Pseudo-peptides derived from isomannide: Inhibitors of serine proteases

Article abstract of DOI:10.1007/s00726-009-0273-4

In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.

Full text of DOI:10.1007/s00726-009-0273-4

Amino Acids (2010) 38:701–709
DOI 10.1007/s00726-009-0273-4
ORIGINAL ARTICLE
Pseudo-peptides derived from isomannide: inhibitors of serine
proteases
´
Thalita G. Barros Æ Sergio Pinheiro Æ J. S. Williamson Æ Amılcar Tanuri Æ
´
M. Gomes Jr Æ Helena S. Pereira Æ R. M. Brindeiro Æ Jose B. A. Neto Æ
O. A. C. Antunes Æ Estela M. F. Muri
Received: 14 November 2008 / Accepted: 11 March 2009 / Published online: 28 March 2009
Ó Springer-Verlag 2009
Abstract In this paper, we describe the synthesis of a
novel class of pseudo-peptides derived from isomannide
and several oxazolones as potential inhibitors of serine
proteases as well as preliminary pharmacological assays
for hepatitis C. Hepatitis C, dengue and West Nile fever are
among the most important flaviviruses that share one
important serine protease enzyme. Serine proteases belong
to the most studied class of proteolytic enzymes and are a
primary target in the drug development field. Several
pseudo-peptides were obtained in good yields from the
reaction of isomannide and oxazolones, and their anti-
HCV potential using the HCV replicon-based assay was
shown.
Keywords Hepatitis C ꢀ Dengue ꢀ Serine protease ꢀ
Isomannide ꢀ Oxazolones
Introduction
The family Flaviviridae comprises more than 60 viruses,
many of which are important human pathogens. Among the
most important flaviviruses are the Hepatitis C virus
(HCV), the West Nile virus (WN) and the Dengue virus.
Chronic HCV infection is associated with liver cirrhosis
and hepatocellular carcinoma (Bruix et al. 1989). Current
therapeutic based on alpha interferon and the nucleoside
analog ribavirin is only partially effective and is limited by
the adverse effects of both agents (Wright et al. 2001).
Dengue virus causes dengue fever and dengue hemorrhagic
fever in millions of people each year in tropical and sub-
tropical regions of the world. Currently, there is no vaccine
or effective antiviral therapy for the four known serologi-
cally related virus types (dengue 1–4) (Yusof et al. 2000).
All flaviviruses have a positive-sense nonsegmented
RNA genome that encodes a single long polyprotein pro-
cessed to yield three structural proteins (C, prM and E) and
seven nonstructural proteins (NS1, NS2A, NS2B, NS3,
NS4A, NS4B and NS5) (Leung et al. 2001). A single virus-
encoded protease comprising 180 amino acids of NS3
(NS3pro) is responsible for the cleavage of both in cis and
in trans, which generates viral proteins that are essential
for viral replication and maturation of infectious virions.
The presence of a trypsin-like serine protease within the
N-terminal one-third of the flavivirus NS3 protein was first
proposed by (Bazan and Fletterick (1989, 1990) and
(Gorbalenya et al. (1989). Their analysis of virus sequence
alignments revealed that structural motifs as well as the
characteristic catalytic triad (His51, Asp75, and Ser135) of
T. G. Barros ꢀ E. M. F. Muri (&)
´
Faculdade de Farmacia, Universidade Federal Fluminense
(UFF), Rua Mario Viana 523, Santa Rosa, Niteroi,
´
RJ 24241000, Brazil
e-mail: estelamuri@yahoo.com.br
T. G. Barros ꢀ S. Pinheiro
´ ´
Instituto de Quımica, Campus do Valonguinho, UFF, Niteroi,
RJ, Brazil
J. S. Williamson
Department of Medicinal Chemistry, School of Pharmacy,
University of Mississippi, University, MS, USA
A. Tanuri ꢀ H. S. Pereira ꢀ R. M. Brindeiro ꢀ J. B. A. Neto
´
Laboratorio de Virologia Molecular, ICB, UFRJ, Rio de Janeiro,
RJ, Brazil
M. Gomes Jr ꢀ O. A. C. Antunes
´
Instituto de Quımica, UFRJ, Rio de Janeiro, RJ, Brazil
H. S. Pereira
Faculdade de Odontologia de Nova Friburgo, Universidade
Federal Fluminense (FOUFF), Nova Friburgo, RJ, Brazil
123

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T. G. Barros et al.
mammalian serine proteases were conserved in all flavi-
viruses. As NS3pro activity is essential for viral replication,
it represents a suitable target for the development of che-
motherapeutic approaches for the treatment of flaviviruses.
As part of our antiviral program for flaviviruses, we describe
in this paper the synthesis and preliminary pharmacological
assays of a series of pseudo-peptides derived from
isomannide, designed as potential inhibitors of the catalytic
triad of serine proteases and as analogs of the compounds
previously published by us (Muri et al. 2004, 2005, 2006).
and the product extracted with diethyl ether. The organic
layer was dried and evaporated. The crude diazide was
purified by column chromatography to give a product of
pale yellow liquid in 78% yield. 1H NMR d (CDCl3,
300 MHz): 4.62 (s, 2H), 4.06 (dd, 2H, J = 3.9, 1.8 Hz),
3.98–3.60 (m, 4H). 13C NMR (CDCl3, 75 MHz): 85.8
(–CH), 71.6 (–CH2), 65.5 (–CH). IR (KBr) m cm-1: 3,340,
2,953, 2,883, 2,503, 2,105, 1,471, 1,258, 1,094, 953, 914,
846. a2D0 = ? 94 (c, 0.1) DMSO.
1,4:3,6-Dianhydro-2,5-diamino-2,5-dideoxy- L-iditol (4).
A mixture of diazide (1.27 mmol, 0.25 g) and 10% Pd/C
(0.127 mmol, 0.140 g) in EtOH (10 mL) was hydrogenated
at 40 psi. After 12 h, the catalyst was filtered off and the
solvent was evaporated giving a product of a hygroscopic
Materials and methods
1
General: chemistry
solid with 93% yield. HNMR d (CDCl3, 300 MHz): 5.41
(s, 2H), 4.82 (dd, 2H, J = 9.3, 4.8 Hz), 4.56 (dd, 2H,
13
J = 9.3, 2.4 Hz), 4.34 (dd, 2H, J = 4.5, 2.4 Hz). NMR
All solvents were purchased as reagent grade, dried, using
standard conditions and stored over molecular sieves.
Purification of products was carried out using silica gel
flash chromatography (Whatman 60, 230–400 mesh).
Routine NMR analyses were carried out on a Varian Unity
Plus-300 spectrometer. Melting points were obtained on a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. High-resolution mass spectra (HRMS) were
performed on a Waters Micromass Q-Tof Micro mass
spectrometer equipped with a lock spray source. The IR
spectra were obtained on a Perkin-Elmer spectrometer
model Spectrum One in liquid film and KBr pellets. The
alpha-D measurements were done on a Perkin-Elmer 341
LC polarimeter.
(CDCl3, 75 MHz): 89.9 (–CH); 75.5 (–CH2); 59.2 (–CH).
IR (KBr) m cm-1: 3,352, 2,952, 1,601, 1,470, 1,050.
a2D0 = ? 27 (c, 0.1) DMSO. HRMS calcd. for C6H13N2O2
145.0977. Found 145.097.
General procedure for formation of the final products
(28-48)
To a solution of amine (1.38 mmol, 0.2 g) in ethyl acetate
(25 mL), was added the corresponding oxazolone
(3.05 mmol). The reaction was refluxed for 24-48 h, after
which the formed precipitate was filtered and washed with
ethyl acetate.
1,4:3,6-Dianhydro-2,5-di-O-p-tosyl-D-mannitol (2). A
solution of p-toluenesulphonyl chloride (27.36 mmol,
5.2 g) in pyridine (40 mL) was added dropwise to a solu-
tion of isomannide (13.68 mmol, 2.0 g) in dry pyridine
(24 mL) and stirred at r.t overnight. The mixture was
cooled and poured on ice-cold 2 N HCl. The product was
extracted with ethyl acetate, dried and filtered. The crude
product was recrystallized from MeOH to give a product of
white solid with 90% yield. 1H NMR d (CDCl3, 300 MHz):
7.80 (d, 4H, J = 8.1 Hz), 7.33 (d, 4H, J = 8.1 Hz), 4.90–
4.75 (m, 2H), 4.55–4.45 (m, 2H), 3.91 (dd, 2H, J = 6.6,
9.3 Hz), 3.72 (dd, 2H, J = 7.5, 9.3 Hz), 2.44 (s, 6H). 13C
NMR (CDCl3, 75 MHz): 145.2 (–C), 132.9 (–C), 129.8
(–CH), 127.8 (–CH), 79.8 (–CH), 77.8 (–CH), 70.0 (–CH2),
21.6 (–CH3). IR (KBr) m cm-1: 3,444, 3,064, 2,956, 2,938,
2,880, 1,596, 1,371, 1,191, 1,173, 1,138, 1,113, 1,075,
1,037, 916, 881,789, 720, 669. a2D0 = ? 96 (c, 0.1) DMSO,
mp 93–94°C.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-3,4-methyl-
1
enedioxycinnamamido]-2,5-dideoxy-L-iditiol (28). HNMR
d (DMSO, 300 MHz): 9.79 (s, 2H), 8.28 (d, 2H, J = 7.2 Hz),
7.98 (d, 4H, J = 7.2 Hz), 7.61–7.47 (m, 6H), 7.15 (d, 2H,
J = 1.8 Hz), 7.10–7.05 (m, 4H), 6.91 (d, 2H, J = 8.1 Hz),
5.99 (s, 4H), 4.55 (s, 2H), 4.30–4.20 (m, 2H), 4.03–3.93
(m, 2H), 3.70 (dd, 2H, J = 9.3, 3.3 Hz). 13CNMR (DMSO,
75 MHz): 165.8 (–C), 165.5 (–C), 147.7 (–C), 147.4 (–C),
133.7 (–C), 131.8 (–CH), 129.0 (–CH), 128.4 (–CH), 128.3
(–C), 127.9 (–CH), 125.0 (–CH), 108.5 (–CH), 108.5 (–CH),
108.4 (–CH), 101.4 (–C), 86.7 (–CH), 71.3 (–CH2), 56.7
(–CH). IR (KBr) m cm-1: 3,233, 3,059, 2,912, 1,633, 1,580,
1,556, 1,504, 1,481, 1,447, 1,353, 1,280, 1,241, 1,035, 692.
a2D0 = ? 77 (c, 0,1) DMSO, mp 246–247°C. HRMS calcd.
for C40H35N4O10 731.2275. Found 731.3270. The product is
a pale yellow solid; 70% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-cinnamam-
ido]-2,5-dideoxy-L-iditiol (29). 1HNMR
1,4:3,6-Dianhydro-2,5-diazido-2,5-dideoxy-L-iditol (3).
d
(DMSO,
NaN3 (30.84 mmol, 2.0 g) was added to a solution of
300 MHz): 9.90 (s, 2H), 8.40 (d, 2H, J = 7.2 Hz), 7.98
(d, 4H, J = 6.9 Hz), 7.60–7.48 (m, 6H), 7.40–7.20 (m, 10H),
7.11 (s, 2H), 4.59 (s, 2H), 4.30–3.90 (m, 2H), 3.98 (dd, 2H,
J = 9.3, 6.0 Hz), 7.73 (dd, 2H, J = 9.3, 3.3 Hz). 13CNMR
ditosylate
2
(7.71 mmol, 3.5 g) in [bmim]?[BF4]-
(46.2 mmol, 9.3 mL), and the mixture was stirred over-
night at 120°C. The mixture was cooled, water was added
123

Pseudo-peptides derived from isomannide
703
(DMSO, 75 MHz): 166.0 (–C), 165.5 (–C), 134.3 (–C), 133.7
(–C), 131.8 (–CH), 130.2 (–C), 129.,4 (–CH), 128.6 (–CH),
128.5 (–CH), 128.4 (–CH), 128.3 (–CH), 127.9 (–CH), 86.7
(–CH), 71.4 (–CH2), 56.7 (–CH). IR (KBr) m cm-1: 3,270,
3,058, 2,950, 1,649, 1,579, 1,514, 1,477, 1,280, 1,206, 1,080,
692. a2D0 = ? 72 (c, 0,1) DMSO, mp 169°C. HRMS calcd.
for C38H35N4O6 643.2478. Found 643.3465. The product is a
pale yellow solid; 40% yield.
700. a2D0 = ? 65 (c, 0,1) DMSO, mp 152–153°C. HRMS
calcd. for C38H33F2N4O6 679,2289. Found 679.3388. The
product is a pale yellow solid; 37% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-4-chloro-
cinnamamido]-2,5-dideoxy-L-iditiol (33). 1HNMR
d
(DMSO, 300 MHz): 9.90 (s, 2H), 8.44 (d, 2H, J = 6.9 Hz),
7.97 (d, 4H, J = 7.2 Hz), 7.65-7.45 (m, 10H), 7.41 (d, 4H,
J = 8.7 Hz), 7.06 (s, 2H), 4.59 (s, 2H), 4.28–4.20 (m, 2H),
3.98 (dd, 2H, J = 9.3, 5.7 Hz), 3.73 (dd, 2H, J = 9.3,
3.3 Hz). 13CNMR (DMSO, 75 MHz): 165.6 (–C), 165.2
(–C), 133.4 (–C), 133.1 (–C), 132.7 (–C), 131.6 (–CH), 130.8
(–CH), 130.7 (–C), 128.3 (–CH), 128.2 (–CH), 127.7 (–CH),
126.6 (–CH), 86.5 (–CH), 71.1 (–CH2), 56.5 (–CH). IR (KBr)
m cm-1: 3,271, 3,061, 2,970, 1,651, 1,580, 1,514, 1,477,
1,280, 1,091, 907, 820, 709. a2D0 = ? 89 (c, 0,1) DMSO, mp
166–169°C. HRMS calcd. for C38H33Cl2N4O6 711.1699.
Found 711.2859. The product is a pale yellow solid; 35%
yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-4-methoxy-
cinnamamido]-2,5-dideoxy-L-iditiol (30). 1HNMR
d
(DMSO, 300 MHz): 9.80 (s, 2H), 8.27 (d, 2H, J =
7.2 Hz), 7.99 (d, 4H, J = 6.9 Hz), 7.65–7.40 (m, 10H),
7.12 (s, 2H), 6.91 (d, 4H, J = 8.7 Hz), 4.57 (s, 2H), 4.30–
4.20 (m, 2H), 3.96 (dd, 2H, J = 9.3, 5.7 Hz), 3.73 (s, 6H,
-CH3), 3.80-3.60 (m, 2H). 13CNMR (DMSO, 75 MHz):
165.9 (–C), 165.7 (–C), 159.7 (–C), 133.8 (–C), 131.7 (–CH),
131.2 (–CH), 128.9 (–CH), 128.4 (–CH), 128.0 (–C), 127.9
(–CH), 126.7 (–C), 114,1 (–CH), 86.8 (–CH), 71.4 (–CH2),
56.7 (–CH), 55.3 (–CH3). IR (KBr) m cm-1: 3,274, 3,061,
2,962, 2,838, 1,650, 1,604, 1,578, 1,512, 1,477, 1,300, 1,255,
1,177, 1,080, 1,028, 829, 707. a2D0 = ? 86 (c, 0,1) DMSO,
mp 167–168°C. HRMS calcd. for C40H39N4O8 703.2689.
Found 703.3768. The product is a pale yellow solid;
46% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-4-bromo-
cinnamamido]-2,5-dideoxy-L-iditiol (34). 1HNMR
d
(DMSO, 300 MHz): 9.89 (s, 2H), 8.44 (d, 2H, J = 6.9 Hz),
7.96 (d, 4H. J = 7.5 Hz), 7.62-7.40 (m, 14H), 7.04 (s, 2H),
4.59 (s, 2H), 4.30–4.20 (m, 2H), 4.00–3.95 (m, 2H), 3.73 (dd,
2H, J = 9.0, 3.0 Hz). 13CNMR (DMSO, 75 MHz): 165.6
(–C), 165.1 (–C), 133.5 (–C), 133.4 (–C), 131.6 (–CH), 131.2
(–CH), 131.0 (–CH), 130.8 (–C), 128.2 (–CH), 127.7 (–CH),
126.6 (–CH), 121.4 (–C), 86.4 (–CH), 71.1 (–CH2), 56.5
(–CH). IR (KBr) m cm-1: 3,266, 3,061, 2,973, 2,882, 1,650,
1,580, 1,513, 1,476, 1,280, 1,074, 1,009, 906, 816, 710.
a2D0 = ? 80 (c, 0,1) DMSO, mp 172–174°C. HRMS calcd.
for C38H33Br2N4O6 799.0689. Found 801.1971. The product
is a pale yellow solid; 40% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-3,4-dimeth-
oxycinnamamido]-2,5-dideoxy-L-iditiol (31). 1HNMR d
(DMSO, 300 MHz): 9.80 (s, 2H), 8.26 (d, 2H,
J = 6.9 Hz), 8.03 (d, 4H, J = 7.2 Hz), 7.61–7.40 (m, 6H),
7.23–7.10 (m, 6H), 6.94 (d, 2H, J = 8.4 Hz), 4.57 (s, 2H),
4.30–4.20 (m, 2H), 4.10–3.90 (m, 4H), 3.73 (s, 6H, -CH3),
3.50 (s, 6H, -CH3). 13CNMR (DMSO, 75 MHz): 165.7
(–C), 165.4 (–C), 149.3 (–C), 148.2 (–C), 133.5 (–C), 131.6
(–CH), 129.4 (–CH), 128.2 (–CH), 127.7 (–CH), 127.6
(–C), 126.7 (–C), 123.4 (–CH), 112.2 (–CH), 111.4 (–CH),
86.6 (–CH), 71.1 (–CH2), 56.5 (–CH), 55.4 (–CH3), 55.0
(–CH3). IR (KBr) m cm-1: 3,271, 3,060, 2,960, 2,838,
1,648, 1,601, 1,579, 1,515, 1,478, 1,332, 1,263, 1,163,
1,143, 1,082, 1,023, 808, 716, 621. a2D0 = ? 72 (c, 0,1)
DMSO, mp 159–160°C. HRMS calcd. for C42H43N4O10
763.2901. Found 763.4067. The product is a pale yellow
solid; 55% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-4-trifluo-
romethylcinnamamido]-2,5-dideoxy-L-iditiol (35). 1HNMR
d (DMSO, 300 MHz): 9.99 (s, 2H), 8.54 (d, 2H,
J = 6.6 Hz), 7.97 (d, 4H, J = 7.2 Hz), 7.77–7.60 (m, 8H),
7.63–7.56 (m, 2H), 7.50–7.47 (m, 4H), 7.09 (s, 2H), 4.62
(s, 2H), 4.29-4.21 (m, 2H), 4.02–3.96 (m, 2H), 3.75
(dd, 2H, J = 9.3, 3.0 Hz). 13CNMR (DMSO, 75 MHz):
165.7 (–C), 165.0 (–C), 138.5 (–C), 133.3 (–C), 132.3 (–C),
131.6 (–CH), 129.6 (–CH), 128.2 (–CH), 127.7 (–CH),
125.7 (–CH), 125.1 (–CH), 112.6 (–C), 110.2 (–C), 86.4
(–CH), 71.1 (–CH2), 56.5 (–C). IR (KBr) m cm-1: 3,274,
3,065, 1,646, 1,580, 1,519, 1,477, 1,324, 1,281, 1,168,
1,126, 1,069, 830, 708. a2D0 = ? 53 (c, 0,1) DMSO, mp
164–166°C. HRMS calcd. for C40H33F6N4O6 779.2226.
Found 779.3478. The product is a pale yellow solid; 40%
yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-4-fluorocin-
1
namamido]-2,5-dideoxy-L-iditiol (32). HNMR d (DMSO,
300 MHz): 9.86 (s, 2H), 8.33 (d, 2H, J = 6.6 Hz), 7.98
(d, 4H, J = 7.5 Hz), 7.70–7.40 (m, 10H), 7.19 (t, 4H,
J = 9.0 Hz), 7.11 (s, 2H), 4.59 (s, 2H), 4.30–4.20 (m, 2H),
3.98 (dd, 2H, J = 9.3, 5.7 Hz), 3.73 (dd, 2H, J = 9.3,
36 Hz). 13CNMR (DMSO, 75 MHz): 165.2 (–C), 163.3
(–C), 160.0 (–C), 133.5 (–C), 131.3 (–C), 131.2 (–C), 130.7
(–CH), 129.8 (–CH), 128.2 (–CH), 127.7 (–CH), 115.4
(–CH), 115.1 (–CH), 86.5 (–CH), 71.1 (–CH2), 56.5 (–CH).
IR (KBr) m cm-1: 3,245, 3,060, 2,950, 1,646, 1,601, 1,580,
1,508, 1,476, 1,366, 1,281, 1,230, 1,159, 1,075, 1,050, 832,
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-2-thiophe-
nylacrylamido]-2,5-dideoxy-L-iditiol (36). 1HNMR
d
(DMSO, 300 MHz): 9.74 (s, 2H), 8.28 (d, 2H, J = 6.9 Hz),
8.06 (d, 2H, J = 6.9 Hz), 7.64-7.51 (m, 12H), 7.42 (d, 2H,
J = 2.7 Hz), 7.09 (dd, 2H, J = 4.8, 3.6 Hz), 4.55 (s, 2H),
123

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T. G. Barros et al.
4.27–4.22 (m, 2H), 3.96 (dd, 2H, J = 9.0, 5.7 Hz), 3.70 (dd,
2H, J = 9.0, 3.3 Hz). 13CNMR (DMSO, 75 MHz): 165.9
(–C), 164.5 (–C), 136.6 (–C), 133.7 (–C), 131.9 (–CH), 131.5
(–CH), 129.9 (–CH), 128.1 (–CH), 127.8 (–CH), 126.8
(–CH), 126.6 (–C), 125.0 (–CH), 86.6 (–CH), 71.1 (–CH2),
56.4 (–CH). IR (KBr) m cm-1: 3,415, 3,262, 3,066, 2,965,
1,648, 1,517, 1,475, 1,282, 1,077, 1,052, 906, 706.
a2D0 = ? ? 81 (c, 0,1) DMSO, mp 194–196°C. HRMS
calcd. for C34H31N4O6S2 655.1685. Found 655.1697. The
product is a pale yellow solid; 55% yield.
for C28H31N4O6 519.2244. Found 519.2244. The product is
a pale yellow solid; 95% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-4-chlorocin-
1
namamido]-2,5-dideoxy-L-iditiol (40). HNMR d (DMSO,
300 MHz): 9.44 (s, 2H), 8.31 (d, 2H, J = 7.2 Hz), 7.56–
7.42 (m, 8H), 6.84 (s, 2H), 4.56 (s, 2H), 4.22–4.15 (m, 2H),
3.95 (dd, 2H, J = 9.3, 6.0 Hz), 3.70 (dd, 2H, J = 9.3,
3.3 Hz), 1.97 (s, 6H). 13CNMR (DMSO, 75 MHz): 169.1
(–C), 165.2 (–C), 133.1 (–C), 132.6 (–C), 130.8 (–CH),
130.7 (–C), 128.3 (–CH), 124.9 (–CH), 86.4 (–C), 71.1
(–CH2), 56.5 (–C), 22.7 (–CH3). IR (KBr) m cm-1: 3,468,
3,273, 2,977, 2,876, 1,650, 1,622, 1,537, 1,488, 1,371,
1,311, 1,280, 1,090, 1,041, 1,012, 822, 739. a2D0 = ? 85
(c, 0,1) DMSO, mp 168–170°C. HRMS calcd. for C28H28
N4O6NaCl2 609.1284. Found 609.1282. The product is a
pale yellow solid; 96% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-benzamido-(Z)-3-pyridyl-
acrylamido]-2,5-dideoxy-L-iditiol (37). 1HNMR d (DMSO,
300 MHz): 9.98 (s, 2H), 8.70 (d, 2H, J = 1.8 Hz), 8.50
(d, 2H, J = 6.9 Hz), 8.44 (dd, 2H, J = 4.8, 1.5 Hz), 7.98–
7.91 (m, 6H), 7.61–7.48 (m, 6H), 7.37 (dd, 2H, J = 8.1,
5.1 Hz), 7.10 (s, 2H), 4.60 (s, 2H), 4.30–4.20 (m, 2H), 3.99
(dd, 2H, J = 9.3, 5.7 Hz), 3.74 (dd, 2H, J = 9.3, 3.3 Hz).
13CNMR (DMSO, 75 MHz): 165.9 (–C), 165.7 (–C), 159.7
(–C), 133.8 (–C), 131.7 (–CH), 131.2 (–CH), 128.9 (–CH),
128.4 (–CH), 128.0 (–C), 127.9 (–CH), 126.7 (–C), 114.1
(–CH), 86.8 (–CH), 71.4 (–CH2), 56.7 (–CH), 55.3 (–CH3).
IR (KBr) m cm-1: 3,404, 3,275, 3,065, 2,967, 1,645, 1,539,
1,515, 1,476, 1,283, 1,193, 1,083, 1,038, 908, 707.
a2D0 = ? 64 (c, 0,1) DMSO, mp 167°C. HRMS calcd. for
C26H33N6O6 645.2462. Found 645.2468. The product is a
pale yellow solid; 45% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-4-bromocin-
1
namamido]-2,5-dideoxy-L-iditiol (41). HNMR d (DMSO,
300 MHz): 9.40 (s, 2H), 8.28 (d, 2H, J = 6.6 Hz), 7.60–7.55
(m, 4H), 7.50–7.44 (m, 4H), 6.82 (s, 2H), 4.56 (s, 2H), 4.22–
4.16 (m, 2H), 3.95 (dd, 2H, J = 9.3, 6.0 Hz), 3.70 (dd, 2H,
J = 9.3, 3.3 Hz), 1.97 (s, 6H). 13CNMR (DMSO, 75 MHz):
169.0 (–C), 165.1 (–C), 133.4 (–C), 131.2 (–CH), 131.0
(–CH), 130.8 (–C), 124.8 (–CH), 121.2 (–C), 86.4 (–CH),
71.1 (–CH2), 56.4 (–C), 22.5 (–CH3). IR (KBr) m cm-1
:
3,275, 2,974, 2,878, 1,651, 1,587, 1,537, 1,485, 1,371, 1,311,
1,278, 1,075, 1,042, 1,009, 816, 706. a2D0 = ? 81 (c, 0,1)
DMSO, mp 175°C. HRMS calcd. for C28H28N4O6NaBr2
697.0273. Found 697.0306. The product is a pale yellow
solid; 58% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-3,4-methyl-
enedioxycinnamamido]-2,5-dideoxy-L-iditiol (38). 1HNMR
d (DMSO, 300 MHz): 9.33 (s, 2H), 8.17 (d, 1H, J =
6.9 Hz), 7.20–6.90 (m, 6H), 6.87 (s, 2H), 6.04 (s, 4H), 4.54
(s, 2H), 4.20–4.10 (m, 2H), 4.00–3.90 (m, 2H), 3.71–3.60
(m, 2H), 1.97 (s, 6H). 13CNMR (DMSO, 75 MHz): 169.1
(–C), 165.3 (–C), 147.3 (–C), 147.2 (–C), 128.2 (–C), 128.0
(–C), 127.1 (–CH), 124.6 (–CH), 108.4 (–CH), 108.2
(–CH), 101.1 (–CH2), 86.5 (–CH), 71.1 (–CH2), 56.4
(–CH), 22.7 (–CH3). IR (KBr) m cm-1: 3,306, 3,244, 2,982,
2,890, 1,650, 1,620, 1,532, 1,502, 1,480, 1,448, 1,373,
1,351, 1,255, 1,090, 1,071, 1,038, 929, 813, 723.
a2D0 = ? 37 (c, 0,1) DMSO, mp 170–173°C. HRMS calcd.
for C30H31N4O10 607.1962. Found 607.2885. The product
is a pale yellow solid; 70% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-4-trifluoro-
methylcinnamamido]-2,5-dideoxy-L-iditiol (42). 1HNMR d
(DMSO, 300 MHz): 9.52 (s, 2H), 8.38 (d, 2H, J = 6.6 Hz),
7.80–7.67 (m, 8H), 6.87 (s, 2H), 4.58 (s, 2H), 4.24–4.18
(m, 2H), 3.97 (dd, 2H, J = 9.3, 5.7 Hz), 3.72 (dd, 2H, J =
9.3, 3.3 Hz), 1.98 (s, 6H). 13CNMR (DMSO, 75 MHz):
169.1 (–C), 165.0 (–C), 138.4 (–C), 132.3 (–C), 129.6
(–CH), 125.8 (–C), 125.1 (–CH), 123.9 (–CH), 122.2 (–C),
86.4 (–CH), 71.1 (–CH2), 56.5 (–CH), 22.7 (–CH3). IR
(KBr) m cm-1: 3,467, 3,283, 2,986, 2,883, 1,628, 1,538,
1,499, 1,374, 1,325, 1,283, 1,164, 1,125, 1,089, 1,069, 839,
729. a2D0 = ? 51 (c, 0,1) DMSO, mp: 209–210°C. HRMS
calcd. for C30H28N4O6NaF6 677.1811. Found 677.1792.
The product is a pale yellow solid; 50% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-cinnamam-
ido]-2,5-dideoxy-L-iditiol (39). 1HNMR
d
(DMSO,
300 MHz): 9.41 (s, 2H), 8.25 (d, 2H, J = 6.9 Hz), 7.53
(d, 4H, J = 7.2 Hz), 7.42–7.20 (m, 6H), 6.88 (s, 2H), 4.57
(s, 2H), 4.23–4.16 (m, 2H), 3.96 (dd, 2H, J = 9.3, 5.4 Hz),
3.71 (dd, 2H, J = 9.3, 3.6 Hz), 1.98 (s, 6H). 13CNMR
(DMSO, 75 MHz): 169.2 (–C), 165.3 (–C), 134.1 (–C),
130.1 (–C), 129.2 (–CH), 128.4 (–CH), 128.3 (–CH), 126.5
(–CH), 86.5 (–CH), 71.2 (–CH2), 56.4 (–CH), 22.7 (–CH3).
IR (KBr) m cm-1: 3,480, 3258, 3,056, 2,975, 2,870, 1,651,
1,537, 1,489, 1,446, 1,373, 1,287, 1,208, 1,088, 1,042, 932.
a2D0 = ? 71 (c, 0,1) DMSO, mp 191–192°C. HRMS calcd.
1,4:3,6-Dianhydro-2,5-bis-n[2-acetamido-(Z)-4-cyanocin-
1
namamido]-2,5-dideoxy-L-iditiol (43). HNMR d (DMSO,
300 MHz): 9.57 (s, 2H), 8.40 (d, 2H, J = 6.6 Hz), 7.82
(d, 4H, J = 8.4 Hz), 7.67 (d, 4H, J = 8.4 Hz), 6.83 (s, 2H),
4.58 (s, 2H), 4.23–4.17 (m, 2H), 3.96 (dd, 2H, J = 9.3,
5.7 Hz), 3.72 (dd, 2H, J = 9.3, 3.0 Hz), 1.98 (s, 6H).
13CNMR (DMSO, 75 MHz): 169.0 (–C), 164.9 (–C), 139.2
(–C), 132.7 (–C), 132.1 (–CH), 129.6 (–CH), 123.5 (–CH),
118.6 (–C), 110.0 (–C), 86.3 (–CH), 71.1 (–CH2), 56.5
123

Pseudo-peptides derived from isomannide
705
(–CH), 22.7 (–CH3). IR (KBr) m cm-1: 3,243, 2,980, 2,890,
2,229, 1,659, 1,534, 1,488, 1,372, 1,316, 1,267, 1,092,
1,042, 1,007, 890, 834. a2D0 = ? 106 (c, 0,1) DMSO, mp
204–206°C. HRMS calcd. for C30H28N6O6Na 591.1968.
Found 591.1971. The product is a pale yellow solid; 56%
yield.
7.20 (d, 2H, J = 1.5 Hz), 7.10 (dd, 2H, J = 8.1, 1.5 Hz),
6.97 (d, 2H, J = 8.7 Hz), 6.92 (s, 2H), 4.55 (s, 2H), 4.22–
4.16 (m, 2H), 3.95 (dd, 2H, J = 9.0, 5.7 Hz), 3.77 (s, 3H),
3.75 (s, 3H), 3.70 (dd, 2H, J = 9.3, 3.3 Hz); 2.00 (s, 6H).
13CNMR (DMSO, 75 MHz): 169.2 (–C), 165.4 (–C), 149.2
(–C), 148.2 (–C), 127.9 (–C), 127.5 (–CH), 126.6 (–C), 123.0
(–CH), 112.3 (–CH), 111.5 (–CH), 86.5 (–CH), 71.2 (–CH2),
56.5 (–CH), 55.4 (–CH3), 55.2 (–CH3), 22.7 (–CH3). IR
(KBr) m cm-1: 3,449, 3,289, 2,967, 2,876, 1,653, 1,619,
1,519, 1,370, 1,262, 1,145, 1,024, 905, 810. a2D0 = ? 87
(c, 0,1) DMSO, mp 173–175°C. HRMS calcd. for
C32H39N4O10 639.2666. Found 639.2687. The product is a
white solid; 40% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-4-methylcin-
1
namamido]-2,5-dideoxy-L-iditiol (44). HNMR d (DMSO,
300 MHz): 9.33 (s, 2H), 8.19 (d, 2H, J = 6.6 Hz), 7.42 (d,
4H, J = 8.4 Hz), 7.19 (d, 4H, J = 8.1 Hz), 6.88 (s, 2H),
4.56 (s, 2H), 4.14 (m, 2H), 3.95 (dd, 2H, J = 9.3, 5.7 Hz),
3.70 (dd, 2H, J = 9.3, 3.3 Hz), 2.30 (s, 6H), 1.98 (s, 6H).
13CNMR (DMSO, 75 MHz): 169.1 (–C), 165.3 (–C), 137.9
(–C), 131.2 (–C), 129.3 (–C), 129.2 (–CH), 128.9 (CH),
126.8 (–CH), 86.4 (–CH), 71.1 (–CH2), 56.4 (–CH), 22.7
(–CH3), 20.7 (–CH3). IR (KBr) m cm-1: 3,243, 2,976, 2,879,
1,651, 1,531, 1,371, 1,320, 1,286, 1,206, 1,185, 1,084, 1,043,
813, 709. a2D0 = ? 83 (c, 0,1) DMSO, mp 174–175°C.
HRMS calcd. for C30H34N4O6Na 569.2373. Found
569.2361. The product is a pale yellow solid; 52% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-2-naphthyl-
acrylamido]-2,5-dideoxy-L-iditiol (45). 1HNMR d (DMSO,
300 MHz): 9.38 (s, 2H), 8.03 (s, 2H), 7.92–7.84 (m, 8H),
7.77 (d, 2H, J = 8.1 Hz), 7.55-7.49 (m, 4H), 7.32 (s, 2H),
4.44 (s, 2H), 3.80 (dd, 2H, J = 9.0, 4.8 Hz), 3.60 (dd, 2H,
J = 9.0, 4.8 Hz), 3.42 (dd, 2H, J = 4.8, 2.7 Hz); 2.01
(s, 6H). 13CNMR (DMSO, 75 MHz): 167.4 (–C), 167.2
(–C), 132.6 (–C), 132.5 (–C), 132.4 (–C), 131.2 (–C), 129.0
(–CH), 128.0 (–CH), 127.3 (–CH), 127.2 (–CH), 126.4
(–CH), 126.1 (–CH), 86.8 (–CH), 72.5 (–CH2), 56.6 (–C),
22.8 (–CH3). IR (KBr) m cm-1: 3,253, 3,054, 2,982, 2,890,
1,650, 2,153, 1,660, 1,634, 1,525, 1,380, 1,322, 1,275,
1,149, 1,050, 1,015, 811, 746, 716. a2D0 = ? 10 (c, 0,1)
DMSO, mp 191–193°C. The product is a pale yellow solid;
86% yield.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-4-fluorocin-
1
namamido]-2,5-dideoxy-L-iditiol (48). HNMR d (DMSO,
300 MHz): 9.37 (s, 2H), 8.23 (d, 2H, J = 6.6 Hz), 7.58
(dd, 4H, J = 8.7, 5.7 Hz), 7.21 (t, 4H, J = 9.0 Hz), 6.89
(s, 2H), 5.56 (s, 2H), 4.24-4.16 (m, 2H), 3.95 (dd, 2H,
J = 9.0, 5.7 Hz), 3.70 (dd, 2H, J = 9.3, 3.3 Hz), 1.98
(s, 6H). 13CNMR (DMSO, 75 MHz): 169.3 (–C), 165.3 (–C),
131.4 (–CH), 130.7 (–C), 129.8 (–C), 125.4 (–CH), 115.5
(–CH), 115.2 (–CH), 86.5 (–CH), 71.2 (–CH2), 56.5 (–CH),
22.7 (–CH3). IR (KBr) m cm-1: 3,468, 3,258, 2,978, 2,872,
1,647, 1,537, 1,426, 1,371, 1,281, 1,230, 1,156, 1,090, 931,
831, 706. a2D0 = ? 74 (c, 0,1) DMSO, mp 228–230°C.
HRMS calcd. for C28H29F2N4O6 555.2055. Found
555.2030. The product is a white solid; 72% yield.
General: biology
HCV replicon
The HCV subgenomic replicon named I389/3-30-Luc-
UbiNeo, a gift from R. Bartenschlager, was previously
described (Mesaik et al. 2004). Briefly, the replicon codes
for HCV NS3 through NS5B nonstructural genes under the
Encephalomyocarditis virus (EMCV) internal ribosome
entry site (IRES) and neomycin resistance under the HCV
IRES. The HCV 50 and 30 nontranslated regions are also
present. The mRNA expresses the Firefly luciferase (Luc)
under HCV IRES. The Luc reporter is used as an indirect
measurement of HCV replication and the activity of the
Luc reporter is proportionally related to HCV RNA levels.
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-2-thiopheny-
lacrylamido]-2,5-dideoxy-L-iditiol (46). 1HNMR
d
(DMSO, 300 MHz): 9.22 (s, 2H), 8.17 (d, 2H, J = 7.2 Hz),
7.69 (d, 2H, J = 6.3 Hz), 7.50 (s, 2H), 7.41 (d, 2H,
J = 3.9 Hz), 7.11 (dd, 2H, J = 4.8, 3.3 Hz), 4.54 (s, 2H),
4.24-4.18 (m, 2H), 3.95 (dd, 2H, J = 9.3, 6.0 Hz), 3.70
(dd, 2H, J = 9.3, 3.3 Hz); 2.05 (s, 6H). 13CNMR (DMSO,
75 MHz): 169.7 (–C), 164.5 (–C), 136.6 (–C), 131.8
(–CH), 129.8 (–CH), 126.9 (–CH), 126.6 (–C), 124.4
(–CH), 86.6 (–CH), 71.1 (–CH2), 56.5 (–CH), 23.3 (–CH3).
IR (KBr) m cm-1: 3,309, 3,220, 2,966, 2,885, 1,651, 1,614,
1,529, 1,423, 1,367, 1,278, 1,213, 1,085, 929, 904, 853,
717. a2D0 = ? 94 (c, 0,1) DMSO, mp 222–224°C. HRMS
calcd. for C24H27N4O6S2 531.1372. Found 531.1385. The
product is a white solid; 70% yield.
Cell culture
Huh-7 cells (Hepatoma cell line) were grown at 37°C in
Dulbecco’s modified Eagle’s medium (DMEM) with 10%
fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin,
and 100 lg/ml streptomycin. Huh-7 cells were transfected
with RNA transcribed from linearized pHCVreplicon plas-
mid (Ribomax Large Scale RNA production, Promega).
Transient transfections were performed as previously
1,4:3,6-Dianhydro-2,5-bis-[2-acetamido-(Z)-3,4-dimeth-
oxycinnamamido]-2,5-dideoxy-L-iditiol (47). 1HNMR d
(DMSO, 300 MHz): 9.33 (s, 2H), 8.13 (d, 2H, J = 7.2 Hz),
123

706
T. G. Barros et al.
described (Muri et al. 2005). Transfected cells were selected
and grown in G418 (Geneticin; Gibco) at 500 lg/ml, which
was absent in all experiments. Cell growth was monitored by
counting the number of viable cells with trypan blue stain-
ing. Cells were treated with recombinant human IFN
(100 IU/ml in DMEM) for 18 h at 37°C, as positive control,
unless stated otherwise.
was envisaged as the center of this new family of pepti-
demimetic compound. The structural analogy of
isomannide with cyclic rigid dipeptides and its C2
symmetry will result in core rigid C2 symmetric peptide-
mimetic compounds (Bencsik et al. 2003; Dietrich and
Lubell 2003).
The pseudo-peptide compounds were prepared by the
reaction of the commercial isomannide 1 and p-toluene-
sulphonyl chloride in pyridine forming the bis-sulphonated
2 with 90% yield after recrystallization with MeOH
(Hockett et al. 1946; Muri et al. 2005). After which, the
compound 2 reacted with sodium azide yielding the
bis-azido compound 3 with inversion of configuration
(Chiappe et al. 2003). Several solvents can be used in the
nucleophilic substitution step such as DMF and DMSO. In
our case, however, the ionic liquid, [bmim]?[BF4]-, was
shown to be superior, improving the reaction yield from
60% (using DMF) to 78%. The ionic liquid, [bmim]?[BF4]
-, was synthesized following the methodology described in
literature. The ionic solvents possess several properties,
such as low melting point, negligible vapor pressure, low
coordinating ability and excellent thermal and chemical
stability, which make them attractive alternatives to tradi-
tional solvents (Suarez et al. 1996; Lancaster et al. 2002;
Earle and Seddon 2000). The reduction of diazido deriva-
tive 3 with hydrogen over palladium on carbon gave the
bis-amino compound 4 a 93% yield (Scheme 1) (Archibald
and Baum 1989).
IC50 and CC50 determinations using the replicon system
Briefly, 5,000 Huh-7 cells containing HCV subgenomic
replicon were plated in 96-well plates in a total volume of
100 ll of growth medium in Dulbecco’s modified Eagle
medium containing 5% (vol/vol) fetal bovine serum with-
out G418. Inhibitors were added 24 h post-plating in
threefold dilutions at a final DMSO concentration of 1%
(vol/vol). After 3 days, the cells were harvested and the
Firefly luciferase signal was quantified using a Luciferase
Assay System (Promega). The 50% inhibitory concentra-
tion (IC50) values were calculated as the concentration of
inhibitor at which a 50% reduction in the levels of Firefly
luciferase signal was observed compared to untreated cells.
Human interferon alpha (IFN-a) and thiophene was inclu-
ded in each run as a positive control. Subconfluent cultures
of the Huh.7 cell line were plated out into 96-well plates
and used for analysis of cell viability (cytotoxicity) or
antiviral activity. The cytotoxicity of each compound was
assessed as a percent of viable cells relative to untreated-
cells using CellTiter-Blue (Promega), a colorimetric assay
used as an indicator for cell viability.
Oxazolones are a class of important heterocyclic com-
pounds, which are useful precursors for the synthesis of
amino acids and peptides; so, we decided to use them to
build the peptidemimetic compounds. Initially, the oxa-
zolones (so called azalactones) (7-27) were synthesized
from commercially available glycine (5) and benzoyl
chloride or acetic anhydride obtaining the benzoylglycine
(6a) with 95% yield and acetylglycine (6b) with 77% yield,
which reacted with different aldehydes in the presence of
anhydrous sodium acetate/acetic anhydride by Erlenmeyer
conditions (Kitazawa et al. 1995; Mesaik et al. 2004). This
methodology gives only the thermodynamically stable
isomer Z (Scheme 2) (Rao 1976; Brocklehurst et al. 1971).
The substituents, yields and melting points of the synthe-
sized oxazolones are shown in Table 1 (Kitazawa et al.
1995; Bautista et al. 2002; Paul et al. 2004; Kuhn 1987;
Slater and Somerville 1966; Wong et al. 1992; Hoshina
et al. 2000; Sen and Shanker 1996; Jendralla et al. 1995;
Etschenberg et al. 1979; Meiwes et al. 1997; Lisichkina
et al. 1999). The last step consisted in the reaction of 4 and
the respective oxazolones 7-27 yielding the final pseudo-
peptide products 28-48. Initially, MeOH, THF and DMF
were tried as solvents, but the reactions showed low yields.
So we decided to use AcOEt, which presented the best
Results and discussion
Chemistry
The peptidemimetic approach in drug research has signif-
icant potential in the design of substrate-based inhibitors.
The introduction of a fused-bicyclic structure has been
applied to the design of modified peptides and peptidomi-
metics in an effort to discover new therapeutics and gain an
improved understanding of the interactions of ligands with
target enzymes and receptors. Examples of such com-
pounds include the bis-tetrahydrofuranyl, Danuravir
(TMC-114), which has recently been approved for the
treatment of HIV/AIDS patients (Ghosh et al. 2007), and
the bicyclic proline derivative VX-950, which hass been
evaluated as an inhibitor of HCV NS3 protease (Lin et al.
2004).
Based on our earlier results on designing and synthesis
of peptides derived from isomannide, this rigid scaffold
123

Pseudo-peptides derived from isomannide
707
RO
anti-HCV drug discovery (Lohmann et al. 1999, 2003;
Bassit et al. 2008; Kanda et al. 2004; He et al. 2008). HCV
replicon-based assay has been previously reported as sen-
sitive and specific for anti-HCV drug screening and
therefore has been considered adequate to evaluate the anti-
HCV potential candidates. The anti-HCV potential of
pseudo-peptides derived from isomannide 28-48 was
evaluated by using the HCV replicon-based assay. The
values of IC50 and CC50 of these compounds are shown in
Table 2, where we can see that the methylenedioxy
derivative 28 showed the most effective activity.
H
H
H
O
N3
H2
N
O
O
O
H
c
b
O
O
NH2
N3
OR
H
H
3
4
1
2
R = -H
R = -SO2C6H4Me-p
a
Scheme 1 Synthesis of bis-amino derivative 4. a TsCl, Py, r.t.,
overnight, 90%. b NaN3, [bmim]?[BF4] -, 12 h, 120°C, 78%. c) H2,
10% Pd/C, EtOH, 40psi, 12 h, 93%
O
H
O
OH
OH
b
a
H2N
R1
N
H
R2
O
O
O
N
R3
Conclusions
6a
6b
R1 = -Ph
1 = -CH3
5
7-27
R
In this work, we present the synthesis of a series of pseudo-
peptides derived from isomannide 28-48 and their anti-
HCV potential using the HCV replicon-based assay. The
compound named 28 was the most effective inhibitor
compound tested with IC50, near to 20 lM. However,
compound 28 was also the most cytotoxic compound tested
here, with CC50 near to 20 lM. Further investigations on
the cytotoxicity of this compound should be carried out
through modifications in its chemical structure in order to
improve the putative selectivity on the HCV protease.
Scheme 2 Synthesis of Oxazolones 7-27. a BzCl, 10% NaOH, HCl
or Ac2O, H2O, 95% (6a) and 77% (6b). b Ac2O, NaOAc, R2CHO
results (Scheme 3) (Gomes et al. 2006; Valdes et al. 2007;
Hernandez Valdes et al. 2004).
Biological results
Subgenomic HCV replicon cell culture systems have
significantly impacted the field of HCV research and
Table 1 Oxazolone derivatives
7-27 produced via Scheme 2
Compound
Names
Mp (°C)
Yield (%)
7
4-(30,40-Methylenedioxybenzylidene)-2-phenyl-5(4H)-oxazolone
4-Benzylidene-2-phenyl-5(4H)-oxazolone
200
50
61
55
50
71
60
66
78
84
40
35
48
58
66
55
84
50
40
55
45
64
8
169–170
162–163
154–155
186
9
4-(40-Methoxybenzylidene)-2-phenyl-5(4H)-oxazolone
4-(30,40-Dimethoxybenzylidene)-2-phenyl-5(4H)-oxazolone
4-(40-Fluorobenzylidene)-2-phenyl-5(4H)-oxazolone
4-(40-Chlorobenzylidene)-2-phenyl-5(4H)-oxazolone
4-(40-Bromobenzylidene)-2-phenyl-5(4H)-oxazolone
4-(40-Trifluoromethylbenzylidene)-2-phenyl-5(4H)-oxazolone
4-(20-Thiophenylmethylidene)-2-phenyl-5(4H)-oxazolone
4-(3’-pyridylmethylidene)-2-phenyl-5(4H)-oxazolone
4-(30,40-Methylenedioxybenzylidene)-2-methyl-5(4H)-oxazolone
4-Benzylidene-2-methyl-5(4H)-oxazolone
4-(40-Chlorobenzylidene)-2-methyl-5(4H)-oxazolone
4-(40-Bromobenzylidene)-2-methyl-5(4H)-oxazolone
4-(4’-Trifluoromethylbenzylidene)-2-methyl-5(4H)-oxazolone
4-(40-Cyanobenzylidene)-2-methyl-5(4H)-oxazolone
4-(40-Methylbenzylidene)-2-phenyl-5(4H)-oxazolone
4-(2’-Naphtylmethylidene)-2-methyl-5(4H)-oxazolone
4-(20-Thiophenylmethylidene)-2-methyl-5(4H)-oxazolone
4-(30,40-Dimethoxybenzylidene)-2-methyl-5(4H)-oxazolone
4-(40-Fluorobenzylidene)-2-methyl-5(4H)-oxazolone
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
199–200
207–208
174–175
176–178
154
183
158
149
135
119
191–192
136
225
120
163–165
149–151
123

708
T. G. Barros et al.
Scheme 3 Synthesis of pseudo-
peptide derivatives 28-48. a
AcOEt, reflux, 24 h
OR2
O
H
H
N
H
O
H2N
O
H
H
O
O
H
a
N
H
+
R2
O
R3
O
H
N
O
N
NH2
R3
R3
N
H
O
4
7-27
28-48
R2
Bautista FM, Campelo JM, Garcia A, Luna D, Marinas JM, Romero
AA (2002) Study on dry-media microwave azalactone synthesis
on different supported KF catalysts: influence of textural and
acid–base properties of supports. J Chem Soc Perkin Trans
2:227–234
Table 2 Activity of different pseudo-peptides derived from iso-
mannide in the HCV Luc/Ubi/Neo replicon
Compound
CC50%(lM)a
IC50%(lM)b
Thiophene
28
[100
[20
NS
5
Bazan JF, Fletterick RJ (1989) Detection of a trypsin-like serine
protease domain in flaviviruses and pestiviruses. Virology
171:637–639. doi:10.1016/0042-6822(89)90639-9
Bazan JF, Fletterick RJ (1990) Structural and catalytic models of
trypsin-like viral proteases. Semin Virol 1:311–322
Bencsik JR, Kercher T, O’Sullivan M, Josey JA (2003) Efficient,
stereoselective synthesis of oxazolo[3, 2-a]pyrazin-5-ones: novel
bicyclic lactam scaffolds from the bicyclocondensation of 3-aza-
1, 5-keto acids and amino alcohols. Org Lett 54:2727–2730. doi:
10.1021/ol030065h
Brocklehurst K, Bywater RP, Palmer RA, Patrick R (1971) Crystal
structure of the stable isomer of a-benzamidocinnamic acid.
Influence of cis–trans isomerism on the kinetics of the hydrolysis
of the products of interaction of a-chymotrypsin with isomeric 4-
benzylidene-2-phenyl-D2-oxazolin-5-ones. J Chem Soc D Chem
Commun: 632–633
[20
NS
29
30
[100
NS
[100
NS
31
32
NS
NS
33
NS
NS
34
[100
[100
NT
[100
95
35
36
NT
37
NS
NS
38
[100
NS
[100
NS
39
Bruix J, Cavalet X, Costa J, Ventura M, Bruguera M, Castillo R,
Barrera JM, Ercilla G, Sanchez-Tapias JM, Vall M, Bru C,
Rodes J (1989) Prevalence of antibodies to hepatitis C virus in
Spanish patients with hepatocellular carcinoma and hepatic
cirrhosis. Lancet 2:1004–1006. doi:10.1016/S0140-6736(89)
91015-5
Chiappe C, Pieraccini D, Saullo P (2003) Nucleophilic displacement
reactions in ionic liquids: substrate and solvent effect in the
reaction of NaN3 and KCN with alkyl halides and tosylates.
J Org Chem 68:6710–6715. doi:10.1021/jo026838h
Dietrich E, Lubell WD (2003) Efficient synthesis of enantiopure
pyrrolizidinone amino acid. J Org Chem 68:6988–6996. doi:
10.1021/jo034739d
Earle MJ, Seddon KR (2000) Ionic liquids: green solvents for the
future. Pure Appl Chem 72:1391–1398. doi:10.1351/pac20007
2071391
40
NS
NS
41
NS
NS
42
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
43
44
45
46
47
48
a
Measured in Huh7 cells
Measured in replicon I389/NS 3-30-LucUbi, Neo ET
b
NT non-tested, NS non-solubility in DMEM
Etschenberg E, Opitz W, Raddatz SDE (1979) Patent 77 2745584
Ghosh AK, Dawson ZL, Mitsuya H (2007) Darunavir, a conceptually
new HIV-1 protease inhibitor for the treatment of drug-resistant
HIV. Bioorg Med Chem 15:7576–7580. doi:10.1016/j.bmc.2007.
09.010
Acknowledgments This work was financially supported by
CAPES, CNPq and FAPERJ. We thank to Paulo Carvalho (University
of Mississippi) and Marcos Nogueira Eberlin (USP) for HRMS.
Gomes M Jr, Hernandez-Valdes R, Marques CESJ, Bastos ML,
Aranda DAG, Antunes OAC (2006) Enantioselective heteroge-
neous catalytic production of alpha-amino acids. React Kinet
Catal Lett 87:19–24. doi:10.1007/s11144-006-0004-8
Gorbalenya AE, Donchenko AP, Kunin EV, Blinov VM (1989)
N-terminal domains of putative helicases of flavi- and pestivi-
ruses may be serine proteases. Nucleic Acids Res 17:3889–3897.
doi:10.1093/nar/17.10.3889
He Y, King MS, Kempf DJ, Lu L, Lim HB, Krishnan P, Kati W,
Middleton T, Molla A (2008) Relative replication capacity and
selective advantage profiles of protease inhibitor-resistant hep-
atitis C virus (HCV) NS3 protease mutants in the HCV genotype
References
Archibald TG, Baum K (1989) Synthesis of polynitro-2, 6-dioxabi-
cyclo[3.3.0]octanes. Synth Commun 19:1493–1498. doi:
10.1080/00397918908051042
Bassit L, Grier J, Bennett M, Schinazi RF (2008) Combinations of 20-
C-methylcytidine analogues with interferon-a 2b and triple
combination with ribavirin in the hepatitis C virus replicon
system. Antivir Chem Chemother 19:25–31
123

Pseudo-peptides derived from isomannide
709
1b replicon system. Antimicrob Agents Chemother 52:1101–
1110. doi:10.1128/AAC.01149-07
replication in cell culture. J Virol 77:3007–3019. doi:10.1128/
JVI.77.5.3007-3019.2003
Hernandez Valdes R, Puzer L, Gomes M, Marques CESJ, Aranda
DAG, Bastos ML, Gemal AL, Antunes OAC (2004) Production
of L-DOPA under heterogeneous asymmetric catalysis. Catal
Commun 5:631–634. doi:10.1016/j.catcom.2004.07.018
Hockett RC, Fletcher HG Jr, Sheffield EL, Goepp RM Jr, Soltzberg S
(1946) The structures of the anhydromannitols of Brigl and
Meiwes J, Schudok M, Kretzschmar G (1997) Asymmetric synthesis
of L-thienylalanines. Tetrahedron Asymmetry 8:527–536. doi:
10.1016/S0957-4166(97)00016-5
Mesaik MA, Rahat S, Khan KM, Zia-Ullah Choudhary MI, Murad S,
Ismail Z, Atta-ur-Rahman Ahmad A (2004) Synthesis and
immunomodulatory properties of selected oxazolone derivatives.
Bioorg Med Chem 12(9):2049–2057. doi:10.1016/j.bmc.2004.
02.034
Gr.ovrddot.uner.
J
10.1021/ja01210a004
Am Chem Soc 68:930–935. doi:
Hoshina H, Kubo K, Morita A, Sakurai T (2000) Formation of
isoquinoline and 1-azetine derivatives via novel photocyclization
of substituted a-dehydrophenylalanines. Tetrahedron 56:2941–
2951. doi:10.1016/S0040-4020(00)00188-5
Jendralla H, Seuring B, Herchen J, Kulitzscher B, Wunner J, Stueber
W, Koschinsky R (1995) Efficient kg-scale synthesis of thrombin
inhibitor CRC 220. Tetrahedron 51:12047–12068. doi:10.1016/
0040-4020(95)00765-Z
Kanda T, Yokosuka O, Imazeki F, Tanaka M, Shino Y, Shimada H,
Tomonaga T, Nomura F, Nagao K, Ochiai T, Saisho H (2004)
Inhibition of subgenomic hepatitis C virus RNA in Huh-7 cells:
ribavirin induces mutagenesis in HCV RNA. J Viral Hepat
11:479–487. doi:10.1111/j.1365-2893.2004.00531.x
Muri EMF, Gomes M Jr, Costa JS, Alencar FL, Sales A Jr, Bastos ML,
Hernandez-Valdes R, Albuquerque MG, Cunha EFF, Alencastro
RB, Williamson JS, Antunes OAC (2004) N- t-Boc-amino acid
esters of isomannide: potential inhibitors of serine proteases.
Amino Acids 27:153–159. doi:10.1007/s00726-004-0121-5
Muri EMF, Gomes M Jr, Albuquerque MG, Cunha EFF, Alencastro
RB, Williamson JS, Antunes OAC (2005) Pseudo-peptides
derived from isomannide as potential inhibitors of serine
proteases. Amino Acids 28:413–419. doi:10.1007/s00726-004-
0146-9
Muri EMF, Gomes MJr, Costa JS, Bastos ML, Hernandez-Valdes R,
Albuquerque MG, Cunha EFF, Alencastro RB, Antunes OAC
(2006) Braz Pat 0401908-3
Kitazawa M, Higuchi R, Takahashi M, Wada T, Sasabe H (1995)
Novel molecular design for second-harmonic generation: azlac-
tone derivatives. J Phys Chem 99:14784–14792. doi:10.1021/
j100040a031
Paul S, Nanda P, Gupta R, Loupy A (2004) Calcium acetate-catalyzed
synthesis of 4-arylidene-2-phenyl-5(4H)-oxazolones under sol-
vent-free conditions. Tetrahedron Lett 45:425–427. doi:
10.1016/j.tetlet.2003.10.125
Kuhn DG (1987) US Patent 4 659 857
Rao YS (1976) Reactions in polyphosphoric acid. I. New stereospe-
cific synthesis of the E isomers of 2-phenyl-4-arylmethylene-2-
oxazolin-5-ones. J Org Chem 41:722–725. doi:10.1021/jo008
66a037
Sen S, Shanker K (1996) Synthesis of some new 2-methyl-4-
(substituted benzylidene) 1-phenyl-1, 2, 4 triazolo[3, 4,-b]1, 3, 4-
thiadiazole as potential AChE inhibitory agents. Boll Chim Farm
135:465–467
Lancaster NL, Salter PA, Welton T, Young GB (2002) Nucleophi-
licity in ionic liquids. 2. Cation effects on halide nucleophilicity
in a series of bis(trifluoromethylsulfonyl)imide ionic liquids.
J Org Chem 67:8855–8861. doi:10.1021/jo026113d
Leung D, Schroder K, White H, Fang N-X, Stoermer MJ, Abbenante
G, Martin JL, Young PR, Fairlie DP (2001) Activity of
recombinant dengue 2 virus NS3 protease in the presence of a
truncated NS2B co-factor, small peptide substrates, and inhib-
itors. J Biol Chem 276:45762–45771. doi:10.1074/jbc.M10736
0200
Slater G, Somerville AW (1966) Syntheses from pyridinecarboxal-
dehydes. I. 6H- and 7H-pyridines. Tetrahedron 22:35–42. doi:
10.1016/0040-4020(66)80098-4
Lin C, Lin K, Luong Y-P, Rao BG, Wei Y-Y, Brennan DL, Fulghum
JR, Hsiao H-M, Ma S, Maxwell JP, Cottrell KM, Perni RB,
Gates CA, Kwong AD (2004) In vitro resistance studies of
hepatitis C virus serine protease inhibitors, VX-950 and BILN
2061: structural analysis indicates different resistance mecha-
nisms. J Biol Chem 279:17508–17514. doi:10.1074/jbc.M3130
20200
Lisichkina IN, Ushakova OM, Alekseeva MO, Peregudov AS,
Belikov VM (1999) The effect of anions in the coordination
sphere of Mg complexes of N-acetyldehydrophenylalanyl-(S)-
valine on the diastereoselectivity of hydrogenation. Russ Chem
Bull 48:1682–1684. doi:10.1007/BF02494812
Suarez PAZ, Dullius JEL, Einloft S, De Souza RF, Dupont J (1996)
The use of new ionic liquids in two-phase catalytic hydrogena-
tion reaction by rhodium complexes. Polyhedron 15:1217–1219.
doi:10.1016/0277-5387(95)00365-7
Valdes RH, Aranda DAG, Alvarez HM, Antunes OAC (2007)
Microwave-promoted ring opening reaction of azlactones. Lett
Org Chem 4:35–38. doi:10.2174/157017807780037360
Wong HNC, Xu ZL, Chang HM, Lee CM (1992) Compounds from
Danshen. 6. A modified synthesis of ( )- b-aryllactic acid.
Synthesis (8):793–797. doi:10.1055/s-1992-26228
Wright M, Main J, Thomas HC (2001) Treatment of chronic viral
hepatitis. Antivir Chem Chemother 12:201–212
Lohmann V, Korner F, Koch J-O, Herian U, Theilmann L,
Bartenschlager R (1999) Replication of subgenomic hepatitis C
virus RNAs in a hepatoma cell line. Science 285:110–113. doi:
10.1126/science.285.5424.110
Lohmann V, Hoffmann S, Herian U, Penin F, Bartenschlager R
(2003) Viral and cellular determinants of hepatitis C virus RNA
Yusof R, Clum S, Wetzel M, Murthy HM, Padmanabhan R (2000)
Purified NS2B/NS3 serine protease of dengue virus type 2
exhibits cofactor NS2B dependence for cleavage of substrates
with dibasic amino acids in vitro. J Biol Chem 275:9963–9969.
doi:10.1074/jbc.275.14.9963
123