised 27 August 2008 rangement of the isoxazolidine nucleus.
Accepted 4 September 2008
Ó 2008 Published by Elsevier Ltd.
Available online 7 September 2008

1,3-Dipolar cycloaddition

1. Introduction and the second are pyrophosphate (product) analogues,16,17
namely diketoacids (DKA) 1. Actually, only three scaffolds have
Hepatitis C Virus (HCV) infection constitutes a global health been reported: phenyl-DKA18 like 1, meconic acid derivatives 219
problem, which affects more than 170 million individuals.1,2 The and carboxypyrimidines 320 (Fig. 1).
NS5B RNA-dependent RNA polymerase (NS5B RdRp) has shown In these last years, we have developed an efficient synthetic
to be the catalytic core of the HCV replication machinery.3,4 This procedure towards the construction of 3-amino-2(5H)furanones
enzyme is not expressed in uninfected cells, and, due to its unique by basic treatment of 3-alkoxycarbonyl substituted isoxazoli-
features, represents an attractive target for the development of safe dines.21 We have assumed that the introduction of a carbonyl
antiviral drugs.5–7 group at the C4 position of the 3-amino-2(5H)furanone skeleton,
The catalytic activity of the enzyme is mediated, in the active as in compounds 4, could produce potential inhibitors of pyrophos-
site, by two magnesium ions, which serve to activate the 3’-OH phate site. In fact, 3-amino-2(5H)furanones 4 may be regarded as
of the elongating RNA and to position the incoming nucleotide-tri- DKA cyclic analogues: the 1,3-diketonic functionality is replaced
phosphate for the nucleophilic attack.8–11
Different classes of NS5B inhibitors have been disclosed and
they can be divided by their mechanism of action into three major
classes: non-nucleoside inhibitors acting at allosteric binding sites,
nucleoside analogues, and pyrophosphate analogues. The allosteric
inhibitors include a variety of heterocyclic systems, which have
been shown to bind to three distinct sites on the polymerase.6,7,12
The others two classes are active-site inhibitors: the first are
modified chain-terminating nucleoside (substrate) analogues13–15

* Corresponding author. Tel.: +39 090356230; fax: +39 0906766562.
E-mail address: UUdiannazzo@pharma.unime.itUU (D. Iannazzo). Figure 1. NS5B pyrophosphate analogues inhibitors and new potential ones.

0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.

N- THF dry. so inducing on the dipolaro- phile an electron withdrawing effect at the double bound and a Compounds 8 and 9. 6a — CO2Me Me 6b — CO2Et CO2Et 6c — H C(O)Me 2. TBAF.N-diisopropilcarbodiimide. 7f Me C(O)Me Me In particular.6-diphenylphenoxide) (ATPH) catalyst the crude reaction mixture showed the only desired C4 substituted compound with Scheme 2. enolized into the corresponding 1. 80 °C. 0 °C. The driving force for The comparison of EC50 of 4a (EC50 = 525 lM). in Huh-7-derived HBI10A cells.b-unsaturated ketones. Thus. activated at 7c Me CO2Et CO2Et C3 by the presence of an estereal group. dation products not easily characterizables. The classic pericyclic reaction of nitrones with a. in the presence of aluminum tris(2. tained by DIBAL-H reduction of 4a. (d) Scheme 3. 4e Me Me C(O)Me 4f Me Ph Ph obtained as mixture of cis/trans isomers. were prepared by reaction 7g Me Ph Ph 4a Me Me CO2Me of dipoles 5 with alkenes 6a–b. 30 min. ATPH is known to give stable complexes with carbonyl compounds such as alkenes 6c and 6d. rt 5 h.26. The inhibitory activity of compounds 4a–f. were separated by med- ium pressure liquid chromatography (MPLC). are reported in Table 2 besides the respective Pearson’s r reported. rt. In this paper we report the synthesis and Compound R1 R2 R3 the anti-HCV activity of 3-amino-2(5H)furanones 4. into 3-methylamino-2. and 8 (EC50 = 19 lM) shows that by replacing the ke- quired to induce an ionic centre at C3 position of the isoxazolidine nucleus which promotes the ring opening of the heterocyclic sys- tem and the subsequent intramolecular lactonization (Scheme 2). Chem. DIBAL-H. Scheme 1. Moreover. (b) DCM dry. isoxazolidine 7d was obtained as single ste. In fact. The regiochemical control towards 4- substituted isoxazolidines 7d and 7e was obtained by using the pinhole Lewis acid as catalyst in mild conditions. Chemical conversion of the isoxazolidine nucleus to 3-amino- a 85% yield. . 2(5H)furanone. while. The reaction of nitrone of N. aniline. diisopropilcarbodiimide. in quantitative yield. The obtained four cycloadducts were separated by been tested directly in a cell-based subgenomic HCV replicon sys- MPLC and the relative stereochemical assignment was performed tem. expressed as EC50. in the presence of ATPH. (c) THF dry.21 promote a competitive side reaction leading to degra. Reagents and conditions: (a) MeOH. con using a cell-based assay. Table 1 keto-enamino functionality. 12 h. toluene. 8 the tetrabutyl ammonium fluoride (TBAF) (75–85% yield). = 132 lM). with or without microwave irradiation affords as major cycloadduct the regioisomer with the ketonic functionality at C5 position. 100 W. carboxamide 8 was obtained by basic treatment of 4a with potas- versed regiochemistry with the presence of a carbonyl group at sium carbonate followed by reaction with aniline in the presence C4 position of the isoxazolidine nucleus. (c) Et2O dry. ATPH 10 mol%.25 The reaction of nitrone 5a with 3-buten-2-one 6c shows a dramatic change of regioselectivity between the catalyzed and uncatalyzed reactions. 7d Me H C(O)Me dipolar cycloaddition of C-alkoxycarbonyl nitrones 5 with suitable 7e Me Me C(O)Me alkenes (Scheme 1 and Table 1). harboring a subgenomic HCV repli- The chemical conversion of 3-alkoxycarbonyl isoxazolidines 7. Reagents and conditions: (a) Microwaves. N. such as 6c and 6d. 78 °C. DIEA. and the relative toxicity. 4e (EC50 this rearrangement is represented by the low critical energy re.e under microwave irradiation. isoxazolidines 7a–c. reflux 6 h. / Bioorg. the N-phenyl these effects. The anti-HCV activity of all the synthesized compounds has see Section 4). while the acid moiety is masked in Substituents on compounds 4–7 the furanose structure. Results and discussion 6d — Me C(O)Me 6e — Ph Ph The synthetic scheme toward 3-amino-2(5H)furanones 4 relies 7a Me Me CO2Me 7b Bn Me CO2Me on the basic treatment of isoxazolidine derivatives 7.25 As a consequence of tained starting from compound 4a (Scheme 3). D. reoisomer. screening of all the compounds was performed up to the fixed con- (5H)furanones 4 have been performed using a mild base. as previously CC50. expressed as carbonyl substituted isoxazolidines the use of NaH. without catalyst the only C5 substituted compound was obtained with a 60% yield. Med. For C4 and 9. such as centration of 103 lM. useful for our biological studies were ob- steric bulk at the carbonyl functionality. 16 (2008) 9610–9615 9611 by the 1-keto-3-imino group. or control of regioselectivity where the ratio 7e/7f is 4:1 (80% yield. values. NaH. 5 h. (b) DMF dry. shows a min. Some of the 5a Me — — newly described compounds have shown a good inhibitory activity 5b Bn — — in a cell-based subgenomic HCV replication assay. 4 h.22–24 The cycloadducts.g. while the desired lactol 9 was ob- 5a with 3-penten-2-one 6d. Iannazzo et al. K2CO3.27 The as mixture of cis and trans isomers. as previously described.3. 4b Bn Me CO2Me The cycloaddition reaction proceeded with high yields (85–90%) 4c Me CO2Et CO2Et and the observed regiochemistry and stereochemistry are in agree. 4d Me H C(O)Me ment with the previously reported results. the inhibition of replication of HCV RNA was measured by NOE measurements. Thus. the expected major cycloadduct arises from a re. easily accessible by 1.

28 4c 229.92 177. lactol 9.16 161. 4f (EC50 = 17 lM. Chem.61 0. present into the N-benzyl substituent.60 182. corresponding to the cleus. . the toxicity of this com.55 224.59 0. a catalytic mechanism has been proposed for polymerases in Inhibitory activity of compounds 4a–f. Iannazzo et al. shows an EC50 of 66 lM. 2 and Table 3) ongoing from ester to amide functional- The biological activity of DKA as inhibitors of NS5B polymerase ities.32 In all the cases. as indi- From these data the amide 8 emerges as the more active com.56 142.08 212. reported in Table 3. besides the complexation energy.77 136..28 On the basis of the considerations that our 3.25 4e 119. The two possible 1:1 Mg2+ complexes for compounds 4a–e. the gain in stability for the 9-C2.35 148.51 0. com- calculations. complete agreement with the biological results.94 0. On 4d 521 0.59 0.19 277. 2). OB and NA atom charges in not complexed compounds Compounda Hf Hf(C1) Hf(C2) DH(react. Med. tution with a hydrogen atom such as in compound 4d The obtained results.93 >1000 — produced in two possible modes. 16 (2008) 9610–9615 Table 2 site.25 8 92.45 0. we have steric hindrance. investigated their ability to complex Mg2+ ions by semiempirical According to the examination of complexation DH values. and the respective Pearson’s r values and in the activation of an incoming nucleophile. optimization was carried out without any symmetry constraints. OB (see Fig. 8 and 9.45 0. show that in all the (EC50 = 521 lM) or ethoxycarbonyl as in 4c (no active) induces a cases the more stable complex. 8 and 9.23 185. Finally. the activity We have also investigated the effect of modifications on the of compounds is directly proportional to the ability of magnesium 2(5H)furanone skeleton. The formation of a cyclic six member complex is al- pound prevents the evaluation of the effect of the aryl ways favoured upon the five membered one.28 a Hf(Mg2+) = 543.56 289.61 0.57 0.81 289.89 these bases we have conducted an in silico study upon the forma- 4f 17 0. pound.58 240. 8 and 9 with Mg2+ ion.24 0. expressed in EC50. thus facilitating its departure from the phosphate.41 0. the interaction of Mg2+ with the lone pairs of the enamine nitrogen When phenyl groups are present at C5 and C4 of furanone nu.95 >1000 — pounds 4a–e. Although there are two magnesium ions in the active pound 4c should show a biological activity similar to that of 4a and 4d.3 lM.16 169.01 143. CC50 = 28 lM). i.89 28 0. The substitution of the methyl group pounds 4a.88 tion and the stabilities of the 1:1 complexes generated by com- 8 19 0. These complexes can be 9 66 0. This modification has produced This trend is also supported by a major electron availability on a 8-fold improvement in potency with respect to 4a. a All the calculations were performed utilizing the new PM6 semiem- CC50 value of >1000 lM ensures to this compound a very high pirical hamiltonian30 as implemented in MOPAC 2007 package31 selectivity index. which at the best of our knowledge is the more ac- tive pyrophosphate inhibitor reported in literature. complexation. IC50 = 0.86 218. and OA. within each compound. since its substi.40 0.97 292.95 280. The second metal 4b >1000 — >1000 — ion also stabilizes the negative charge that appears on the leaving 4c >1000 — >1000 — 3’-oxygen. if compared to 4a-C2. Table 3 Enthalpies of formation (kcal/mol) for C1 and C2 Mg2+ complexes of compounds 4a–e.13 145. arises from significative loss of activity. hydrophobic effect plays an important role with the preference of a hydrophobic substituent at C5 with respect to a polar one.29 Nucleophilic at- Compound EC50 (lM) Pearson’s r CC50 (lM) Pearson’s r tack would then generate a trigonal bipyramidal transition state 4a 525 0. full geometry is an important requisite for the biological activity.23 4d 110. for com- substituents on the activity. calculated the enthalpies for the complexation reaction. the at the nitrogen atom (R1) with a benzyl group leads to a complete stability of Mg2+ complex (C2) follows the trend 8 > 4e > 4a. the biological To obtain the relative stability for each type of complex we have activity changes in the order C(O)NHR > C(O)R > C(O)OR.31 220.25 4b 136.38 236. which does not exhibit any biological activity can be the recent characterization of the affinity of the enzyme for metal ascribed to the net p-Mg2+ stabilizing interaction due to the phenyl ions suggests that magnesium is the cation that is used in vivo dur. Moreover.09 kcal/mol. however.60 0. it is likely to con- ing polymerization. 8 and 9. The better value of the complexation energy showed by com- is related to their ability to chelate Mg2+ and Mn2+ ions. tone functionality with an estereal or an amide ones. (1). however.44 0. The results in Table 2 suggest that R2 = methyl using Winmostar as GUI interface. sider that this conformation is precluded in the enzymatic site for amino-2(5H)furanones can be regarded as DKA mimetics.86 142.88 0.82 819 0. oxygen.20 Moreover.15 lM).27 141. corresponding to the two different sites of complexation (Fig. / Bioorg.6-dihydroxy-2- DHðreact:Þ ¼ HfðcomplexÞ  ½Hfðisolated form of the ligandÞ þ HfðMg2þ Þ  ð1Þ (2-thienyl)pyrimidine-4-carboxylic acid (EC50 = 9. and of the oxygen of the carbonyl group at C4. arises from the increasing of negative charges on both NA and OB atoms due to the loss of the attractive conjugation with the lactone Figure 2. complex-1 (C1) and complex-2 (C2). in lack of activity (see Table 2).28 0.45 0.25 9 176.19 0.44 0. chosen as model com.78 147. However the observed lack of antiviral activity of 4c (R1 = R2 = CO2Et) suggested that.e.92 >1000 — that would be stabilized by both metal ions.12 303.) for C1 DH(react. cated in Eq. 4e and 8. pound 4b.15 153.95 145. which one metal ions is involved in both positioning the substrate expressed in CC50.59 0.) for C2 OA q(e) OB q(e) NA q(e) 4a 161.59 0. relative toxicity.81 >1000 — 4e 132 0.9612 D. Thus. pound and its EC50 is comparable with that of 5. which differ for the R3 substituent. complex 2 site.61 139.

125 MHz) d 14.040–0. 13C NMR (125 MHz.1. The analytical and spectroscopical data were previously d 14.55 (dd. 1H.and (Z)-C-ethoxycarbonyl-N-benzyl nitrone 5b. J = 7.5.6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acid 4. 70.2. Melting points were determined with a Kofler apparatus at room temperature. Iannazzo et al.85 (d. the mixture was filtered on a Celite and are reported uncorrected.1 Hz).1005. isoxazolidine (7a) J = 6. 3. 13C NMR dry toluene (5 mL) in a pressure tube equipped with a stir bar (125 MHz. J = 6.25 (d. 4.1 Hz).1158.13 (dd.and (Z)-C-ethoxycarbonyl-N-methyl nit- rone 5a and (E). 3H). J = 7. 210. Thin-layer chromatographic separations were performed on Merck 4. 16 (2008) 9610–9615 9613 3. inhibitors of subgenomic HCV RNA in the replicon assay. HRMS rate 6b. 1. 4.1 Hz). 43. 2.3 lM)20 which.5.1155.21 (m. CDCl3) d from N-methyl-C-ethoxycarbonyl nitrone 5a and dimethyl fuma- 4.1 Hz).1 Hz).5 Hz).2. J = 5. Found: ratus and heated at 90 W. 3H. 2. Focused Microwave System. 43. 3. 2. 2. 3H). 3H. J = 6.4).30 The synthesis of compounds 7b as cis/trans mixture.20 (q. The synthesis of compounds 7a as cis/trans mixture.2. Model Discover.0.7. 1H.1 Hz).86 (d.22 (q. 1H. 3. isoxazolidine (7d) phy was accomplished on Merck silica gel (200–400 mesh). yellow oil.0 mL min1.5.1 Hz). 3-Ethoxycarbonyl-5-acetyl- scribed procedures: (E). 3.2. Found: 215.8 mmol in 10 mL of CH2Cl2) was added Solvents and reagents were used as received from commercial dropwise during 20 min. 4. 30. 42. Flash chromatogra.1 Hz). 15. 43. light yellow oil. Found: 215. CDCl3) d 15.6-diphenylphenol (290 mg.063 mm and the eluting solvents were (ddd.2. 3H).2.3.1 Hz). CDCl3) d 15.1001. Then.4. 2. 4. 2.7 Hz). Found: 201. 13C NMR reaction under microwave irradiation was carried out using a (CDCl3. 2.9 Hz).5–7. 43.4. A solution of nitrone 5 (0.6.85 (d. 2H. 3. a solution of nitrone 6a (3. started J = 8. 67.80 (s.1158. J = 7.2 Hz).45 (d. J = 7. light yellow oil. yellow oil.82 (s.81 (s.32 (t. Nuclear magnetic reso. The analytical and spectroscopic data were previously (methylamino)-5-oxo-N-phenylfuran-3-carboxamide 8 emerges reported. The analytical and spectroscopical data were previously Calcd for (M+) C10H17NO4: 215. 4. HRMS Calcd for (M+) from N-methyl-C-ethoxycarbonyl nitrone 5a and methyl crotonate C10H17NO4: 215. at the best of our knowledge. 208. evaporated and the resulting solid was purified by MPLC on a silica Second eluted compound.5 mmol) and alkene 6 (1. 1H). Experimental trimethylaluminum (0. was inserted into the cavity of a discover Microwave System appa- 170. 3 H). Light yellow oil. 13C NMR re.5. The iden.7. The 4.5-dihydro-2-methyl-4.4-dimethyl isoxazolidine (7f) 4. HRMS Calcd for (M+) C10H17NO4: 215.0 and 8. 1H NMR gel with cyclohexane/ethyl acetate (80:20).95 (m.5 Hz). 25.23 Calcd for (M+) C10H17NO4: 215.3.2 mL.40 (quintet. 171. 1H NMR 7g (CDCl3.4SR)-3-Ethoxycarbonyl-4-acetyl-2-methyl silica gel 60-F254 precoated aluminum plates. (CDCl3.22 4.25 (s. 17. 500 MHz) d 1. 3H. 1H.5. 5.4. 99.1. 25. yield 43%. 13C NMR (125 MHz.1 Hz).3. 0.5. 65.5. The reaction of N-methyl-C-ethoxycarbonyl nitrone 5a and but- corded at 75 or 125 MHz) were obtained on Varian Instruments 3-en-2-one 6c affords 7d as single stereoisomer (yield 85%). 3H). isoxazolidine (7c) 3H).7 Hz). CDCl3) 6a.9.90 (m.85 (s.3. Conclusions 4. 61.29 (t. Found: 215. HRMS Calcd for (M+) C9H15NO4: 201.0. parative separations were carried out by MPLC Büchi C-601 using J = 7. the filtrate was evaporated in vacuo and the residue subjected with a Perkin-Elmer elemental analyzer. Second eluted compound: yield 8%.0 and 7. 3H. and are referenced in ppm relative to TMS or the solvent signal.6. HRMS reported.7. started 61. 3H). 2. from N-methyl-C-ethoxycarbonyl nitrone 5a and trans stilbene have been reported.2. 2. 65.4.3. The analytical and spectroscopical data were previously reported.12 (d. J = 7.1. J = 7.0 Hz). The synthesis of compounds 7a as cis/trans mixture. 33.5. ture of two cis/trans isomers (global yield 80%). 1H). 2. 1H.2. 61. From this study. D.1158. after 30 min. 2M solution in toluene) and the solu- tion was left stirring for 30 min at this temperature.5-dimethoxycarbonyl 2-methyl 500 MHz) d 1. J = 7. 59.2 Hz). 208.0 Hz).30 (d. 1H NMR (CDCl3.7 and 8. 2.9.0. tification of samples from different experiments was secured by The reaction of N-methyl-C-ethoxycarbonyl nitrone 5a and mixed melting points and superimposable NMR spectra.1158. started pounds.5. nance spectra (1H NMR recorded at 300 or 500 MHz. 1H.27 (t. reported. 2H. 1H NMR (CDCl3. First eluted compound. J = 6. 500 MHz) d 1. 1H.30 (q.32 (d.0.17 (dd.38 mmol) in dry dichloromethane (20 mL) at 0 °C was added under N2 atmosphere 4. J = 8. 2H. 3-Ethoxycarbonyl-4-methoxycarbonyl-2. Pre.3.43 (d. 28.1. yield 21%. 28. pent-3-en-2-one 6d affords the regioisomers 7e and 7f as a mix- The following compounds were prepared according to de. 4. 2H. 3H. from N-benzyl-C-ethoxycarbonyl nitrone 5b and methyl crotonate 1H. 169. 1. 2. 1. General procedure for the synthesis of isoxazolidines 7a–c. 4.6. 1H NMR (CDCl3. J = 7. 210. 1. 1H). The mixture was 215.25 (q. 3-Ethoxycarbonyl-4-methoxycarbonyl-2-benzyl-5- First eluted compound: yield 8%. is the more active pyrophosphate inhibitor in the replicon assay. To a solution of 2.7 Hz). 171.1 Hz). 500 MHz) d 1.30 (s. J = 7.31 (d. Elemental analysis was performed pad. J = 5.2. methyl isoxazolidine (7b) 500 MHz) d 1. 15.1.9. 98. 2. 61. 6e.1156.5 mmol) in 1H. 3H. started (s. General procedure for the synthesis of isoxazolidines 7d–f (EC50 = 9. 4.3. CEM Corp.7 Hz). 65.7 Hz).2. to MPLC chromatography.0.5-diphenyl-2-methyl isoxazolidine (7g) The synthesis and the biological activity of a new class of com.22 (q. J = 6. J = 5. J = 6. 3H.2. for 20–30 min. 1H. Then.8 mmol of alkene 6c or 6d was added at 0 °C and. 2H.4 and 8. 70. 3-Ethoxycarbonyl-4-acetyl-2. 3-Ethoxycarbonyl-4. 3H).74 (d. J = 5. 71.5.20 (s. J = 6.2.1 Hz). Chem. 1H.0. J = 5.0 and 6.5. 65. 4.3 Hz).45 (dd. 61. The reaction mixture was stirred for 12 h sources. 1H. J = 5.1160.7.0. 3H. 17. J = 8. delivered by a pump at the flow-rate of 3. 3.0. Med.87 Merck silica gel 0.40 (d. (3RS. 3H).27 4.5-dimethyl J = 5. 2.20 (s. 34.5. 4.78 (s. 62.9 Hz). 1H.23 as the most active compound and its EC50 is comparable with that of 5. 13C NMR (125 MHz. General 3.2. 80 °C.4. / Bioorg.49 (d. 171.23 . 3-Ethoxycarbonyl-4. 6a. 3H). 62. 194.5-dimethyl isoxazolidine (7e) 4. The synthesis of compounds 7a as cis/trans mixture.0.3. 2. 3H. 4. 3.

500 MHz) d 1.2 ratio and in a quantitative yield as yellow oil. J =4.30 (t. 3. 2 mmol. 3H).79 (s.10 (s.10 (d.30 (m. J = 8.1008. mp 120–124 °C. 3H. 27. Cell culture (br s.3. 29. 500 MHz) d 1. 6. HRMS Calcd for (M+) 1H). 94.9. 97.4. Life Technologies. quenched by the addition of brine and extracted three times with red for 4 h at room temperature. 3. 3H. J = 7. 102.5-Diphenyl-3-methylamino-2(5H)furanone (4f) 4.0.7. 57. 5. 3H. 1 mmol) were added N. 1H. 13C NMR (CDCl3.8. 3H).2.3.0. 138. 3H).30 (d. 129. At the end of this time. Found: 169. St.38 (t. Synthesis of Methyl 2. NH). 3. light yellow solid. were grown as described for Huh-7 cells. Anti-hepatitis C virus assay Yield 78%. 4-Acetyl-3-methylamino-2(5H)furanone (4d) 4.0691. 3H). yellow oil. 3. 175. C11H15NO6: 257. 2(5H)furanone (4a) 125 MHz) d 21. The reaction mixture was then EtOAc. HCV subgenomic replicon have been previously described.26 They 3H. 3 mmol) at room tempera- Method B. 3H. Pero.4. 1H NMR (CDCl3.02 (br s. The residue was purified by MPLC using sure.80 (s.3 Hz). 13 mmol) and DIEA (500 lL. 7.2. MO) and serially diluted in D-MEM in a way that oxo-N-phenylfuran-3-carboxamide (8) DMSO concentration was never higher than 1%. 5. J = 0.4. J = 7. at the same temperature for 5 h.1. 5H).5. 7. trations ranging from 102 to 1 U/mL. 500 MHz) 154. 2H. 158. ified minimal essential medium (D-MEM. 79.3.8 mg of neomycin sulfate (G418.9 Hz).4.2 Hz). J = 7.7. 1.0739.N-diisopropilcarbodiimide using CHCl3/MeOH (99:1) as eluent.35 (d. 1H). EuroClone. 500 MHz) d 1. 128. 29.5.0582.14 (q.1. 171. 6. 29. J = 7. 3.95 (br s. 1H).6. 70. J = 5.62 (d.1001. mp 80–82 °C.7. 125 MHz) d 21.2 Hz).45 (s. NH) with MeOH (1 mL) and water (1 mL) and the resulting precipitate 7.1 Hz).21 (d. but the medium was sup- J = 6.1. 109. concentrated in vacuo. Found: 265. 13C NMR (CDCl3.1.4.05 (dd.1. 5. light yellow oil. 168.2 Hz). J = 6.4. 124. 1H NMR (CDCl3.5-dihydro-2-methyl-4-(methylamino)-5. 46.05 (d. 50. 3H).30 (q. 7.2 Hz). 61.7.8. 1. The organic layers were dried over anhydrous Na2SO4 and quenched with water (0. 1H. Germany) were grown in Dulbecco’s mod- Found: 155.3. 109. 4.9 Hz). 13C NMR (CDCl3.97 (q. 98. 1H). 3H). 500 MHz) d 1. 1H). 13C NMR (CDCl3. (br s.6. 5-Methyl-4-acetyl-3-methylamino-2(5H)furanone (4e) Paisley.8.7. Found: 185. 1H.1 Hz).5 Hz).95 (br s.4. 3H. supplemented with 10% fetal bovine serum (FBS. J = 6. was utilised. 117. J = 6. Minor 2H. 5.79 (s. 3H. 5H).8.20 (br s.22 (d. J = 6. 1H). 50.3. J = 4. Med. 1H. 13C NMR (CDCl3.6.27 Briefly.1 Hz).1.2 Hz). 5. C17H15NO2: 265.6. (500 mg. 3H. 142. 1H. HRMS Calcd for (M+) with different concentrations of the compounds or control diluent. 3H. To a solution of the crude material in dry DMF (3 mL) and ani- the solvent was removed and the residue was purified by MPLC line (103 mg. 3H). anomer: 1H NMR (CDCl3.8. 2H).1103.70 (br s.9614 D. 1H NMR (CDCl3. 4.2.1 mmol) was dissolved in methanol was performed in triplicate. 127. The filtrate was evaporated 87. the mixture was stirred at 50 °C for 6 h. 61. 5-Methyl-4-methoxycarbonyl-3-methylamino. Louis.0.4.8. 1 mmol) and the mixture was stir. 1 mmol) in anhydrous ether 2(5H)furanone (4b) (30 mL) at 78 °C. 112. 30. 3. NH).94 4.9.7. 10H). CO. 75.10 NH).1 Hz).5 Hz).57 (d.and b-anomers of for (M+) C14H15NO4: 261.5. yellow oil. 500 MHz) d 2. 129. 127.5.1. Ma- jor anomer: 1H NMR (CDCl3. white solid. 10. The reaction mixture was stirred for 5 h and then was (10 mL) was added NaH (24 mg.2. 1H NMR (CDCl3. yellow oil. The residue was purified by MPLC using CHCl3/MeOH (99:1) ethyl acetate/cyclohexane 4:6 as eluent to give 8 as yellow oil as eluent. 1. 128.25 (d. 3H. was filtered under reduced pressure.1 Hz).5. The inhibitor . 74. Scotland. 51.3. d 1.7.3 (br s.0.7.5 Hz). 1H).0.7. Compounds were dissolved in DMSO (Sigma Chemicals 4. 126.2. trypsin–EDTA. 79.80 (br 4. 166. (q. 3.2 Hz).9. 126. 4. 3H.5-Diethoxycarbonyl-3-methylamino-2(5H)furanone (4c) 3. BAL-H (2 mL.1.1.5. Mainz.13 (s. 75. 1H. 125 MHz) d 13. 500 MHz) d 2.95 (d. 5-Methyl-4-methoxycarbonyl-3-benzylamino. 106. 6. To a solution of 4a (185 mg.0579.1. 142.5. Found: 261. 1H). 124. 125 MHz) d 20.1 mmol. 1M) was added and the mixture was stirred J = 6. were assayed for NS3 protein expression with the anti-NS3 10E5/ 24 MAb. 1H. Final concentra- tions of the compounds were 103. 121.5.1 Hz).7. s.2 Hz). 4. 1H).1 mL. 51. J = 7. 45.1105. 13C NMR (CDCl3. 165.1 and 12. Huh-7 cells. J = 5.10–7. HRMS Calcd for (M+) Life Technologies). 13. 4. The assay Compound 4a (205 mg. (d. 1. HRMS Calcd 4. HRMS Calcd in vacuo to obtain an inseparable mixture of a.40 (m. 125 MHz) d 24.2 Hz).2.7.7 Hz).35 (d. 83. plemented with the addition of 0. IFN-a at concen- (3 mL) and treated with a 10% aqueous solution of potassium car. Iannazzo et al.70 (br s. say. 3. 125 MHz) d 22. J = 7.9. 165. 29. under nitrogen atmosphere a solution of DI- Yield 80%. 173. 162. The crude was treated Method A. HBI10A cells. 2.0735. J = 8.1.. as previously described. The disappearance of the starting material was 2(5H)furanones 4a–e monitored by TLC (CHCl3/MeOH 7:3). 132. 3H. The effect of compounds on HCV viral replication was moni- 4.0894. To a solution of isoxazolidine 7f (1 mmol) in dry THF ture. 171. lactol 9 in 1:1. Found: 257. 4. J = 7. As a positive control. 167. Yield 85%.0899.4 Hz).5 mL) and evaporated under reduced pres. / Bioorg. 6.61 (s.20–7. 202. Italy).3.1. 158.1004. 3H. 86.5 Hz).1.0688. 5.96 (s. J = 0.7. 167. J = 5. 8. Cells were passaged 1:5 twice a week using 1 C8H11NO3: 169.4. HRMS Calcd for (M+) C7H9NO3: 155.5-dihydro-5-hydroxy-2-methyl-4- for (M+) C8H11NO4:185. 129.20 (q. bonate (3 mL).4. originally obtained from Ralf Bartenschlager (Uni- 158.0. 5.35 (d. (methylamino)furan-3-carboxylate (9) 4.1006.6. 4. 3H. 8. 1H NMR (CDCl3.6. 1M in THF) and reduced pressure. 6. 13C NMR (CDCl3. 500 MHz) d 1.7. 26. 13C NMR (CDCl3. 1H.4. 128. UK).25 (q.0.10 Found: 246. 168. 6. 67. 131.4.3 (q. 3H. The mixture was neutralized with 2 N HCl and then evaporated in vacuo. 167. 500 MHz) d 1. J = 7. Synthesis of 2. 4. 3.20 (dq.5.12 (br s. 45 (d. To a solution of isoxazolidine 7a–e (1 mmol) in dry with chloroform and the organic extracts were concentrated under THF (10 mL) was added TBAF ( 1H NMR (CDCl3. (yield 85%).3.95 (s. versity of Mainz. 16 (2008) 9610–9615 4. General procedure for the synthesis of 3-amino. J = 6. 136. 125 MHz) d 26.8. 5. 5. J = 7. The solution was then quenched 1H. and 1 lM.5 Hz).1. 13C NMR (CDCl3.0. 4. 124. 1H NMR (CDCl3. either treated 128.6.0. Huh-7-derived HBI10A cells expressing an Yield 75%.5.5 Hz).77 (s.5. 7. 109.4.0. tored in HBI10A cells by a cell-enzyme-linked immunosorbent as- 125 MHz) d 32.3 Hz). 3H). 125 MHz) d 21.1 and 12. 120. Chem. 1H.20 (s. 129.5 and Yield 79%.0.30 (m. HRMS Calcd for (M+) C13H14N2O3: 246. 4. Biological assays Yield 82%.9. 4.4. 1H. 6. J = 6.2.95 (dd. 3.4.

coefficient. Mastino. was also calculated for each significant value of EC50. Chem. U. PCT Int. ables. V.. EMBO J. Z. B... 2004.. L.Chem. M. Moradpour. 1989. L. 2004.1007/s00894-007-0233-4. S. J. C. S. Makeyev. Chiacchio. X... Appl. Atenni. R. J. S. S. Lauer. L.. A. 18202– was calculated by fitting the data to the Hill equation: fraction 18210. Narjes. Romeo. Padron. Preparation of antiviral azanucleoside derivatives as inhibitors RNA-dependent viral polymerases. Pawlotsky. Muraglia.. V. where Ai is the 9. C. I. Shim. This work was partially supported by M. Matassa.. Bartholomew. Fay. Malancona.. 31–43. J. Koch. G. S... De Clercq. J. Biol. F.html..5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy.. De Francesco. 103. Pace.. 5284–5297. R. 115–133.. Kanemasa. Bonatti. 207–219. CO. Uccella. R.. J.. DeFrancesco. 3669–3679. Chiacchio.. L.. I. A. Dubuisson. De Francesco. Hong. Samples were then incubated for a further 4 h at 37 °C before 19. A. Casuscelli. 2004. Rescifina. 3673–3674.. 1693–1705. A. A. Paonessa. Neuner. Z. Carroll. Di Marco. J. product–moment correlation coefficient (Pearson’s r). 39.. S. S. Colarusso. Rowley. Di Bella.R. Chem. Biswal.. B. Burlein. Curr. F. 2007.. Chem. W. U. 4. J. Conte. 5. Chemistry. and n is the Hill 2001.I. 47. Antiviral Res. Tomas. I. P.. Romeo. Naim. M. Bedard. T. J. C. 8605–8612. H.. Bennett. Investig. J. 25. Wu.. Butcher. 2000.. M. Chan. 2547– examined function and x is the drug concentration.. R. phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] were added to each Tomei. MEM + 5% FBS in a 96 well plate and after 4 h incubation at 37 °C 16. G. J... Romeo. Grimes. E. / Bioorg. 7. L. S. G. Di Bella. Med. 49. Biol. S.. Narjes. G. 2000. where y is the value of the Francesco. 20 lL of 2000. 2002... A. Dande. Virol.101).net/Featuresof_PM6. 410.. 47. Thomson. Nazionale: Sintesi Stereselettiva e valutazione biologica di comp. M. G.. Leone. 3868–3875. J. Stuart.. Bilimoria. Love. J. G. Wang. 2000. 18. 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