3-Amino-2(5H)furanones as inhibitors of subgenomic hepatitis C virus RNA replication

Article abstract of DOI:10.1016/j.bmc.2008.09.006

A new class of compounds able to block the replication of subgenomic HCV RNA in liver cells is described. 3-Amino-2(5H)furanones 4 may be regarded as diketoacid analogues and were obtained by basic rearrangement of the isoxazolidine nucleus.

Full text of DOI:10.1016/j.bmc.2008.09.006

Bioorganic & Medicinal Chemistry 16 (2008) 9610–9615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3-Amino-2(5H)furanones as inhibitors of subgenomic hepatitis C virus
RNA replication
a
a
a
b
Daniela Iannazzo a, , Anna Piperno , Giovanni Romeo , Roberto Romeo , Ugo Chiacchio ,
*
Antonio Rescifina b, Emanuela Balestrieri c, Beatrice Macchi c, Antonio Mastino d, Riccardo Cortese e
a Dipartimento Farmaco-Chimico, Università di Messina, Via SS. Annunziata, Messina 98168, Italy
b Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, Catania 95125, Italy
c Dipartimento di Neuroscienze, Università di Roma ‘‘Tor Vergata”, Via Montpellier 1 and IRCSS S. Lucia, Roma 00133, Italy
d Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, Università di Messina, Salita Sperone 31, Messina 98168, Italy
e 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:
Received 8 May 2008
Revised 27 August 2008
Accepted 4 September 2008
Available online 7 September 2008
A new class of compounds able to block the replication of subgenomic HCV RNA in liver cells is described.
3-Amino-2(5H)furanones 4 may be regarded as diketoacid analogues and were obtained by basic rear-
rangement of the isoxazolidine nucleus.
Ó 2008 Published by Elsevier Ltd.
Keywords:
DKA
HCV
Furanones
1,3-Dipolar cycloaddition
Isoxazolidines
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
problem, which affects more than 170 million individuals.1,2 The
NS5B RNA-dependent RNA polymerase (NS5B RdRp) has shown
to be the catalytic core of the HCV replication machinery.3,4 This
enzyme is not expressed in uninfected cells, and, due to its unique
features, represents an attractive target for the development of safe
antiviral drugs.5–7
The catalytic activity of the enzyme is mediated, in the active
site, by two magnesium ions, which serve to activate the 3’-OH
of the elongating RNA and to position the incoming nucleotide-tri-
phosphate for the nucleophilic attack.8–11
been reported: phenyl-DKA18 like 1, meconic acid derivatives 219
and carboxypyrimidines 320 (Fig. 1).
In these last years, we have developed an efficient synthetic
procedure towards the construction of 3-amino-2(5H)furanones
by basic treatment of 3-alkoxycarbonyl substituted isoxazoli-
dines.21 We have assumed that the introduction of a carbonyl
group at the C4 position of the 3-amino-2(5H)furanone skeleton,
as in compounds 4, could produce potential inhibitors of pyrophos-
phate site. In fact, 3-amino-2(5H)furanones 4 may be regarded as
DKA cyclic analogues: the 1,3-diketonic functionality is replaced
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.
doi:10.1016/j.bmc.2008.09.006

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

9612
D. Iannazzo et al. / Bioorg. Med. Chem. 16 (2008) 9610–9615
Table 2
site, a catalytic mechanism has been proposed for polymerases in
which one metal ions is involved in both positioning the substrate
and in the activation of an incoming nucleophile.29 Nucleophilic at-
tack would then generate a trigonal bipyramidal transition state
that would be stabilized by both metal ions. The second metal
ion also stabilizes the negative charge that appears on the leaving
3’-oxygen, thus facilitating its departure from the phosphate. On
these bases we have conducted an in silico study upon the forma-
tion and the stabilities of the 1:1 complexes generated by com-
pounds 4a–e, 8 and 9 with Mg2+ ion. These complexes can be
produced in two possible modes, complex-1 (C1) and complex-2
(C2), corresponding to the two different sites of complexation
(Fig. 2).
Inhibitory activity of compounds 4a–f, 8 and 9, expressed in EC50, relative toxicity,
expressed in CC50, and the respective Pearson’s r values
Compound
EC50
525
>1000
>1000
521
132
17
(
lM)
Pearson’s r
CC50
(
l
M)
Pearson’s r
4a
4b
4c
4d
4e
4f
8
0.92
—
—
0.81
0.82
0.89
0.95
0.93
>1000
>1000
>1000
>1000
819
28
>1000
>1000
—
—
—
—
0.89
0.88
—
19
66
9
—
To obtain the relative stability for each type of complex we have
calculated the enthalpies for the complexation reaction, as indi-
cated in Eq. (1).
tone functionality with an estereal or an amide ones, the biological
activity changes in the order C(O)NHR > C(O)R > C(O)OR.
From these data the amide 8 emerges as the more active com-
pound and its EC50 is comparable with that of 5,6-dihydroxy-2-
D
H
¼ HfðcomplexÞ ꢀ ½Hfðisolated form of the ligandÞ þ HfðMg2þÞꢁ
ð1Þ
ðreact:Þ
(2-thienyl)pyrimidine-4-carboxylic
IC50 = 0.15 M), which at the best of our knowledge is the more ac-
tive pyrophosphate inhibitor reported in literature.20 Moreover, a
acid
(EC50 = 9.3 lM;
l
All the calculations were performed utilizing the new PM6 semiem-
pirical hamiltonian30 as implemented in MOPAC 2007 package31
using Winmostar as GUI interface.32 In all the cases, full geometry
optimization was carried out without any symmetry constraints.
The obtained results, reported in Table 3, show that in all the
cases the more stable complex, within each compound, arises from
the interaction of Mg2+ with the lone pairs of the enamine nitrogen
and of the oxygen of the carbonyl group at C4, corresponding to the
complex 2 site. The formation of a cyclic six member complex is al-
ways favoured upon the five membered one. Moreover, for com-
pounds 4a, 4e and 8, which differ for the R3 substituent, the
stability of Mg2+ complex (C2) follows the trend 8 > 4e > 4a, in
complete agreement with the biological results, i.e., the activity
of compounds is directly proportional to the ability of magnesium
complexation.
CC50 value of >1000 lM ensures to this compound a very high
selectivity index. The results in Table 2 suggest that R2 = methyl
is an important requisite for the biological activity, since its substi-
tution with
(EC50 = 521
a
hydrogen atom such as in compound 4d
l
M) or ethoxycarbonyl as in 4c (no active) induces a
significative loss of activity.
When phenyl groups are present at C5 and C4 of furanone nu-
cleus, 4f (EC50 = 17 lM; CC50 = 28 lM), the toxicity of this com-
pound prevents the evaluation of the effect of the aryl
substituents on the activity. The substitution of the methyl group
at the nitrogen atom (R1) with a benzyl group leads to a complete
lack of activity (see Table 2).
We have also investigated the effect of modifications on the
2(5H)furanone skeleton. Thus, lactol 9, chosen as model com-
This trend is also supported by a major electron availability on
OB (see Fig. 2 and Table 3) ongoing from ester to amide functional-
ities. The better value of the complexation energy showed by com-
pound 4b, which does not exhibit any biological activity can be
pound, shows an EC50 of 66 lM. This modification has produced
a 8-fold improvement in potency with respect to 4a.
The biological activity of DKA as inhibitors of NS5B polymerase
is related to their ability to chelate Mg2+ and Mn2+ ions; however,
the recent characterization of the affinity of the enzyme for metal
ions suggests that magnesium is the cation that is used in vivo dur-
ing polymerization.28 On the basis of the considerations that our 3-
amino-2(5H)furanones can be regarded as DKA mimetics, we have
investigated their ability to complex Mg2+ ions by semiempirical
calculations. Although there are two magnesium ions in the active
ascribed to the net p
-Mg2+ stabilizing interaction due to the phenyl
present into the N-benzyl substituent; however, it is likely to con-
sider that this conformation is precluded in the enzymatic site for
steric hindrance.
According to the examination of complexation
DH values, com-
pound 4c should show a biological activity similar to that of 4a and
4d. However the observed lack of antiviral activity of 4c
(R1 = R2 = CO2Et) suggested that, besides the complexation energy,
hydrophobic effect plays an important role with the preference of a
hydrophobic substituent at C5 with respect to a polar one.
Finally, the gain in stability for the 9-C2, if compared to 4a-C2,
arises from the increasing of negative charges on both NA and OB
atoms due to the loss of the attractive conjugation with the lactone
oxygen.
Figure 2. The two possible 1:1 Mg2+ complexes for compounds 4a–e, 8 and 9.
Table 3
Enthalpies of formation (kcal/mol) for C1 and C2 Mg2+ complexes of compounds 4a–e, 8 and 9, and OA, OB and NA atom charges in not complexed compounds
Compounda
Hf
Hf(C1)
Hf(C2)
D
H(react.) for C1
D
H(react.) for C2
OA q(e)
OB q(e)
NA q(e)
4a
4b
4c
4d
4e
8
ꢀ161.58
ꢀ136.55
ꢀ229.92
ꢀ110.97
ꢀ119.95
ꢀ92.12
240.38
224.31
177.16
292.56
280.19
303.81
218.08
236.27
220.60
169.77
289.61
277.86
289.78
212.35
ꢀ141.13
ꢀ182.23
ꢀ136.01
ꢀ139.56
ꢀ142.95
ꢀ147.16
ꢀ148.15
ꢀ145.24
ꢀ185.94
ꢀ143.40
ꢀ142.51
ꢀ145.28
ꢀ161.19
ꢀ153.88
ꢀ0.45
ꢀ0.45
ꢀ0.41
ꢀ0.44
ꢀ0.45
ꢀ0.44
ꢀ0.57
ꢀ0.59
ꢀ0.59
ꢀ0.59
ꢀ0.59
ꢀ0.60
ꢀ0.61
ꢀ0.61
ꢀ0.25
ꢀ0.28
ꢀ0.23
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.28
9
ꢀ176.86
a
Hf(Mg2+) = 543.09 kcal/mol.

D. Iannazzo et al. / Bioorg. Med. Chem. 16 (2008) 9610–9615
9613
4.2.4. 3-Ethoxycarbonyl-4,5-diphenyl-2-methyl isoxazolidine
(7g)
3. Conclusions
The synthesis of compounds 7a as cis/trans mixture, started
from N-methyl-C-ethoxycarbonyl nitrone 5a and trans stilbene
6e. The analytical and spectroscopic data were previously
reported.23
The synthesis and the biological activity of a new class of com-
pounds, inhibitors of subgenomic HCV RNA in the replicon assay,
have been reported. From this study, 2,5-dihydro-2-methyl-4-
(methylamino)-5-oxo-N-phenylfuran-3-carboxamide
as the most active compound and its EC50 is comparable with that
of 5,6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acid
(EC50 = 9.3
M)20 which, at the best of our knowledge, is the more
8 emerges
4.3. General procedure for the synthesis of isoxazolidines 7d–f
l
To a solution of 2,6-diphenylphenol (290 mg, 0.38 mmol) in dry
dichloromethane (20 mL) at 0 °C was added under N2 atmosphere
trimethylaluminum (0.2 mL, 2M solution in toluene) and the solu-
tion was left stirring for 30 min at this temperature. Then,
3.8 mmol of alkene 6c or 6d was added at 0 °C and, after 30 min,
a solution of nitrone 6a (3.8 mmol in 10 mL of CH2Cl2) was added
dropwise during 20 min. The reaction mixture was stirred for 12 h
at room temperature. Then, the mixture was filtered on a Celite
pad, the filtrate was evaporated in vacuo and the residue subjected
to MPLC chromatography.
active pyrophosphate inhibitor in the replicon assay.
4. Experimental
4.1. General
Solvents and reagents were used as received from commercial
sources. Melting points were determined with a Kofler apparatus
and are reported uncorrected. Elemental analysis was performed
with a Perkin-Elmer elemental analyzer. Nuclear magnetic reso-
nance spectra (1H NMR recorded at 300 or 500 MHz, 13C NMR re-
corded at 75 or 125 MHz) were obtained on Varian Instruments
and are referenced in ppm relative to TMS or the solvent signal.
Thin-layer chromatographic separations were performed on Merck
silica gel 60-F254 precoated aluminum plates. Flash chromatogra-
phy was accomplished on Merck silica gel (200–400 mesh). Pre-
parative separations were carried out by MPLC Büchi C-601 using
Merck silica gel 0.040–0.063 mm and the eluting solvents were
delivered by a pump at the flow-rate of 3.5–7.0 mL minꢀ1. The
reaction under microwave irradiation was carried out using a
CEM Corp. Focused Microwave System, Model Discover. The iden-
tification of samples from different experiments was secured by
mixed melting points and superimposable NMR spectra.
The reaction of N-methyl-C-ethoxycarbonyl nitrone 5a and but-
3-en-2-one 6c affords 7d as single stereoisomer (yield 85%).
4.3.1. (3RS,4SR)-3-Ethoxycarbonyl-4-acetyl-2-methyl
isoxazolidine (7d)
Light yellow oil. 1H NMR (CDCl3, 500 MHz) d 1.27 (t, 3H,
J = 7.1 Hz), 2.25 (s, 3H), 2.78 (s, 3H), 3.74 (d, 1H, J = 5.4), 3.87
(ddd, 1H, J = 5.0, 5.4 and 8.7 Hz), 4.13 (dd, 1H, J = 5.0 and 8.7 Hz),
4.17 (dd, 1H, J = 8.7 and 8.5 Hz), 4,22 (q, 2H, J = 7.1 Hz). 13C NMR
(CDCl3, 125 MHz) d 14.0, 28.4, 42.9, 59.4, 61.7, 67.4, 70.1, 171.5,
194.0. HRMS Calcd for (M+) C9H15NO4: 201.1001. Found: 201.1005.
The reaction of N-methyl-C-ethoxycarbonyl nitrone 5a and
pent-3-en-2-one 6d affords the regioisomers 7e and 7f as a mix-
ture of two cis/trans isomers (global yield 80%).
The following compounds were prepared according to de-
scribed procedures: (E)- and (Z)-C-ethoxycarbonyl-N-methyl nit-
rone 5a and (E)- and (Z)-C-ethoxycarbonyl-N-benzyl nitrone 5b.27
4.3.2. 3-Ethoxycarbonyl-4-acetyl-2,5-dimethyl isoxazolidine
(7e)
4.2. General procedure for the synthesis of isoxazolidines 7a–c,
7g
First eluted compound; yield 43%, light yellow oil; 1H NMR
(CDCl3, 500 MHz) d 1,29 (t, 3H, J = 7.1 Hz), 1.43 (d, 3H, J = 6.3 Hz),
2.20 (s, 3H), 2.80 (s, 3H), 3.45 (dd, 1H, J = 5.0 and 7.5 Hz), 3.49 (d,
1H, J = 5.0 Hz), 4.20 (q, 2H, J = 7.1 Hz), 4.21 (m, 1H). 13C NMR
(125 MHz, CDCl3) d 15.5, 17.2, 28.9, 43.3, 61.5, 62.0, 65.6, 71.2,
170.0, 210.0. HRMS Calcd for (M+) C10H17NO4: 215.1158. Found:
215.1155.
A solution of nitrone 5 (0.5 mmol) and alkene 6 (1.5 mmol) in
dry toluene (5 mL) in a pressure tube equipped with a stir bar
was inserted into the cavity of a discover Microwave System appa-
ratus and heated at 90 W, 80 °C, for 20–30 min. The mixture was
evaporated and the resulting solid was purified by MPLC on a silica
gel with cyclohexane/ethyl acetate (80:20).
Second eluted compound; yield 21%, light yellow oil; 1H NMR
(CDCl3, 500 MHz) d 1,32 (t, 3H, J = 7.2 Hz), 1.40 (d, 3H, J = 6.1 Hz),
2.20 (s, 3H), 2.81 (s, 3H), 3.45 (d, 1H, J = 5.0 Hz), 3.55 (dd, 1H,
J = 5.0 and 6.1 Hz), 4.25 (q, 2H, J = 7.2 Hz), 4.40 (quintet, 1H,
J = 6.1 Hz). 13C NMR (125 MHz, CDCl3) d 15.7, 17.5, 30.0, 43.5,
61.6, 62.0, 65.6, 70.5, 169.5, 210.0. HRMS Calcd for (M+)
C10H17NO4: 215.1158. Found: 215.1156.
4.2.1. 3-Ethoxycarbonyl-4-methoxycarbonyl-2,5-dimethyl
isoxazolidine (7a)
The synthesis of compounds 7a as cis/trans mixture, started
from N-methyl-C-ethoxycarbonyl nitrone 5a and methyl crotonate
6a. The analytical and spectroscopical data were previously
reported.22
4.3.3. 3-Ethoxycarbonyl-5-acetyl-2,4-dimethyl isoxazolidine
(7f)
4.2.2. 3-Ethoxycarbonyl-4-methoxycarbonyl-2-benzyl-5-
methyl isoxazolidine (7b)
The synthesis of compounds 7b as cis/trans mixture, started
from N-benzyl-C-ethoxycarbonyl nitrone 5b and methyl crotonate
6a. The analytical and spectroscopical data were previously
reported.23
First eluted compound: yield 8%; yellow oil. 1H NMR (CDCl3,
500 MHz) d 1.25 (d, 3H, J = 6.9 Hz), 1.31 (d, 3H, J = 7.1 Hz), 2,30
(s, 3H), 2.85 (s, 3 H), 2.86 (d, 1H, J = 6.1 Hz), 2.90 (m, 1H), 4.12 (d,
1H, J = 6.9 Hz), 4.30 (q, 2H, J = 7.1 Hz). 13C NMR (125 MHz, CDCl3)
d 14.9, 15.2, 25.5, 34.2, 43.5, 61.5, 65.4, 98.2, 171.0, 208.5. HRMS
Calcd for (M+) C10H17NO4: 215.1158. Found: 215.1160.
Second eluted compound: yield 8%; yellow oil. 1H NMR (CDCl3,
500 MHz) d 1,30 (d, 3H, J = 7.1 Hz), 1.32 (d, 3H, J = 8.7 Hz), 2.30 (s,
3H), 2.82 (s, 3H), 2.85 (d, 1H, J = 6.7 Hz), 2.95 (m, 1H), 3.85 (d, 1H,
J = 8.7 Hz), 4,22 (q, 2H, J = 7.1 Hz). 13C NMR (125 MHz, CDCl3) d
14.5, 15.2, 25.3, 33.7, 43.2, 61.7, 65.3, 99.0, 171.1, 208.1. HRMS
Calcd for (M+) C10H17NO4: 215.1158. Found: 215.1155.
4.2.3. 3-Ethoxycarbonyl-4,5-dimethoxycarbonyl 2-methyl
isoxazolidine (7c)
The synthesis of compounds 7a as cis/trans mixture, started
from N-methyl-C-ethoxycarbonyl nitrone 5a and dimethyl fuma-
rate 6b. The analytical and spectroscopical data were previously
reported.23

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D. Iannazzo et al. / Bioorg. Med. Chem. 16 (2008) 9610–9615
4.4. General procedure for the synthesis of 3-amino-
2(5H)furanones 4a–e
bonate (3 mL). The disappearance of the starting material was
monitored by TLC (CHCl3/MeOH 7:3). The mixture was neutralized
with 2 N HCl and then evaporated in vacuo. The crude was treated
with chloroform and the organic extracts were concentrated under
reduced pressure.
To a solution of the crude material in dry DMF (3 mL) and ani-
line (103 mg, 1 mmol) were added N,N-diisopropilcarbodiimide
(500 mg, 13 mmol) and DIEA (500 lL, 3 mmol) at room tempera-
Method A. To a solution of isoxazolidine 7a–e (1 mmol) in dry
THF (10 mL) was added TBAF (1.1 mL, 1.1 mmol, 1M in THF) and
the mixture was stirred at 50 °C for 6 h. At the end of this time,
the solvent was removed and the residue was purified by MPLC
using CHCl3/MeOH (99:1) as eluent.
Method B. To a solution of isoxazolidine 7f (1 mmol) in dry THF
(10 mL) was added NaH (24 mg, 1 mmol) and the mixture was stir-
red for 4 h at room temperature. The reaction mixture was then
quenched with water (0.5 mL) and evaporated under reduced pres-
sure. The residue was purified by MPLC using CHCl3/MeOH (99:1)
as eluent.
ture. The reaction mixture was stirred for 5 h and then was
quenched by the addition of brine and extracted three times with
EtOAc. The organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by MPLC using
ethyl acetate/cyclohexane 4:6 as eluent to give 8 as yellow oil
(yield 85%); 1H NMR (CDCl3, 500 MHz) d 1.45 (s, 3H), 1.62 (d, 3H,
J = 8.5 Hz), 3.21 (d, 3H, J = 7.4 Hz), 5.30 (q, 1H, J = 8.5 Hz), 6.80 (br
s, 1H), 6.95 (br s, 1H), 7.20–7.40 (m, 5H). 13C NMR (CDCl3,
125 MHz) d 21.3, 29.9, 74.5, 106.4, 120.4, 124.8, 126.6, 129.1,
154.3, 162.8, 168.1. HRMS Calcd for (M+) C13H14N2O3: 246.1004.
Found: 246.1008.
4.4.1. 5-Methyl-4-methoxycarbonyl-3-methylamino-
2(5H)furanone (4a)
Yield 85%; white solid, mp 80–82 °C; 1H NMR (CDCl3, 500 MHz)
d 1.35 (d, 3H, J = 7.1 Hz), 3.25 (d, 3H, J = 6.2 Hz), 3.77 (s, 3H), 5.10
(q, 1H, J = 6.1 Hz), 6.20 (br s, 1H, NH). 13C NMR (CDCl3, 125 MHz)
d 20.1, 29.8, 51.0, 75.1, 109.4, 142.1, 165.0, 167.5. HRMS Calcd
for (M+) C8H11NO4:185.0688. Found: 185.0691.
4.6. Synthesis of Methyl 2,5-dihydro-5-hydroxy-2-methyl-4-
(methylamino)furan-3-carboxylate (9)
4.4.2. 5-Methyl-4-methoxycarbonyl-3-benzylamino-
2(5H)furanone (4b)
To a solution of 4a (185 mg, 1 mmol) in anhydrous ether
(30 mL) at ꢀ78 °C, under nitrogen atmosphere a solution of DI-
BAL-H (2 mL, 2 mmol, 1M) was added and the mixture was stirred
at the same temperature for 5 h. The solution was then quenched
with MeOH (1 mL) and water (1 mL) and the resulting precipitate
was filtered under reduced pressure. The filtrate was evaporated
Yield 80%; yellow oil; 1H NMR (CDCl3, 500 MHz) d 1. 45 (d, 3H,
J = 6.2 Hz), 3.80 (s, 3H), 4.95 (dd, 1H, J = 6.1 and 12.5 Hz), 5.05 (dd,
1H, J = 6.1 and 12.5 Hz), 5.20 (q, 1H, J = 6.2 Hz), 6.95 (br s, 1H, NH)
7.30 (m, 5H). 13C NMR (CDCl3, 125 MHz) d 21.2, 46.3, 51.3, 61.2,
87.3, 112.1, 126.9, 128.1, 129.2, 138.7, 165.0, 167.4. HRMS Calcd
for (M+) C14H15NO4: 261.1001. Found: 261.1006.
in vacuo to obtain an inseparable mixture of a- and b-anomers of
lactol 9 in 1:1.2 ratio and in a quantitative yield as yellow oil. Ma-
jor anomer: 1H NMR (CDCl3, 500 MHz) d 1.22 (d, 3H, J = 7.2 Hz),
3.10 (d, 3H, J =4.9 Hz), 3.79 (s, 3H), 5.20 (dq, 1H, J = 0.5 and
7.2 Hz), 5.95 (d, 1H, J = 0.5 Hz), 6.70 (br s, 1H). 13C NMR (CDCl3,
125 MHz) d 22.2, 30.5, 50.5, 79.2, 97.5, 109.5, 158.6, 173.0. Minor
anomer: 1H NMR (CDCl3, 500 MHz) d 1.35 (d, 3H, J = 7.1 Hz), 3.10
(d, 3H, J = 4.9 Hz), 3.79 (s, 3H), 4.97 (q, 1H, J = 7.1 Hz), 6.05 (br s,
1H), 6.70 (br s, 1H). 13C NMR (CDCl3, 125 MHz) d 24.4, 29.7, 50.1,
79.9, 98.0, 109.5, 158.6, 166.3.
4.4.3. 4,5-Diethoxycarbonyl-3-methylamino-2(5H)furanone (4c)
Yield 79%; yellow oil; 1H NMR (CDCl3, 500 MHz) d 1.30 (t, 3H,
J = 7.1 Hz), 1.38 (t, 3H, J = 7.2 Hz), 3.05 (d, 3H, J = 5.2 Hz), 4.14 (q,
2H, J = 7.1 Hz), 4.25 (q, 2H, J = 7.2 Hz), 5.10 (s, 1H), 8.02 (br s,
NH). 13C NMR (CDCl3, 125 MHz) d 13.7, 13.8, 29.7, 57.4, 61.8,
86.7, 124.9, 129.1, 168.0, 70.2, 171.5. HRMS Calcd for (M+)
C11H15NO6: 257.0899. Found: 257.0894.
4.4.4. 4-Acetyl-3-methylamino-2(5H)furanone (4d)
Yield 82%; light yellow solid; mp 120–124 °C; 1H NMR (CDCl3,
500 MHz) d 2.13 (s, 3H), 3.30 (d, 3H, J = 5.7 Hz), 4.96 (s, 2H), 7.94
(br s, NH). 13C NMR (CDCl3, 125 MHz) d 26.7, 27.3, 67.7, 121.0,
158.7, 175.3, 202.1, HRMS Calcd for (M+) C7H9NO3: 155.0582.
Found: 155.0579.
4.7. Biological assays
4.7.1. Cell culture
Huh-7 cells, originally obtained from Ralf Bartenschlager (Uni-
versity of Mainz, Mainz, Germany) were grown in Dulbecco’s mod-
ified minimal essential medium (D-MEM, EuroClone, Pero, Italy),
supplemented with 10% fetal bovine serum (FBS, Life Technologies,
Paisley, Scotland, UK). Huh-7-derived HBI10A cells expressing an
HCV subgenomic replicon have been previously described.26 They
were grown as described for Huh-7 cells, but the medium was sup-
plemented with the addition of 0.8 mg of neomycin sulfate (G418,
Life Technologies). Cells were passaged 1:5 twice a week using 1ꢂ
trypsin–EDTA.
4.4.5. 5-Methyl-4-acetyl-3-methylamino-2(5H)furanone (4e)
Yield 75%; light yellow oil; 1H NMR (CDCl3, 500 MHz) d 1.57 (d,
3H, J = 6.3 Hz), 2.20 (s, 3H), 3.35 (d, 3H, J = 5.5 Hz), 5.3 (q, 1H,
J = 6.3 Hz), 8.3 (br s, 1H). 13C NMR (CDCl3, 125 MHz) d 21.4, 26.8,
45.2, 75.9, 117.9, 142.5, 167.0, 193.1. HRMS Calcd for (M+)
C8H11NO3: 169.0739. Found: 169.0735.
4.4.6. 4,5-Diphenyl-3-methylamino-2(5H)furanone (4f)
Yield 78%; yellow oil; 1H NMR (CDCl3, 500 MHz) d 2.61 (s, 3H),
4.12 (br s, 1H), 5.95 (s, 1H), 7.10–7.30 (m, 10H). 13C NMR (CDCl3,
125 MHz) d 32.0, 83.7, 94.5, 124.6, 127.6, 127.7, 128.0, 128.6,
128.8, 129.0, 131.1, 132.2, 136.1, 171.0. HRMS Calcd for (M+)
C17H15NO2: 265.1103. Found: 265.1105.
4.7.2. Anti-hepatitis C virus assay
The effect of compounds on HCV viral replication was moni-
tored in HBI10A cells by a cell-enzyme-linked immunosorbent as-
say, as previously described.27 Briefly, HBI10A cells, either treated
with different concentrations of the compounds or control diluent,
were assayed for NS3 protein expression with the anti-NS3 10E5/
24 MAb. Compounds were dissolved in DMSO (Sigma Chemicals
CO., St. Louis, MO) and serially diluted in D-MEM in a way that
DMSO concentration was never higher than 1%. Final concentra-
4.5. Synthesis of 2,5-dihydro-2-methyl-4-(methylamino)-5-
oxo-N-phenylfuran-3-carboxamide (8)
tions of the compounds were 103, 102, 10, and 1
was performed in triplicate. As a positive control, IFN-
trations ranging from 102 to 1 U/mL, was utilised. The inhibitor
l
M. The assay
at concen-
Compound 4a (205 mg, 1.1 mmol) was dissolved in methanol
(3 mL) and treated with a 10% aqueous solution of potassium car-
a

D. Iannazzo et al. / Bioorg. Med. Chem. 16 (2008) 9610–9615
9615
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concentration that reduced by 50% the expression of NS3 (EC50
)
was calculated by fitting the data to the Hill equation: fraction
inhibition = 1 ꢀ (Ai ꢀ b)/(A0 ꢀ b) = [I]n/([I]n + EC50), where Ai is the
absorbance value of HBI10A cells supplemented with the appropri-
ate compound [I] concentration, A0 is the absorbance value of
HBI10A cells incubated with control diluent, b is the absorbance
value of Huh-7 cells plated at the same density in the same micro-
titer plates and incubated with control diluent, and n is the Hill
coefficient. The EC50 values were calculated according to the
best-fit curve, y value versus logx, where y is the value of the
examined function and x is the drug concentration. The Pearson
product–moment correlation coefficient (Pearson’s r), that reflects
the degree and direction of linear relationship between two vari-
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4.7.3. Cytotoxicity assay
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a MTS assay. HBI10A cells were seeded at 1 ꢂ 104 cell/100
lL of D-
MEM + 5% FBS in a 96 well plate and after 4 h incubation at 37 °C
cells the compounds were added at the final concentration of
103,102, 10 and 1
lM. After 20 h incubation at 37 °C, 20 lL of
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This work was partially supported by M.I.U.R. (Rome) (Progetto
Nazionale: Sintesi Stereselettiva e valutazione biologica di comp-
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