Broad-spectrum antiviral activity including human immunodeficiency and hepatitis C viruses mediated by a novel retinoid thiosemicarbazone derivative

Article abstract of DOI:10.1016/j.ejmech.2011.02.014

Aromatic aldehyde-derived thiosemicarbazones 4-6, the S-substituted modified thiosemicarbazones 7/8, and a vitamin A-derived (retinoid) thiosemicarbazone derivative 12 were investigated as inhibitors of human hepatitis C virus (HCV) subgenomic RNA replicon Huh7 ET (luc-ubi-neo/ET) replication. Compounds 4-6 and 12 were found to be potent suppressors of HCV RNA replicon replication. The trifluoromethoxy-substituted thiosemicarbazone 6 and the retinoid thiosemicarbazone derivative 12 were even superior in selectivity to the included reference agent recombinant human alpha-interferon-2b, showing potencies in the nanomolar range of concentration. In addition, compounds 5, 6, 8 and 12 were tested as inhibitors of cytopathic effect (CPE) induced by human varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV). Compounds 4-6, 8 and 12 were additionally examined as inhibitors of CPE induced by cowpox virus and vaccinia virus. Thiosemicarbazone 4 was inhibitory on cowpox and vaccinia virus replication comparable in potency and selectivity to the reference agent cidofovir. Retinoid thiosemicarbazone derivative 12 was active as micromolar inhibitor of VZV, HCMV, and, in addition, human immunodeficiency virus type 1 (HIV-1) replication. These results indicate that thiosemicarbazone derivatives are appropriate lead structures to be evaluated in targeted antiviral therapies for hepatitis C (STAT-C), and that the vitamin A-related thiosemicarbazone derivative 12 emerges as a broad-spectrum antiviral agent, co-suppressing the multiplication of important RNA and DNA viruses.

Full text of DOI:10.1016/j.ejmech.2011.02.014

European Journal of Medicinal Chemistry 46 (2011) 1656e1664
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Broad-spectrum antiviral activity including human immunodeficiency and
hepatitis C viruses mediated by a novel retinoid thiosemicarbazone derivative
Andreas J. Kesel*
Chammünsterstr. 47, D-81827 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 November 2010
Received in revised form
Aromatic aldehyde-derived thiosemicarbazones 4e6, the S-substituted modified thiosemicarbazones
7/8, and a vitamin A-derived (retinoid) thiosemicarbazone derivative 12 were investigated as inhibitors
of human hepatitis C virus (HCV) subgenomic RNA replicon Huh7 ET (luc-ubi-neo/ET) replication.
Compounds 4e6 and 12 were found to be potent suppressors of HCV RNA replicon replication. The
trifluoromethoxy-substituted thiosemicarbazone 6 and the retinoid thiosemicarbazone derivative 12
were even superior in selectivity to the included reference agent recombinant human alpha-interferon-
6
February 2011
Accepted 9 February 2011
Available online 15 February 2011
2b, showing potencies in the nanomolar range of concentration. In addition, compounds 5, 6, 8 and 12
Keywords:
were tested as inhibitors of cytopathic effect (CPE) induced by human varicella-zoster virus (VZV) and/or
human cytomegalovirus (HCMV). Compounds 4e6, 8 and 12 were additionally examined as inhibitors of
CPE induced by cowpox virus and vaccinia virus. Thiosemicarbazone 4 was inhibitory on cowpox and
vaccinia virus replication comparable in potency and selectivity to the reference agent cidofovir. Retinoid
thiosemicarbazone derivative 12 was active as micromolar inhibitor of VZV, HCMV, and, in addition,
human immunodeficiency virus type 1 (HIV-1) replication. These results indicate that thiosemicarbazone
derivatives are appropriate lead structures to be evaluated in targeted antiviral therapies for hepatitis C
Thiosemicarbazone derivatives
Hepatitis C virus (HCV)
HCV Huh7 ET replicon
Retinoid thiosemicarbazone
HCV replicon replication inhibitors
HIV-1 inhibitor
(
STAT-C), and that the vitamin A-related thiosemicarbazone derivative 12 emerges as a broad-spectrum
antiviral agent, co-suppressing the multiplication of important RNA and DNA viruses.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1
. Introduction
respectively. The pegylated alpha-interferons are recommended for
combination with ribavirin [8], and presently constitute the most
comprehensive treatment regimen for chronic hepatitis C.
Since the therapy of hepatitis C with pegylated alpha-interferon
(PEG-IFN-a)/ribavirin is hampered by serious side effects (flu-like
symptoms, fever, headache, myalgia, bone marrow depression,
neuropsychiatric complications including psychosis/suicide, fatigue,
alopecia, etc.), and non-responsiveness of HCV virological status
(therapy-refractory chronic disease) in a significant proportion of
Human hepatitis C virus (HCV) infection constitutes a major
global public health problem with approximately 170 million
people infected worldwide (ca. 3% of world population) [1]. Infec-
tion with the hepatotropic (liver-targeting) Hepacivirus (Flavivir-
idae) HCV can result in chronic disease involving liver cirrhosis and/
or primary human hepatocellular carcinoma (HCC) [2]. The latter
characteristic shares resemblance to the Orthohepadnavirus (Hep-
adnaviridae) human hepatitis B virus (HBV), a completely different
pathogen which also can manifest itself chronically with progres-
sion to HCC [3].
PEG-IFN-a/ribavirin-treated patients [8,9], recentefforts are focusing
on the development of IFN-a/ribavirin-independent interventions
for HCV disease, the so-called specific(ally) targeted antiviral thera-
pies for hepatitis C (STAT-C) [10e12].
Present standard-of-care for HCV infected patients is the therapy
Ò
with the combination of recombinant interferon-
A) or interferon-
REBETOL ) [4,5]. The two interferons are available in poly(ethy-
lene glycol) (PEG)-conjugated (pegylated) forms, peginterferon-
a
a
2a (ROFERON
The STAT-C therapies target HCV protein NS3-NS4A protease
[10,11], and, recently, NS3 RNA helicase enzymatic functions
[13e15], or the NS5B RNA-dependent RNA polymerase enzymatic
function [10,11]. As an example for a clinically examined NS3-NS4A
protease inhibitor should serve the substance BILN 2061, being
a complex macrocylic compound [16]. BILN 2061 now is super-
seded by the HCV NS3-NS4A protease inhibitors in clinical devel-
opment telaprevir, boceprevir, danoprevir (RG7227/ITMN-191),
TMC435350 [10,11], and narlaprevir (SCH 900518) [17]. Recently,
Ò
Ò
a
2b (INTRON A) and ribavirin (COPEGUS ,
Ò
Ò
Ò
2a (PEGASYS ) [6] and peginterferon-a 2b (PEG-INTRON ) [7],
*
Tel.: þ49 (0)89 453 64500.
E-mail address: andreas.kesel@munich.netsurf.de.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.014

A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
1657
the new picomolar HCV replication inhibitor BMS-790052 was
reported to target HCV NS5A protein [18]. The HCV NS5A protein
still is enigmatic in its function, but, obviously, is indispensable for
HCV replication [18]. This indicates that the HCV proteome offers
more proteins which could serve as targets for antiviral chemo-
therapy than anticipated until recently.
This work presents thiosemicarbazone-related compounds
being strong (sub-micromolar) inhibitors of HCV replicon replica-
tion in Huh7 ET cells. Their discovery was stimulated by the
disclosure of an U.S. patent application [19], proposing thio-
semicarbazone derivatives for the treatment of chronic human
hepatitis C, a strategy utilizing the powerful biological effects
mediated by the inclusion of sulfur in the constant substitution
motif of various aromatic aldehyde-derived thiosemicarbazones.
On the other hand, the implementation of the retinoid (vitamin
A-derived) diterpenoid polyene side chain in this constant thio-
semicarbazone motif, as will be demonstrated, proofed to be very
supportive for inhibition of HCV replication. Retinoids, derived
from all-trans retinol, all-trans retinal, and/or all-trans retinoic acid,
are generally esteemed for their powerful antineoplastic/chemo-
preventive [20], immunopotentiating [21], and antiviral actions
Fig. 1. Synthesis of the thiosemicarbazones 4e6 from the aromatic aldehydes 1e3.
Reagents and conditions: (a) thiosemicarbazide, EtOH/H
and 6) and reflux.
2
O (with addition of HOAc for
5
S-tetradecyl isothiosemicarbazone 7 was synthesized starting with
, by refluxing the sodium salt of 4 with 1-bromotetradecane in
4
EtOH/H
2
O (Fig. 2). The 1,3-thiazole derivative 8 was prepared from
0
4
via Hantzsch thiazole synthesis using 2-bromo-4 -fluoroac-
2
etophenone in acetone/EtOH/H O (Fig. 2).
The third group of compounds is solely composed of a retinoid
thiosemicarbazone derivative synthesized from vitamin A. All-trans
retinyl acetate 9 was saponified to all-trans retinol 10 (Fig. 3), and
subsequently oxidized to all-trans retinal 11 by influence of
manganese(IV) oxide MnO (Fig. 3). Compound 11 was reacted to
2
the intermediate all-trans retinal thiosemicarbazone by refluxing
with thiosemicarbazide. Two molecules all-trans retinal thio-
[22]. These effects could synergistically supplement the direct
antiviral action of the thiosemicarbazone motif. It seems to be quite
remarkable that an anti-infective action of vitamin A was discov-
ered as early as 1928 by Green and Mellanby [23]. Therefore, it
seemed to be consequent to additionally investigate several of the
synthesized test compounds as inhibitors of human varicella-zoster
virus (VZV) [Herpesviridae, Alphaherpesvirinae, Varicellovirus,
human herpesvirus 3 (HHV-3)] and/or human cytomegalovirus
semicarbazone split off ammonia NH
the dimeric retinoid thiosemicarbazone derivative trisodium salt
2 (Fig. 3). Compound 12 was recrystallized from aqueous acetone
3
in presence of NaOH to give
1
to give an intermediate monosodium salt. This could be re-trans-
formed, joined by efficacious purity increase, into 12 by impact of
NaOH at room temperature (Fig. 3).
The CHNS/O elemental analysis of 12 gave erratic sulfur and
2 4
oxygen results due to the formation of incombustible Na SO from
(
HCMV) [Herpesviridae, Betaherpesvirinae, Cytomegalovirus, human
herpesvirus 5 (HHV-5)] replication. These herpesviruses constitute
major human pathogens, especially in the immunocompromised
host [24], causing, for example, herpetic retinitis, a potential threat
of vision loss after organ transplantation or during human immu-
nodeficiency virus (HIV) infection [(H)CMV-retinitis emerging
during the progression of acquired immunodeficiency syndrome
the coinciding presence of sulfur and sodium in both substances,
being already reported for combustion analysis of sulfur-containing
sodium salts [28]. The dimeric structure of 12 was in turn
confirmed by electrospray ionization mass spectrometry (ESI-MS)
in negative ion mode (see Supplementary data, Fig. S1). Due to
specific mass spectrometric calibration, the m/z values are 0.43
atomic mass units (amu ¼ Da) higher than given in the spectrum
(
AIDS)]. To supplement these pathogens affecting immunosup-
pressed patients, cowpox virus and vaccinia virus (Poxviridae,
Chordopoxvirinae, Orthopoxvirus) were included in these assays as
paradigms for infections potentially evolving in immunocompro-
mised patients [25]. These orthopoxviruses serve also as surrogates
for variola major (smallpox) virus (Poxviridae, Chordopoxvirinae,
Orthopoxvirus) which recently attained renewed interest as
potential bioterrorist attack agent. In view of the emerging broad-
spectrum antiviral activity of the retinoid (vitamin A-derived) thi-
osemicarbazone derivative, this compound was additionally tested
as inhibitor of human immunodeficiency virus type 1 (HIV-1)
propagation in freshly donated (primary) peripheral blood mono-
nuclear cells (PBMCs).
(see Supplementary data, Fig. S1). Therefore, the dehydrated,
partially desodiated molecular radical anion (calcd m/z 719.4037)
ꢀ
ꢁ
þ
þ
was detected at m/z 719.3995 [M ꢁ 2Na þ 2H ] (see
2
. Results and discussion
2.1. Synthesis and chemistry of the antiviral test substances
The first group of compounds constitutes the thiosem-
icarbazones 4e6 of the aromatic aldehydes 1e3. For synthesis
Fig. 1) the aromatic aldehydes were converted with an equimolar
(
amount of thiosemicarbazide in aqueous ethanol (EtOH), in the
case of the synthesis of 5 and 6 by addition of glacial acetic acid. The
isolation of 4e6 was achieved by crystallization at low temperature,
in the case of 6 under addition of water. Compound 4, as the only
one of the synthesized compounds in this work, was already
described in literature [26,27].
Fig. 2. Synthesis of the modified thiosemicarbazones 7 and 8 from compound 4.
2 2
Reagents and conditions: (a) (i) NaOH, EtOH/H O, (ii) 1-bromotetradecane, EtOH/H O
The second group of compounds represents modified thio-
semicarbazones derived from compound 4. The alkane-modified
0
and reflux; (b) (i) 2-bromo-4 -fluoroacetophenone, acetone, (ii) NaOH, EtOH/H
reflux.
2
O and

1658
A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
are given (Table 1), the initial HCV RNA replicon doseeresponse
assay and the more accurate HCV RNA replicon confirmatory RNA-
based assay. In the doseeresponse assaycytotoxicity (CC50%/90%) was
determined by a colorimetric method, in the RNA-based assay by
quantification of ribosomal RNA. The thiosemicarbazones 4e6 all
shared good activity in these assays in the low micromolar range of
concentration. Compound 6 was the most effective compound of
4e6 with regard to the selectivity index SI50%. This trifluoro-
methoxy-substituted derivative 6 expressed activity in nanomolar
range of concentration, and considerable selectivity. Since the
sulfur-modified thiosemicarbazone derivatives 7 and 8 showed no
or low activity, respectively, it could be concluded that the sulfur
atom should be free from S-substitution to achieve significant anti-
HCV replicon activity.
High activity was seen with the retinoid thiosemicarbazone
derivative 12. This trisodium salt 12 was capable of reaching 90%
inhibition grade of HCV replicon RNA replication, and was superior
to the reference agent interferon-a 2b with regard to the selectivity
indices. To mention, the definition of international units (IU) of
Ò
recombinant human interferon-
a
2b (INTRON A) is the one given
Fig. 3. Synthesis of the retinoid thiosemicarbazone derivative 12 from all-trans retinyl
acetate 9. Reagents and conditions: (a) NaOH, EtOH/H O and reflux; (b) MnO , EtOAc,
RT; (c) (i) thiosemicarbazide, EtOAc/EtOH/H and reflux, (ii) acetone/H and
recrystallization; (d) NaOH, acetone/H O, RT.
by Schering Corporation [32], corresponding to the World Health
2
2
2
O
2
O
Organization (WHO) international biological standards for human
Ò
2
interferon-a [33]. Recombinant human interferon-a 2b (INTRON
A) incorporates a molecular weight of 19,271 Da (g/mol), and an
8
Supplementary data, Fig. S2). The base peak was found at m/z
61.0480. Like often seen in ESI-MS spectra [29], the retinoid
polyene chain was partially hydrogenated (þnH (see
Supplementary data, Fig. S2). Concomitant loss of NaSH and H
activity of 2.6 ꢂ 10 IU/mg [32].
3
2
)
2.3. Thin-layer-chromatographic mobility shift assay of compound
2
S
12
was also observed (see Supplementary data, Fig. S2). The structure
of the molecule radical (an)ion at m/z 719 verified the dimeric
constitution of 12, since no peak would have arisen in this region if
The potential binding of the anti-HCV compound 12 to cationic
elements of proteins, notably the basic amino acids
L-lysine and
12 was a derivative (analogue) of a monomeric diterpenoid (C20
)
L-arginine, was investigated to gain first hints for the mechanism
retinoid. The dimeric retinoid structure of 12 was further supported
of antiviral action of 12. It was anticipated that the antiviral action
of 12 is mediated by hydrophobic and/or ionic interaction of 12
with viral proteins at positively charged residues because of the
hydrophobic/anionic nature of 12. The methodology of thin-layer-
chromatographic shift retention of two selected potential binding
partners [34], when going into mutual complex formation, seemed
to be suitable for this purpose. Possible associations of 12 with
1
13
by means of H NMR and C NMR spectroscopy experiments on 12,
because in the 1H NMR spectrum of 12 (see Supplementary data,
Figs. S9 and S10) all possible imino and/or amino signals were
missing. The NMR signal assignments of the retinoid polyene
1
system in 12 were performed with help of literature data on
H
NMR spectra [30] and 13C NMR spectra [30,31] of retinoids, espe-
cially on Schiff bases of all-trans retinal 11 [30].
basic amino acids [
peptide [the synthetic growth-promoting [35] tripeptide GHK
(glycyl- -histidinyl- -lysine)] were investigated. Three different
thin-layer chromatography eluents were applied [90%, 60%, 30%
v/v) aqueous acetone]. In 90% aqueous acetone the lipophilic 12
L-lysine (K) and L-arginine (R)] and a basic tri-
2.2. Antiviral activities of the synthesized test compounds against
L
L
HCV
(
The HCV replicon replication-inhibiting biological activities, the
effective concentrations 50%/90% (EC50%/90%), the cytotoxic concen-
trations 50%/90% (CC50%/90%) and the selectivity indices 50%/90%
was eluted maximally, whereas in 30% aqueous acetone the
hydrophilic amino acids and the tripeptide GHK were eluted
better. Compound 12 was detected by its yellow color, the amino
acids and the tripeptide by classical ninhydrin staining (which did
(
SI50%/90%) of 4e8 and 12 are summarized (Table 1). Two sets of data
Table 1
Anti-HCV activity of the thiosemicarbazone derivatives 4e8 and 12.
Compound
HCV doseeresponse assay (concentrations in
mM)
HCV RNA-based confirmatory assay (concentrations in mM)
EC50%
1.71
0.28
0.08
EC90%
8.61
5.93
0.50
CC50%
>20
>20
8.85
M
>20
CC90%
>20
>20
>20
SI50%
SI90%
>2.3
>3.4
>40
EC50%
1.12
0.25
0.08
Almost inactive
Almost inactive
EC90%
>20
>20
CC50%
>20
12.21
1.44
CC90%
>20
>20
5.08
SI50%
>17.9
48.8
SI90%
a
4
5
6
7
8
>11.7
>71.4
110.6
e
a
e
0.61
18.0
8.3
a
31.4% inhibition at 20
6.32
0.85
m
a
a
>20
4.0
>20
>20
>3.2
15.2
e
1
2
12.93
>5.0
0.36
1.97
11.17
>20
31.0
>10.2
Concentrations in IU/mL
Concentrations in IU/mL
Positive control
rIFN- 2b
EC50%
0.14
EC90%
0.55
CC50%
>2
CC90%
>2
SI50%
>14.3
SI90%
>3.6
EC50%
0.15
EC90%
1.21
CC50%
>2
CC90%
>2
SI50%
>13.3
SI90%
>1.7
a
HCV ¼ human hepatitis C virus; EC50% ¼ effective inhibitory concentration 50%; EC90% ¼ effective inhibitory concentration 90%; CC50% ¼ host cellular toxic concentration 50%;
2b;
CC90% ¼ host cellular toxic concentration 90%; SI50% ¼ selectivity index CC50%/EC50%; SI90% ¼ selectivity index CC90%/EC90%; rIFN-
a
2b ¼ recombinant human interferon-
a
IU ¼ international units (1 IU corresponds to 3.85 pg rIFN-
a 2b; 1 IU/mL to 200 fM rIFN-a 2b protein concentration).
a
Not determined due to the lack of activity.

A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
1659
Table 2
Selected anti-VZV, anti-HCMV and anti-orthopoxviral activities of the thiosemicarbazone derivatives 4e6, 8 and 12.
Compound
VZV CPE reduction assay (
m
M)
HCMV CPE reduction assay (
m
M)
Cowpox virus CPE reduction assay (
virus CPE reduction assay)
mM) (in parentheses: vaccinia
EC50%
EC90%
CC50%
SI50%
EC50%
EC90%
CC50%
SI50%
EC50%
EC90%
CC50%
SI50%
>40.0 (>20.8)
>2.6 (>2.7)
>5.5 (>1.2)
>8.3 (>4.1)
<4.9 (<4.9)
4
5
6
8
nd
>300
nd
nd
>300
nd
nd
>300
nd
e
nd
nd
nd
>300
290
e
7.5 (14.4)
115 (110)
54.3 (250.0)
36.0 (73.6)
>2.4 (>2.4)
20.6 (34.5)
294 (239)
>300 (>300)
>300 (84.9)
>2.4 (>2.4)
>300 (>300)
>300 (>300)
>300 (>300)
>300 (>300)
11.8 (11.8)
a
a
e
>300
>60
>300
>300
>60
>300
e
e
<4.8
d
a
16.6
26.5
17.0
>300
>18.1
>300
b
b
b
b
b
b
13.6b
5.7b
12
4.7
33.3
7.1
2.4
>4.0
Positive controls (mM)
ACV
GCV
CDV
1.1
nd
nd
nd
nd
nd
>100
nd
nd
>90.9
e
nd
0.04
nd
nd
nd
nd
nd
>100
nd
e
nd (inactive)
nd (nearly inactive)
6.7 (9.8)
nd (inactive)
nd (nearly inactive)
18.4 (20.0)
nd
nd
e
>2500
e
e
e
>317 (>317)
>47.3 (>32.3)
VZV ¼ human varicella-zoster virus; HCMV ¼ human cytomegalovirus; CPE ¼ cytopathic effect; EC50% ¼ effective inhibitory concentration 50%; EC90% ¼ effective inhibitory
concentration 90%; CC50% ¼ host cellular toxic concentration 50%; SI50% ¼ selectivity index CC50%/EC50%; ACV ¼ acyclovir; GCV ¼ ganciclovir; CDV ¼ cidofovir; nd ¼ not done.
a
Not determined due to the lack of activity.
Plaque reduction assay employed.
b
not influence the color of 12). It was observed that K and GHK, but
not R, suffered a chromatographic shift retention when in contact
with 12, each relative to the single controls K, GHK, and R. Chro-
matographic shift retention means that the corresponding amino
acid K and the tripeptide GHK did not leave the start point of the
thin-layer chromatography sheet when mixed and incubated
For Orthopoxvirus inhibition byaromatic thiosemicarbazones the
basic structureeactivity relationship (SAR) is known from earlier
studies, and was comprehensively reviewed [39]. Whereas the thi-
osemicarbazone 4 showed a moderate inhibition of Orthopoxvirus
multiplication comparable to the included reference anti-DNA-viral
agent cidofovir, the retinoid thiosemicarbazone derivative 12 was
found to be a micromolar, considerably active inhibitor of VZV and
HCMV replication. In the VZV and HCMV assays concerning 12 the
veryaccurate plaque reduction method was applied, since in the CPE
reduction assays the antiviral activity of 12 could not be defined
exactly. The mechanism of action of 12 on VZV (and, independently,
HCMV) replication is assumed to be clearly different from its bio-
logical effect(s) exerted onto HCV replication cycle, since there is
a large quantitative difference of the EC50% inhibitory concentrations
required in these test systems to inhibit viral replication.
(
5 min) with 12 prior to application, which strongly pointed to
selective non-eluting, precipitating salt formation of 12 with K and
GHK, but not R. Taken together the thin-layer-chromatographic
mobility shift experiment (see Supplementary data, Fig. S3)
revealed 12 to bind selectively to the terminal lysine
residue in -lysine (K) and the tripeptide GHK (glycyl- -histidinyl-
-lysine), but not to the -arginine (R) -guanidinium moiety or a-
3-amino
L
L
L
L
d
amino group, suggesting that 12 is capable of binding to basic
GHK- and K-containing sequence, respectively, stretches in viral
and/or host cellular proteins. It should be noted that the tripeptide
sequence GHK is not a typical HCV protein sequence, but was
selected for chemical reasons.
2.5. Antiretroviral activity of retinoid thiosemicarbazone derivative
12
2.4. Antiviral activities of the synthesized test compounds against
The vitamin A-related retinoid thiosemicarbazone derivative 12
selected herpes- and orthopoxviruses
exhibited considerable to moderate activity against HIV-1 (IC50%
1.4e5.5 mM), strain lymphadenopathy-associated virus type 1 (LAV-
Since thiosemicarbazones were reported to show antiviral
activity on Poxviridae including variola major (smallpox) virus [36]
and Arenaviridae [37], selected thiosemicarbazones were also
investigated against other viral targets than HCV. The selected
inhibition data of the thiosemicarbazone derivatives 5, 6, 8 and 12
on VZV and/or HCMV replication, and of 4e6, 8 and 12 on cowpox
virus and vaccinia virus replication are summarized (Table 2).
Parameter for antiviral effect was reduction of the virus-induced
cytopathic effect (CPE) by added test compounds. CPE and cyto-
toxicity were estimated colorimetrically by quantification of
neutral red uptake [38]. Compound 7 was omitted in these assays
because it showed no antiviral activity on HCV and was poorly
soluble in dimethyl sulfoxide (DMSO) which resulted in technical
difficulties.
1 ¼ Lai/HTLV-IIIB) [40], as determined in freshly donated PBMCs
(Table 3). The antiretroviral activity of 12 slightly varied between
different batches of donor PBMCs, pointing to a dependency on
primary donor PBMCs idiosyncrasy (individual biochemical status).
The in vitro activity of 12 in PBMCs can be regarded as close to in vivo
situation as possible. Antiretroviral testing in immortalized cell lines
such as CEM, MT-2, MT-4, HUT-78 or H-9 often does not reflect the
true disease situation found in HIV-1-infected individuals. Further-
more, in immortalized, transformed, cancerous cell lines the selec-
tive antineoplastic activity of 12 mimicks high cytotoxicity which is
not significantly developed in primary cell lines like PBMC. It seems
generally necessary to determine antiviral activities of 12 in primary
cell lines like (freshly explanted and cultivated) human foreskin
fibroblast (HFF) cells or PBMCs to quantify its true pharmacological
Table 3
Antiretroviral activity of the retinoid (vitamin A-derived) thiosemicarbazone derivative 12.
Compound
Anti-HIV-1 activity (
m
M) experiment 1
Anti-HIV-1 activity (mM) experiment 2
EC50%
1.4
EC90%
6.5
CC50%
28.5
SI50%
EC50%
5.5
EC90%
14.0
CC50%
28.5
SI50%
12
20.4
5.2
Positive controla
AZT
(mM)
0.0044
0.031
>100
>22,727
0.0044
0.031
>100
>22,727
HIV-1 ¼ human immunodeficiency virus type 1; EC50% ¼ effective inhibitory concentration 50%; EC90% ¼ effective inhibitory concentration 90%; CC50% ¼ host cellular toxic
0
0
concentration 50%; SI50% ¼ selectivity index CC50%/EC50%; AZT ¼ zidovudine (3 -azido-3 -deoxythymidine).
a
The given values for the positive control AZT were averaged from four independent determinations.

1660
A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
impact. Importantly, compound 12 was able to reach 90% inhibition
of HIV-1 replication, at concentrations between 6.5 M and 14.0 M,
suggesting that an effective in vivo end-point inhibition of HIV-1
multiplication could be possible by utilization of 12.
Organics (Geel, Belgium), 4-(dimethylamino)benzaldehyde pro
analysi, thiosemicarbazide pro analysi and 1-bromotetradecane
from Merck-Schuchardt OHG (Hohenbrunn, Germany), vitamin
A-acetate (purity 95%) oily concentrate (approx. 0.327 g all-trans
retinyl acetate/g oil) from AppliChem GmbH (Darmstadt,
m
m
3
. Conclusions and implications
2
Germany). The (deprotected) tripeptide GHK (H N-glycyl-L-histi-
dinyl- -lysine-COOH) was custom-synthesized by standard peptide
L
The anti-HCV thiosemicarbazone derivatives reported here are
synthesis protocols and purchased from Sigma Genosys Ltd.
(Haverhill, United Kingdom). The GHK was supplied as tris(tri-
structurally different from the thiosemicarbazones disclosed in the
U.S. patent [19], representing mainly (alkyl-, alkoxy-, halogen-)
substituted benzaldehyde thiosemicarbazones [19]. However, Fin-
kielsztein et al. published in 2008 the effect of thiosemicarbazone
derivatives on bovine viral diarrhea virus (BVDV) multiplication in
Madin-Darby bovine kidney (MDBK) cells [41]. BVDV (Flaviviridae,
Pestivirus) is a veterinary virus generally regarded as surrogate
model for HCV. The thiosemicarbazone derivatives investigated by
Finkielsztein et al., especially indan-1-one-derived thiosemicar-
bazones, were inhibitory on BVDV replication to various degrees
fluoroacetate) salt (C14
H
24
N
6
O
4
ꢂ 3F CeCOOH, M ¼ 682.45 g/mol),
3
lyophilized directly from the final preparative chromatography
eluent. Thin-layer chromatography (TLC) was performed on Merck
TLC silica gel 60-coated 20 cm ꢂ 20 cm aluminium sheets F254 (with
fluorescence indicator 254 nm) (Merck KGaA, Darmstadt,
Germany) in a Desaga standard TLC chamber (Desaga GmbH,
Wiesloch, Germany).
4.2. Analytical methods
[
41]. Nevertheless, these indan-1-one-derived thiosemicarbazones
were antivirally active in the rather high concentration range (EC50%
.7e250 M), indicating that this specific type of thiosemicarbazone
The FT-IR spectra were recorded on JASCO FT/IR-4100 type A and
FT/IR-6100 type A spectrophotometers (JASCO Inc., Easton, MD,
USA).
1
m
does not offer special advantages regarding inhibition of Pestivirus
and/or, very probably, Hepacivirus replication.
The 1H NMR (400.132 MHz or 700.430 MHz) and 13C NMR
(100.623 MHz or 176.120 MHz) spectroscopy experiments were
recorded at a temperature of 300.0 K using BRUKER Avance 400 or
700 NMR spectrometers (Bruker Biospin, Rheinstetten, Germany),
respectively. The spectra were referenced to the center of the NMR
The HCV replicon replication inhibition mediated by the thio-
semicarbazone derivatives 4e6 and 12 could be of considerable
interest in direction of further studies aiming at simplification of
STAT-C drugs for treating hepatitis C. Compounds 6 and 12 were
clearly superior to the positive control recombinant human inter-
1
13
solvent signal ( H NMR: CDCl
NMR: CDCl 77.00 ppm, DMSO-d
shifts are specified as singlet (s), broad singlet (br s), doublet (d),
3
7.26 ppm, DMSO-d
6
2.50 ppm;
C
feron-
a
2b with regard to their selectivity indices 50%.
3
6
39.52 ppm). Given chemical
Furthermore, antineoplastic and apoptosis-inducing effects of
d
1
2 in the National Cancer Institute (NCI)-60 cancer cell line panel
triplet (t), quartet (q), doublet of doublet (dd), and multiplet (m).
The mass spectrometric experiments were performed on an
electrospray mass spectrometer (ESI-MS) QSTAR (PerkinElmer
Sciex Instruments, PerkinElmer, Waltham, MA, USA), equipped
with hybrid quadrupole-time-of-flight (HQ-ToF) MS/MS mass
analysator, at TopLab GmbH, Martinsried, Germany. The ion spray
source was Ion Sprayer (Sciex, Toronto, Canada) with offline elec-
trospray ionization (non-heated nebulizer gas). The applied
instrument method was offline (direct injection without preceding
liquid chromatography) ESI-ToF-ToF MS. Sample preparation for
ESI-ToF-ToF MS was conducted in pure acetonitrile. For the offline
injection ES380 Medium NanoES spray capillaries (Proxeon Bio-
systems, Odense, Denmark) were applied. In the applied negative
ion mode the ion spray voltage was ꢁ800 V, the declustering
potential ꢁ39 V, the focusing potential ꢁ170 V, and the second
declustering potential was ꢁ10 V. The investigated mass range was
m/z 100e1000.
[42] were recently confirmed (data not shown). Therefore, it is
possible that 12 exerts retinoid-chemopreventive effects on liver
cells, and tumor promotion-inhibiting and/or selective antineo-
plastic effects on HCC cells, which could be of considerable interest
in general evaluation of therapy options for human hepatitis C. It
should be emphasized that the HCV molecular target(s) of 4e6 and
12 remain(s) to be elucidated. Nevertheless, it is tempting to
speculate that these compounds target either the HCV proteins
and/or the host cellular machinery used by HCV for its replication
strategy, or directly the hepaciviral (þ)-ssRNA genome of the
enveloped Hepacivirus HCV.
My studies show that thiosemicarbazone derivatives, especially
when trifluoromethoxy-substituted or vitamin A-derived (reti-
noid), warrant further study as cost-effective, easily synthesized
and in vitro effective STAT-C drugs for experimental treatment of
hepatitis C disease conditions. Whether they could prove to be
analogously efficacious in vivo remains to be demonstrated in
future experiments.
The antiviral activities of compound 12 (versus HCV, VZV, HCMV
and HIV-1) could be interpreted for 12 as being a broad-spectrum
antiviral agent. Other RNA and DNA viruses are currently under
investigation as targets for the antiviral action(s) of the vitamin
A-related retinoid thiosemicarbazone derivative 12. Its promising
in vitro efficacy could be of considerable interest in future viro-
logical examinations, both in vitro and in vivo.
Standard elemental analyses (C, H, N, S) were performed on
LECO Instruments CHNS 932 (LECO, Mönchengladbach, Germany).
Further elemental analyses (C, H, N, S, O) were conducted on the
EURO EA CHNS/O elemental analyzer (HEKAtech GmbH, Wegberg,
Germany).
4.3. Chemical synthesis
4.3.1. (E)-4-(Dimethylamino)benzaldehyde thiosemicarbazone (4)
A modified literature procedure [26,27] leading to a high-purity
product with improved yield was used: 4-(dimethylamino)benz-
aldehyde (10.00 g, 67.03 mmol) and thiosemicarbazide (6.11 g,
67.05 mmol) were suspended in 68% (v/v) aqueous ethanol
4
. Experimental
4.1. Materials
(
410 mL). The mixture was refluxed for 2.5 h. The deep yellow
ꢃ
Chemicals were of analytical or best available quality
solution was cooled to room temperature for 5 h, cooled at 5 C for
0
ꢃ
throughout. 2-Bromo-4 -fluoroacetophenone, 4-bromo-2-fluorob-
enzaldehyde and 4-(trifluoromethoxy)benzaldehyde 99% were
purchased from SigmaeAldrich (St. Louis, MO, USA) or Acros
2 h, and then frozen at ꢁ25 C for 12 h. The evolving light yellow
crystals of 4 (13.86 g, 93%) were filtered and dried. The given
ꢁ
1
analytical data were not previously published. FT-IR (cm ): 3258,

A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
1661
3
0
3
159, 2891, 1578, 1362, 1228, 1064, 947, 817. 1H NMR (DMSO-d
6
)
(CH
3
)
2
), 3.00 (2H, t; J ¼ 7.6 Hz; 1 -CH
2 2
), 5.29 (2H, br s; b-NH
3
3
3
d
2.95 (6H, s; 4-N(CH
3
)
2
), 6.69 (2H, d; J ¼ 8.8 Hz; H-3, H-5), 7.58
amino), 6.61 (2H, d; J ¼ 8.8 Hz; H-3, H-5), 7.54 (2H, d; J ¼ 8.8 Hz;
(
2H, d; 3J ¼ 8.8 Hz; H-2, H-6), 7.75 (1H, br s; H
2
NeC]S, H
), 11.18 (1H, br s; NeH
), 111.66 (C-3, C-5),
-CH]N), 151.35 (C-4),
S: C, 54.03; H, 6.35; N, 25.20;
S, 14.42. Found: C, 54.09; H, 6.37; N, 24.91; S, 14.43.
A
), 7.94
H-2, H-6), 8.21 (1H, s;
22.70 (13 -CH
29.51 (9 -CH
a
-CH]N); C NMR (CDCl
13
3
)
d
*
14.13 (14 -CH
0
3
),
0
0
0
**
*
0
0
*
(
1H, s;
a
-CH]N), 7.99 (1H, br s; H
2
NeC]S, H
B
2
), 28.82 (11 -CH
) , 29.55 (8 -CH
) , 29.70 (3 -, 4 -CH
2
) , 29.16 (10 -CH
) , 29.62 (7 -CH
) , 30.16 (12 -CH
2
) , 29.37 (2 -CH
) , 29.67 (5 -, 6 -
2
) ,
13
0
*
*
0
*
0
0
imino); C NMR (DMSO-d
21.38 (C-1), 128.58 (C-2, C-6), 143.33 (
77.01 (C]S). Anal. Calcd for C10
6
)
d
39.75 (4-N(CH
3
)
2
2
2
2
*
*
0
0
0
0
1
1
a
CH
(4-N(CH
(C-4),154.93 (
2
2
2
), 31.93 (1 -CH
2
), 40.23
H
14
N
4
3 2
) ), 111.77 (C-3, C-5), 123.08 (C-1), 129.14 (C-2, C-6), 151.66
*
**
a
2
-CH]N),159.55 (ReN]C(eSC14H29-n)eNH ); ‘ ’, ‘ ’
These assignments are tentative and interchangeable (they could
not be assigned unequivocally to individual aliphatic carbons in the
4.3.2. (E)-4-Bromo-2-fluorobenzaldehyde thiosemicarbazone (5)
4
-Bromo-2-fluorobenzaldehyde (10.00 g, 49.26 mmol) and thi-
42 4
n-tetradecyl chain). Anal. Calcd for C24H N S: C, 68.85; H, 10.11; N,
osemicarbazide (4.49 g, 49.27 mmol) were suspended in 75% (v/v)
aqueous ethanol (400 mL) and glacial acetic acid (10 mL) was
added. The mixture was refluxed for 3 h. The very light yellow
solution was cooled at 5 C for 1 h, and then frozen at ꢁ25 C for
13.38; S, 7.66. Found: C, 68.92; H, 10.25; N, 13.15; S, 7.39.
4.3.5. 4-[(E)-{(2E)-[4-(4-Fluorophenyl)-1,3-thiazol-2(3H)-ylidene]
hydrazinylidene}methyl]-N,N-dimethylaniline hemihydrate (8)
ꢃ
ꢃ
0
5
.5 h. The evolving yellowish-white crystals of 5 (11.83 g, 87%) were
Compound
4
(3.25 g, 14.62 mmol) and 2-bromo-4 -fluo-
ꢁ
1
filtered and dried. FT-IR (cm ): 3437, 3264, 3150, 1591, 1538, 1465,
roacetophenone (3.18 g, 14.65 mmol) were dissolved in acetone
(150 mL). A yellowish-white precipitate evolved soon after disso-
lution. Then sodium hydroxide (0.59 g, 14.75 mmol) dissolved in
water (5 mL) was added. Immediately, the color changed to deep
brown. After addition of 60% (v/v) aqueous ethanol (150 mL) the
stirred suspension was refluxed for 20 min. The black suspension
1
3
6 or-
1404, 1372, 848, 810, 691. H NMR (DMSO-d ) d 7.45 (1H, d; J
3
19
1
4
tho ¼ 8.3 Hz; H-5), 7.61 (2H, dd; J( F, H) ¼ 10.2 Hz; Jmeta ¼ 1.8 Hz;
H-3), 8.14 (1H, br s; H NeC]S, H ), 8.21 (1H, s; -CH]N), 8.21 (1H,
t; Jortho ¼ 8.3 Hz; H-6), 8.31 (1H, br s; H NeC]S, H ), 11.59 (1H, br
119.16 (d; J( F, C) ¼
2
A
a
3
2
B
13
2 19 13
6
s; NeH imino); C NMR (DMSO-d ) d
2
19 13
3
ꢃ
ꢃ
2
4.9 Hz; C-3), 121.38 (d; J( F, C) ¼ 9.5 Hz; C-1), 123.14 (d;
J
(pH 5e6) was cooled at 5 C for 1 h, afterwards frozen at ꢁ25 C for
1
9
13
4 19 13
(
(
F, C) ¼ 10.3 Hz; C-4), 127.91 (d; J( F, C) ¼ 3.7 Hz; C-5), 128.21
3 19 13 3 19 13
12 h. The evolving brown, amorphous product 8 (3.93 g, 77%) was
ꢁ1
d; J( F, C) ¼ 2.9 Hz; C-6), 133.65 (d; J( F, C) ¼ 4.4 Hz;
a
-CH]
filtered and dried. FT-IR (cm ): 3287, 2901, 1600, 1560, 1521, 1486,
1
19 13
1
N), 160.38 (d; J( F, C) ¼ ꢁ254.7 Hz; C-2), 178.23 (s; C]S). Anal.
Calcd for C SBrF: C, 34.80; H, 2.56; N, 15.22; S, 11.61. Found: C,
5.02; H, 2.55; N, 15.24; S, 11.75.
1351, 1047, 840, 816, 736, 701. H NMR (DMSO-d
6
)
d
2.95 (6H, s; 4-N
3
0
8
H
7
N
3
(CH
3
)
2
), 6.74 (2H, d;
J
ortho ¼ 8.8 Hz; H-3, H-5), 7.23 (3H, m; H-5 ,
0
0
00
3
3
H-3 , H-5 ), 7.48 (2H, d;
J
ortho ¼ 8.8 Hz; H-2, H-6), 7.89 (2H, d;
3
00
00
J
ortho ¼ 8.6 Hz; H-2 , H-6 ), 7.93 (1H, s;
a
-CH]N), 11.81 (1H, br s;
13
4.3.3. (E)-4-(Trifluoromethoxy)benzaldehyde thiosemicarbazone
NeH imino); C NMR (DMSO-d
6
)
d
2
39.76 (s; 4-N(CH ), 102.64 (s;
3 2
)
0
19 13
00
00
(6)
C-5 ), 111.90 (s; C-3, C-5),115.34 (d; J( F, C) ¼ 21.2 Hz; C-3 , C-5 ),
3
19 13
00
00
4
-(Trifluoromethoxy)benzaldehyde (5.00 g, 26.30 mmol) and
121.88 (s; C-1), 127.41 (d; J( F, C) ¼ 8.1 Hz; C-2 , C-6 ), 127.53 (s;
4 19 13
00
thiosemicarbazide (2.40 g, 26.34 mmol) were suspended in 75% (v/
v) aqueous ethanol (200 mL) and glacial acetic acid (5 mL) was
added. The mixture was refluxed for 2 h. The colorless solution was
C-2, C-6), 131.45 (d; J( F, C) ¼ 2.9 Hz; C-1 ), 142.40 (s;
a
-CH]N),
0
1 19 13
149.40 (s; C-4 ), 151.00 (s; C-4), 161.51 (d; J( F, C) ¼ ꢁ244.4 Hz; C-
0
0
0
17 4 2
4 ), 168.52 (s; C-2 ). Anal. Calcd for C18H N SF$0.5H O: C, 61.87; H,
ꢃ
ꢃ
pre-cooled at 5 C for 2 h, and frozen at ꢁ25 C for 1 h. Then water
5.19; N, 16.03; S, 9.18. Found: C, 61.96; H, 4.97; N, 15.87; S, 9.19.
(
50 mL) was added, and the crystallizing white mixture was frozen
ꢃ
at ꢁ25 C for 2 h. Afterwards, water (100 mL) was added, and the
4.3.6. (2E)-2-(All-trans retinylidene)-N-{[(2E)-2-(all-trans
retinylidene)hydrazinyl]carbonothioyl}hydrazinecarbothioamide
trisodium salt hemihendecahydrate (12)
ꢃ
crystallizing mixture was frozen at ꢁ25 C for 19 h. The evolved
white, glistening, feathery crystals of 6 (6.78 g, 98%) were filtered
ꢁ1
and dried. FT-IR (cm ): 3407, 3247, 3157, 3027, 1599, 1536, 1505,
Vitamin A-acetate 9 oily concentrate (approx. 0.327 g all-trans
retinyl acetate/g oil) (40.00 g, 39.82 mmol) was melted into
a round-bottomed flask, and was suspended in 90% (v/v) aqu-
eous ethanol (150 mL) containing sodium hydroxide (5.00 g,
125.00 mmol). The yellow mixture was refluxed for 25 min to
achieve saponification to all-trans retinol 10. The clear yellow
1
3
1
215, 1142, 822. H NMR (DMSO-d
6
)
d
7.38 (2H, d; Jortho ¼ 8.3 Hz;
3
H-3, H-5), 7.95 (2H, dd;
CH]N), 8.08 (1H, br s; H
), 11.51 (1H, br s; NeH imino); C NMR (DMSO-d
J
ortho ¼ 8.3 Hz; H-2, H-6), 8.07 (1H, s;
a-
2
NeC]S, H
A
), 8.25 (1H, br s; H
119.97 (q; J
3
), 121.06 (s; C-3, C-5), 129.09 (s; C-2,
2
NeC]S,
13
1
H
(
B
6
)
d
1
9
13
F, C) ¼ ꢁ256.6 Hz; ReOeCF
3
19 13
ꢃ
C-6), 133.53 (s; C-1), 140.48 (s;
a
-CH]N), 149.03 (d; J( F, C) ¼
OSF : C, 41.06;
H, 3.06; N,15.96; S,12.18. Found: C, 40.98; H, 3.04; N,15.87; S,12.20.
solution was cooled at 5 C for 30 min, and was mixed with glacial
2
.2 Hz; C-4), 178.15 (s; C]S). Anal. Calcd for C
9
H
8
N
3
3
acetic acid (7.2 mL, 125.90 mmol) resulting in pH 5e7. This solution
was diluted with water (200 mL), and was extracted with ethyl
acetate (EtOAc, 200 mL). The isolated EtOAc phase was washed
with water (200 mL). Black, precipitated (active) manganese
4.3.4. (E)-S-Tetradecyl 4-(dimethylamino)benzaldehyde
isothiosemicarbazone (7)
2
dioxide powder (63.00 g, MnO content z 90%) was suspended in
Compound 4 (6.50 g, 29.24 mmol) and 1-bromotetradecane
8.11 g, 29.25 mmol) were suspended in 90% (v/v) aqueous ethanol
the isolated EtOAc phase, which contained mainly 10, and the
mixture was stirred at a temperature of 20 C under light and air
ꢃ
(
(
200 mL). Then sodium hydroxide (1.20 g, 30.00 mmol), dissolved
exclusion for 5 h. The suspension was occasionally shaken. After-
wards the black suspension was filtered through three layers of
filter paper, and the black filter residue was rinsed with EtOAc
in water (5 mL), was added and the mixture was refluxed for 3 h.
The yellow solution was cooled at 5 C for 1.5 h, then frozen at
ꢃ
ꢃ
ꢁ
25 C for 9 h. The crude product was filtered and dried. This crude
(150 mL). To remove the finely suspended MnO
filtrate, transferred into a separation funnel, was mixed with
a freshly prepared solution of -ascorbic acid (10.00 g, 56.78 mmol)
2
residues, the
product (10.51 g) was recrystallized from acetone (190 mL) under
ꢃ
hot filtration. The filtrate was cooled at 5 C for 4 h, then frozen at
L
ꢃ
ꢁ
25 C for 10 h. The light yellow, feathery crystals of 7 (8.69 g, 71%)
and sodium hydroxide (4.54 g, 113.50 mmol) in water (200 mL).
The mixture was shaken vigorously for 1 min. A heavy precipitate
of manganese(II) compounds evolved in the aqueous phase,
which was dissolved by addition of glacial acetic acid (6.5 mL,
113.66 mmol) and repeated shaking. Finally, the separated EtOAc
ꢁ1
were filtered and dried. FT-IR (cm ): 3437, 3316, 2914, 2848, 1616,
1
1
598, 1578, 1553, 1528, 1471, 1295, 1182, 816, 806, 716. H NMR
3
0
0
(
CDCl
3
)
d
0.81 (3H, t; J ¼ 7.1 Hz; 14 -CH
3
), 1.18 (20H, br s; 4 to
0
0
0
1
3 -CH
2
), 1.33 (2H, m; 3 -CH
2
), 1.62 (2H, m; 2 -CH
2
), 2.93 (6H, s; 4-N

1662
A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
phase was washed twice with water (2 ꢂ 200 mL). The EtOAc
phase, containing mainly all-trans retinal 11, was mixed with
a solution of thiosemicarbazide (5.73 g, 62.88 mmol) in 90% (v/v)
aqueous ethanol (220 mL, prepared by short refluxing). The solu-
tion was refluxed for 20 min. The clear, deep yellow solution was
RT, and dilution with acetone (1 mL); Lane 6: 47 mM L-arginine
monohydrochloride in water; Lane 7: solution of lane 1 (0.4 mL) and
solution of lane 6 (0.1 mL) mixed, 5 min incubation at RT, and dilu-
tion with acetone (1 mL). The TLC plates were investigated by UV-C
light 254 nm, no fluorescence deletion could be detected other than
seen in visible light. The yellow spots of compound 12 could be seen
in daylight. The amino acid and tripeptide spots were visualized by
spraying the TLC plates with a 14 mM solution of ninhydrin [2,2-
dihydroxy-1H-indene-1,3(2H)-dione] in acetone (30 mL) containing
ꢃ
then frozen at ꢁ25 C for 4 h. After adding 100 ml of water, the
volume of the cloudy yellow solution was reduced in vacuo until
heavy crystallization started. The crystallizing suspension was
ꢃ
additionally frozen at ꢁ25 C for 3 h. The evolved orange-yellow
ꢃ
crystals were filtered and dried. The orange-yellow filtrate was
glacial acetic acid (1 mL), and heating of the sprayed plates (80 C,
evaporated to a volume of ca. 150 mL, until it crystallized again. For
1 min). The TLC plates with the yellow spots of 12, the reddish-violet
spots of the amino acids and the brownish-yellow spots of the tri-
peptide GHK were dried, electronically scanned in situ, and
computer-processed (Adobe PhotoDeluxe Business Edition 1.0,
Adobe Systems Inc., San Jose, CA, USA) to give the pictures of the
originally visible appearances of the chromatograms.
ꢃ
completion of crystallization, it was frozen at ꢁ25 C for 1 h. The
evolved orange, fine crystalline precipitate of all-trans retinal thi-
osemicarbazone was filtered and dried. All crude products were
combined (12.00 g) and dissolved in acetone (120 mL) by gentle
ꢃ
warming. After 1 h freezing at ꢁ25 C water (60 mL) was added,
ꢃ
and the crystallizing suspension was frozen at ꢁ25 C for 4 h. The
evolved orange-yellow crystals were filtered and dried (10.50 g).
This material was dissolved in acetone (130 mL) by gentle warming.
A solution of sodium hydroxide (1.76 g, 44.00 mmol) in water
4.5. Biological activity
The biologically employed test compounds were initially dis-
solved in dimethyl sulfoxide (DMSO) at the stock concentration
10 mg/mL, and then appropriately diluted with cell culture medium
to achieve a final DMSO content (v/v) of less than 1% in the cell
culture fluid. This DMSO content was non-toxic to cells.
(
7 mL) was added under stirring. Immediately a lemon yellow
precipitate of crude compound 12 formed. The precipitating
ꢃ
suspension was cooled at 5 C for 2 h. The evolved lemon yellow
precipitate of the crude 12 was filtered and dried. The crude
product 12 (11.40 g, 66%) was suspended in 80% (v/v) aqueous
acetone (600 mL). The suspension was refluxed for 15 min. After-
4.5.1. HCV replicon system
ꢃ
wards, the orange solution was cooled at 5 C for 5 h, and then was
The cell line Huh7 ET (luc-ubi-neo/ET), which contains a new
HCV RNA replicon with a stable luciferase (luc) reporter was used
being similar to the cell line 5.1 [43], but contains additional
modifications that make the cell line more robust and provide
stable luc expression for antiviral screening. The underlying human
hepatoma cell line Huh7 was established from a human HBsAg-
negative primary hepatocellular carcinoma in 1982 [44]. The HCV
ꢃ
frozen at ꢁ25 C for 6 h. The evolved yellow-orange crystalline
material (fine needles and accompanying shiny plates) (9.58 g) was
filtered and dried. A part of this material (2.00 g) was dissolved in
acetone (70 mL) by gentle warming.
A solution of sodium
hydroxide (0.25 g, 6.25 mmol) in water (1 mL) was added under
stirring. Immediately a lemon yellow precipitate formed. The
ꢃ
0
0
precipitating suspension was frozen at ꢁ25 C for 2 h. The evolved
RNA replicon Huh7 ET contains the 5 -non-translated region (5 -
0
lemon yellow precipitate of 12 (2.13 g, 99% for this procedure) was
NTR) internal ribosome entry site (IRES) of HCV (5 ) which drives
ꢁ
1
filtered and dried over CaCl
2
in vacuo. FT-IR (cm ): 3357, 2922,
the production of a firefly luciferase (luc), ubiquitin (ubi), and
neomycin phosphotransferase (neo) fusion protein. Ubiquitin
cleavage releases the luc and neo gene products. The murine
1
2
d
6
850, 1548, 1449, 1371, 1304, 992, 970, 817. H NMR (DMSO-d )
1.02 (12H, s; H
3
C-16, H
3
C-17), 1.44 (4H, m; H
2
C-2), 1.58 (4H, m;
0
H
2
C-3), 1.69 (6H, s; H
3
C-18), 1.93 (6H, s; H
3
C-19), 1.96 (6H, s; H
3
C-
encephalomyocarditis virus (EMCV, Picornaviridae, Cardiovirus) 5 -
3
2
0), 2.00 (4H, t; J ¼ 6.1 Hz; H
2
C-4), 6.14 (4H, s; H-7, H-8), 6.21 (4H,
IRES element controls the translation of the HCV non-structural
proteins NS3 to NS5. The NS3 protein cleaves the HCV polyprotein
to release the mature NS3, NS4A, NS4B, NS5A and NS5B proteins
3
m; H-10, H-14), 6.42 (2H, d; J ¼ 15.2 Hz; H-12), 6.63 (2H, dd;
3
3
3
13
J ¼ 15.2 Hz; J ¼ 11.4 Hz; H-11), 8.02 (2H, d; J ¼ 10.6 Hz; H-15);
NMR (DMSO-d 12.48 (H C-19), 12.65 (H C-20), 18.73 (H C-3),
1.52 (H C-18), 28.82 (H C-16, H C-17), 32.58 (H C-4), 33.84 (C-1),
9.21 (H C-2), 123.61 (HC-11), 125.52 (HC-7), 128.71 (C-5), 131.17
HC-10), 131.41 (HC-14), 134.61 (C-6), 135.33 (C-9), 137.41 (HC-8),
37.67 (HC-12), 140.96 (HC-15), 160.50 (C-13), 180.90 (C]S thio-
carbonyl). HRMS (ESI) [m/z (%)]: 719.3995 (7.82) [calcd for
C
0
6
)
d
3
3
2
that are required for HCV replication. At the 3 end of the replicon is
0
2
3
(
1
3
3
3
2
the authentic 3 -NTR of HCV. The details of this test system can be
2
obtained upon request from the NIAID’s Antimicrobial Acquisition
and Coordinating Facility (AACF) (http://www.niaid-aacf.org/
protocols/HCV.htm) and were described in literature [43,45].
ꢀ
ꢁ
þ
þ
C
3
C
42
H
58
N
5
S
2
Na (M ꢁ 2Na þ 2H ) 719.4037], 665.3147 (2.27),
61.0480 (100.00), 326.9493 (23.13). Anal. Calcd for
Na $5.5H O: C, 58.45; H, 7.82; N, 8.11; S, 7.43; O, 10.20.
4.5.2. HCV RNA replicon doseeresponse assay
HCV RNA replicon-derived luc activity was used to measure
antiviral activity. Compound CC50% and CC90% values (cytotoxicity)
were calculated using CytoTox-1 (Promega, Madison, WI, USA),
a colorimetric assay used as an indicator of cell numbers and
cytotoxicity when the luc assay system is employed. All experi-
ments were performed in quadruplicate and treated statistically by
AACF. The details of this test system can be obtained upon request
from the NIAID’s Antimicrobial Acquisition and Coordinating
Facility (AACF) (http://www.niaid-aacf.org/protocols/HCV.htm)
and were described in literature [46,47].
42
H
56
N
5
3
S
2
2
Found: C, 58.39; H, 7.66; N, 8.16; S, 5.99; O, 10.83.
4.4. Thin-layer-chromatographic mobility shift assay
Thin-layer chromatography (TLC) mobility shift experiments
were performed on retinoid thiosemicarbazone derivative 12 with
the following methodology: 90% ¼ TLC eluent 90% (v/v) aqueous
acetone; 60% ¼ TLC eluent 60% (v/v) aqueous acetone; 30% ¼ TLC
eluent 30% (v/v) aqueous acetone; Lane 1: 5.8 mM 12 in acetone;
Lane 2: 147 mM GHK (glycyl-
solution of lane 1 (0.4 mL) and solution of lane 2 (0.2 mL) mixed,
min incubation at RT, and dilution with acetone (1 mL); Lane 4:
1 mM -lysine dihydrochloride in water; Lane 5: solution of lane 1
0.4 mL) and solution of lane 4 (0.1 mL) mixed, 5 min incubation at
L
-histidinyl-
L
-lysine) in water; Lane 3:
4.5.3. HCV RNA replicon confirmatory RNA-based assay
The RNA-based assay was used to examine the effects of
compounds at five half-log concentrations each. Human interferon-
5
9
(
L
a
2b was included in each run as a positive control compound.
Compound EC50% and EC90% values (antiviral activity) were derived

A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
1663
from HCV RNA levels assessed as HCV RNA using TaqManÒ real-
time polymerase chain reaction (RT-PCR). Ribosomal RNA (rRNA)
levels determined via TaqMan RT-PCR (Applied Biosystems Inc.,
were conducted in triplicate and treated statistically. The RT
0
0
inhibitor AZT (zidovudine, 3 -azido-3 -deoxythymidine; RETRO-
Ò
Ò
VIR ) served as a positive control. Cytotoxcicity on PBMCs exerted
Foster City, CA, USA) were used as an indication of cell numbers (for
testing drug cytotoxicity) in the RNA-based assay. Compound
selectivity indices SI50% and SI90% values were calculated. All
experiments were performed in quadruplicate and treated statis-
tically by AACF. The details of this test system can be obtained upon
request from the NIAID’s Antimicrobial Acquisition and Coordi-
nating Facility (AACF) (http://www.niaid-aacf.org/protocols/HCV.
htm) and were described in literature [46,47].
by the test compounds was determined as described by Stuyver
et al. [53], by application of the CellTiter 96 AQueous One Solution
Ò
Cell Proliferation Assay (Promega, Madison, WI, USA). Briefly, the
phenazine ethosulfate (PES)-coupled reduction of the tetrazolium
salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium (MTS) to a purple, water-soluble
formazan by living, undamaged cells was measured.
Acknowledgments
4.5.4. VZV and HCMV CPE reduction assays
VZV (strain Ellen) and HCMV (strain AD169) were assayed in
Thanks are due to the National Institute of Allergy and Infectious
Diseases (NIAID) and the Antimicrobial Acquisition and Coordi-
nating Facility (AACF) of the Southern Research Institute (SRI)
primary, freshly explanted, human foreskin fibroblast (HFF) cells.
Cultivation of HFF cells was performed as reported earlier [48]. The
parameter for antiviral activity was reduction of CPE. CPE and
cytotoxicity were estimated colorimetrically by quantification of
neutral red uptake [38]. Cells not damaged by virus-induced CPE or
test compound took up a greater amount of dye (into lysosomes)
which was read on a computerized microplate autoreader. The
standard antiherpetic drugs acyclovir [9-[(2-hydroxyethoxy)
(
Birmingham, AL, USA) for performing the HCV screening. Grate-
fully acknowledged is the NIAID grant contract N01-AI-30047
AACF). HCV data were generated by Dr. Zhuhui Huang and Dr.
(
Michael G. Murray at SRI, Frederick, MD, USA. VZV, HCMV and
Orthopoxvirus data were generated by Dr. Mark N. Prichard at
University of Alabama, Birmingham, AL, USA. Special acknowledg-
ment goes to Dr. Joseph A. Maddry at SRI for the generous
management of AACF screenings. I am deeply indepted to Prof. Dr.
R.F. Schinazi at the Center for AIDS Research, Emory University
School of Medicine (Atlanta, GA, USA) for providing the HIV-1 data.
Thankfully appreciated are the analytical services of Dr. Thomas M.
Halder and Dipl.-Chem. Volker Krey (TopLab GmbH), Dr. Christoph
Freudenberger and Eva-Maria May (Currenta GmbH) and Klaus
Hecker (HEKAtech GmbH). Especially thankfully appreciated is the
generous and patient involvement of Dr. Wolfram Taiber and Dr.
Martin Elhardt (CEM Chemieschule Dr. Erwin Elhardt, München).
Ò
methyl]-9H-guanine; ZOVIRAX ] (for VZV) and ganciclovir [9-[(1,3-
Ò
dihydroxy-2-propoxy)methyl]-9H-guanine; CYTOVENE , CYM-
Ò
EVENE ] (for HCMV) were included as reference agents. All
experiments were conducted in triplicate and treated statistically
by AACF. The details of this test system can be obtained from AACF
http://niaid-aacf.org/protocols/Herpes.htm) and were described in
(
literature [49].
4
.5.5. VZV and HCMV plaque reduction assays
VZV (strain Ellen) and HCMV (strain AD169) were assayed in HFF
cell agarose overlays. In contrast to the CPE reduction assays in
which drug was added 1 h prior to infection, in these classical plaque
reduction assays the drug was added 1 h after infection. The details
of this test system can be obtained from AACF (http://niaid-aacf.org/
protocols/Herpes.htm) and were described in literature [49].
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2011.02.014.
4
.5.6. Orthopoxvirus (cowpox virus, vaccinia virus) CPE reduction
assay
Cowpox virus (strain Brighton Red) and vaccinia virus (strain
References
Copenhagen) were assayed in HFF cells. The parameter for antiviral
activity was reduction of CPE. CPE and cytotoxicity were estimated
colorimetrically by quantification of neutral red uptake. The anti-
DNA-viral agent cidofovir [1-[(2S)-3-hydroxy-2-(phosphonome-
[1] P. Marcellin, Hepatitis B and hepatitis C in 2009, Liver Int. 29 (s1) (2009) 1e8.
[2] T.J. Liang, T. Heller, Pathogenesis of hepatitis C-associated hepatocellular
carcinoma, Gastroenterology 127 (Suppl. 1) (2004) S62eS71.
[
3] R.P. Beasley, L.-Y. Hwang, C.-C. Lin, C.-S. Chien, Hepatocellular carcinoma and
hepatitis B virus e a prospective study of 22 707 men in Taiwan, Lancet 318
(II) (1981) 1129e1133.
Ò
thoxy)-1-propyl]-1H-cytosine; VISTIDE ] was included as positive
[
4] M.-Y. Lai, J.-H. Kao, P.-M. Yang, J.-T. Wang, P.-J. Chen, K.-W. Chan, J.-S. Chu,
D.-S. Chen, Long-term efficacy of ribavirin plus interferon alfa in the treatment
of chronic hepatitis C, Gastroenterology 111 (1996) 1307e1312.
control. All experiments were conducted in triplicate and treated
statistically by AACF. The details of this test system can be obtained
upon request from the NIAID’s Antimicrobial Acquisition and
Coordinating Facility (AACF) (http://niaid-aacf.org/protocols/
orthopox.htm) and were described in literature [50].
[5] J.G. McHutchison, S.C. Gordon, E.R. Schiff, M.L. Shiffman, W.M. Lee, V.K. Rustgi,
Z.D. Goodman, M.-H. Ling, S. Cort, J.K. Albrecht, for The Hepatitis Interven-
tional Therapy Group, Interferon alfa-2b alone or in combination with riba-
virin as initial treatment for chronic hepatitis C, N. Engl. J. Med. 339 (1998)
1485e1492.
[
6] M.W. Fried, M.L. Shiffman, K.R. Reddy, C. Smith, G. Marinos, F.L. Gonçales Jr.,
D. Häussinger, M. Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman,
J. Yu, Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infec-
tion, N. Engl. J. Med. 347 (2002) 975e982.
4
.5.7. HIV-1 replication reverse transcriptase (RT) assay
HIV-1 [strain lymphadenopathy-associated virus type 1 (LAV-
) ¼ Lai/HTLV-IIIB] [40] was assayed in human, primary, peripheral
1
blood mononuclear cells (PBMCs). The parameter for antiviral
activity was reduction of RT activity in the cell supernatant after
Triton X-100-mediated lysis of released virions, as measured by 5a-
tritiated thymidine 5 -triphosphate [[ H]-dTTP] incorporation into
poly(rA)$poly(dT) directed by the primed RNA template poly(rA)$
oligo(dT). It should be noted that the assay did not detect RT
inhibition by potential RT inhibitors per se, but indirectly quantified
the amount of released HIV-1 virions which contain the RT. The
detailed assay methodology was reported by Schinazi et al. [51], as
based on an older assay system of Spira et al. [52]. The experiments
[7] M.P. Manns, J.G. McHutchison, S.C. Gordon, V.K. Rustgi, M. Shiffman,
R. Reindollar, Z.D. Goodman, K. Koury, M.-H. Ling, J.K. Albrecht, The Interna-
tional Hepatitis Interventional Therapy Group, Peginterferon alfa-2b plus
ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment
of chronic hepatitis C: a randomised trial, Lancet 358 (2001) 958e965.
0
3
[
8] D.E. Baker, Pegylated interferon plus ribavirin for the treatment of chronic
hepatitis C, Rev. Gastroenterol. Disord. 3 (2003) 93e109.
9] D.E. Baker, Pegylated interferons, Rev. Gastroenterol. Disord. 1 (2001) 87e99.
[
[10] T. Asselah, Y. Benhamou, P. Marcellin, Protease and polymerase inhibitors for
the treatment of hepatitis C, Liver Int. 29 (Suppl. 1) (2009) 57e67.
[
11] A.D. Kwong, L. McNair, I. Jacobson, S. George, Recent progress in the devel-
opment of selected hepatitis C virus NS3.4A protease and NS5B polymerase
inhibitors, Curr. Opin. Pharmacol. 8 (2008) 522e531.

1664
A.J. Kesel / European Journal of Medicinal Chemistry 46 (2011) 1656e1664
[
12] T. Shimakami, R.E. Lanford, S.M. Lemon, Hepatitis C: recent successes and
[34] A. Ruzin, A. Severin, F. Ritacco, K. Tabei, G. Singh, P.A. Bradford, M.M. Siegel,
S.J. Projan, D.M. Shlaes, Further evidence that a cell wall precursor [C55
continuing challenges in the development of improved treatment modalities,
Curr. Opin. Pharmacol. 9 (2009) 537e544.
-
MurNAc-(Peptide)-GlcNAc] serves as an acceptor in a sorting reaction, J. Bac-
teriol. 184 (2002) 2141e2147.
[
13] C.-S. Chen, C.-T. Chiou, G.S. Chen, S.-C. Chen, C.-Y. Hu, W.-K. Chi, Y.-D. Chu,
L.-H. Hwang, P.-J. Chen, D.-S. Chen, S.-H. Liaw, J.-W. Chern, Structure-based
discovery of triphenylmethane derivatives as inhibitors of hepatitis C virus
helicase, J. Med. Chem. 52 (2009) 2716e2723.
[35] L. Pickart, J.H. Freedman, W.J. Loker, J. Peisach, C.M. Perkins, R.E. Stenkamp,
B. Weinstein, Growth-modulating plasma tripeptide may function by facili-
tating copper uptake into cells, Nature 288 (1980) 715e717.
[36] D.J. Bauer, K.R. Dumbell, P. Fox-Hulme, P.W. Sadler, The chemotherapy of
variola major infection, Bull. World Health Organ. 26 (1962) 727e732.
[37] C.C. García, B.N. Brousse, M.J. Carlucci, A.G. Moglioni, M. Martins Alho,
G.Y. Moltrasio, N.B. D’Accorso, E.B. Damonte, Inhibitory effect of thio-
semicarbazone derivatives on Junin virus replication in vitro, Antivir. Chem.
Chemother. 14 (2003) 99e105.
[38] G. Repetto, A. del Peso, J.L. Zurita, Neutral red uptake assay for the estimation
of cell viability/cytotoxicity, Nat. Protoc. 3 (2008) 1125e1131.
[39] C.J. Pfau, in: P.E. Came, L.A. Caliguiri (Eds.), The Thiosemicarbazones, Hand-
book of Experimental Pharmacology, vol. 61, Springer-Verlag, Berlin, 1982, pp.
147e204.
[
14] G. Manfroni, J. Paeshuyse, S. Massari, S. Zanoli, B. Gatto, G. Maga, O. Tabarrini,
V. Cecchetti, A. Fravolini, J. Neyts, Inhibition of subgenomic hepatitis C virus
RNA replication by acridone derivatives: identification of an NS3 helicase
inhibitor, J. Med. Chem. 52 (2009) 3354e3365.
[
15] S. Kandil, S. Biondaro, D. Vlachakis, A.-C. Cummins, A. Coluccia, C. Berry,
P. Leyssen, J. Neyts, A. Brancale, Discovery of a novel HCV helicase inhibitor by
a de novo drug design approach, Bioorg. Med. Chem. Lett. 19 (2009) 2935e2937.
16] D. Lamarre, P.C. Anderson, M. Bailey, P. Beaulieu, G. Bolger, P. Bonneau, M. Bös,
D.R. Cameron, M. Cartier, M.G. Cordingley, A.-M. Faucher, N. Goudreau,
S.H. Kawai, G. Kukolj, L. Lagacé, S.R. LaPlante, H. Narjes, M.-A. Poupart,
J. Rancourt, R.E. Sentjens, R. St George, B. Simoneau, G. Steinmann, D. Thibeault,
Y.S. Tsantrizos, S.M. Weldon, C.-L. Yong, M. Llinàs-Brunet, An NS3 protease
inhibitor with antiviral effects in humans infected with hepatitis C virus, Nature
[
[40] S.-Y.P. Chang, B.H. Bowman, J.B. Weiss, R.E. Garcia, T.J. White, The origin of
HIV-1 isolate HTLV-IIIB, Nature 363 (1993) 466e469.
4
26 (2003) 186e189 (Corrigendum published: Nature 426 (2003) 314).
[41] L.M. Finkielsztein, E.F. Castro, L.E. Fabián, G.Y. Moltrasio, R.H. Campos,
L.V. Cavallaro, A.G. Moglioni, New 1-indanone thiosemicarbazone derivatives
active against BVDV, Eur. J. Med. Chem. 43 (2008) 1767e1773.
[42] M.R. Grever, S.A. Schepartz, B.A. Chabner, The National Cancer Institute:
cancer drug discovery and development program, Semin. Oncol. 19 (1992)
622e638.
[43] N. Krieger, V. Lohmann, R. Bartenschlager, Enhancement of hepatitis C virus
RNA replication by cell culture-adaptive mutations, J. Virol. 75 (2001)
4614e4624.
[44] H. Nakabayashi, K. Taketa, K. Miyano, T. Yamane, J. Sato, Growth of human
hepatoma cell lines with differentiated functions in chemically defined
medium, Cancer Res. 42 (1982) 3858e3863.
[45] V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager,
Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line,
Science 285 (1999) 110e113.
[46] S. Ma, J.E. Boerner, C. TiongYip, B. Weidmann, N.S. Ryder, M.P. Cooreman,
K. Lin, NIM811, a cyclophilinS200 inhibitor, exhibits potent in vitro activity
against hepatitis C virus alone or in combination with alpha interferon,
Antimicrob. Agents Chemother. 50 (2006) 2976e2982.
[47] H.-J. Kim, A. Sharon, C. Bal, J. Wang, M. Allu, Z. Huang, M.G. Murray, L. Bassit,
R.F. Schinazi, B. Korba, C.K. Chu, Synthesis and anti-hepatitis B virus and anti-
hepatitis C virus activities of 7-deazaneplanocin A analogues in vitro, J. Med.
Chem. 52 (2009) 206e213.
[48] D.C. Quenelle, M.N. Prichard, K.A. Keith, D.E. Hruby, R. Jordan, G.R. Painter,
A. Robertson, E.R. Kern, Synergistic efficacy of the combination of ST-246 with
CMX001 against orthopoxviruses, Antimicrob. Agents Chemother. 51 (2007)
4118e4124.
[49] E.R. Kern, N.L. Kushner, C.B. Hartline, S.L. Williams-Aziz, E.A. Harden, S. Zhou,
J. Zemlicka, M.N. Prichard, In vitro activity and mechanism of action of
methylenecyclopropane analogs of nucleosides against herpesvirus replica-
tion, Antimicrob. Agents Chemother. 49 (2005) 1039e1045.
[
[
[
17] X. Tong, A. Arasappan, F. Bennett, R. Chase, B. Feld, Z. Guo, A. Hart, V. Madison,
B. Malcolm, J. Pichardo, A. Prongay, R. Ralston, A. Skelton, E. Xia, R. Zhang,
F.G. Njoroge, Preclinical characterization of the antiviral activity of SCH
900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus
NS3 protease, Antimicrob. Agents Chemother. 54 (2010) 2365e2370.
18] M. Gao, R.E. Nettles, M. Belema, L.B. Snyder, V.N. Nguyen, R.A. Fridell,
M.H. Serrano-Wu, D.R. Langley, J.-H. Sun, D.R. O’Boyle II, J.A. Lemm, C. Wang,
J.O. Knipe, C. Chien, R.J. Colonno, D.M. Grasela, N.A. Meanwell, L.G. Hamann,
Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent
clinical effect, Nature 465 (2010) 96e100.
19] S. Altamura, U. Koch, Treating Hepatitis
semicarbazone Compounds, U.S. Patent Appl. 2003/0176503 A1, September
8, 2003.
C Viral Infections with Thio-
1
[
[
[
[
[
[
[
20] R.M. Niles, Recent advances in the use of vitamin A (Retinoids) in the
prevention and treatment of cancer, Nutrition 16 (2000) 1084e1090.
21] R.D. Semba, Vitamin A and immunity to viral, bacterial and protozoan infec-
tions, Proc. Nutr. Soc. 58 (1999) 719e727.
22] A.C. Ross, C.B. Stephensen, Vitamin A and retinoids in antiviral responses,
FASEB J. 10 (1996) 979e985.
23] H.N. Green, E. Mellanby, Vitamin A as an anti-infective agent, Br. Med. J. II
(
1928) 691e696.
24] A.J. Eid, R.R. Razonable, New developments in the management of cytomeg-
alovirus infection after solid organ transplantation, Drugs 70 (2010) 965e981.
25] E. De Clercq, Vaccinia virus inhibitors as a paradigm for the chemotherapy of
poxvirus infections, Clin. Microbiol. Rev. 14 (2001) 382e397.
26] D. Chattopadhyay, S.K. Mazumdar, T. Banerjee, T.C.W. Mak, Crystal and
molecular structure of 4-(N,N-dimethylamino)benzaldehyde thiosemicar-
bazone, J. Cryst. Spectrosc. Res. 18 (1988) 701e712.
27] Y. Yu, L.-R. Lin, K.-B. Yang, X. Zhong, R.-B. Huang, L.-S. Zheng, p-Dimethylamino-
benzaldehyde thiosemicarbazone: a simple novel selective and sensitive fluo-
rescent sensor for mercury(II) in aqueous solution, Talanta 69 (2006) 103e106.
28] A.A. Sirotenko, Microdetermination of carbon in compounds containing alkali
metals, Mikrochim. Acta (Wien) 40 (1952) 30e31.
[
[50] E.R. Kern, M.N. Prichard, D.C. Quenelle, K.A. Keith, K.N. Tiwari, J.A. Maddry,
0
[
[
J.A. Secrist III, Activities of certain 5-substituted 4 -thiopyrimidine nucleosides
against orthopoxvirus infections, Antimicrob. Agents Chemother. 53 (2009)
572e579.
29] J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse, Electrospray
ionization for mass spectrometry of large biomolecules, Science 246 (1989)
[51] R.F. Schinazi, J.-P. Sommadossi, V. Saalmann, D.L. Cannon, M.-Y. Xie, G.C. Hart,
0
0
64e71.
G.A. Smith, E.F. Hahn, Activities of 3 -azido-3 -deoxythymidine nucleotide
dimers in primary lymphocytes infected with human immunodeficiency virus
type 1, Antimicrob. Agents Chemother. 34 (1990) 1061e1067.
[
30] C. Pattaroni, J. Lauterwein, A study of N-butyl-(all-trans-retinylidene)amine
1
13
and its protonated Species by H- and C-NMR spectroscopy, Helv. Chim.
Acta 64 (1981) 1969e1984.
[52] T.J. Spira, L.H. Bozeman, R.C. Holman, D.T. Warfield, S.K. Phillips, P.M. Feorino,
Micromethod for assaying reverse transcriptase of human T-cell lymphotropic
virus type III/lymphadenopathy-associated virus, J. Clin. Microbiol. 25 (1987)
97e99.
[
[
[
31] G. Englert, A 13C-NMR study of cis-trans isomeric vitamins A, carotenoids and
related compounds, Helv. Chim. Acta 58 (1975) 2367e2390.
32] Product Information IntronÒ A: Interferon Alfa-2b, Recombinant, for Injection.
Schering Corporation, Kenilworth, NJ, USA, 2006.
[53] L.J. Stuyver, S. Lostia, M. Adams, J.S. Mathew, B.S. Pai, J. Grier, P.M. Tharnish,
Y. Choi, Y. Chong, H. Choo, C.K. Chu, M.J. Otto, R.F. Schinazi, Antiviral activities
33] A. Meager, R. Gaines Das, K. Zoon, A. Mire-Sluis, Establishment of new and
replacement World Health Organization International Biological Standards for
human interferon alpha and omega, J. Immunol. Methods 257 (2001) 17e33.
0
0
0
0
and cellular toxicities of modified 2 ,3 -dideoxy-2 ,3 -didehydrocytidine
analogues, Antimicrob. Agents Chemother. 46 (2002) 3854e3860.