5-Acetyl-2-arylbenzimidazoles as antiviral agents. Part 4

Article abstract of DOI:10.1016/j.ejmech.2012.03.038

Within a project aimed at discovering new Flaviviridae inhibitors, new variously substituted 2-phenylbenzimidazoles were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against viruses representatives of the three genera of the Flaviviridae family, i.e.: Pestivirus (BVDV), Flavivirus (YFV) and Hepacivirus (HCV). Title compounds were also tested against RNA viruses representative of other single-stranded, positive-sense (ssRNA⁺) negative-sense (RNA^-), or double-stranded (dsRNA) genomes, as well as against representatives of two DNA virus families. Nine compounds showed activity against BVDV (EC₅₀ = 0.8-8.0 μM), compound 31 being the most potent (EC₅₀ = 0.80 μM) and selective (SI = CC₅₀/EC₅₀ = >100). When tested in an HCV replicon assay, compound 31 resulted again the most potent, displaying an EC₅₀ value of 1.11 μM and an SI of 100. Besides inhibiting BVDV, two compounds (35 and 38) showed a moderate activity also against YFV (EC ₅₀ = 13 μM). Interestingly, 35 was moderately active also against RSV (EC₅₀ = 25 μM).

Full text of DOI:10.1016/j.ejmech.2012.03.038

European Journal of Medicinal Chemistry 53 (2012) 83e97
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
5-Acetyl-2-arylbenzimidazoles as antiviral agents. Part 4
,
b
Gabriella Vitale a, Paola Corona a, Mario Loriga a, Antonio Carta a, Giuseppe Paglietti a , Gabriele Giliberti ,
*
,
Giuseppina Sanna b, Pamela Farci b, Maria Elena Marongiu b, Paolo La Colla b
**
a Dipartimento di Scienza del Farmaco, Università degli Studi di Sassari, Via Muroni 23/a, 07100 Sassari, Italy
b Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche, Università degli Studi di Cagliari, Cittadella
Universitaria, 09042 Monserrato (Ca), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Within a project aimed at discovering new Flaviviridae inhibitors, new variously substituted 2-
phenylbenzimidazoles were synthesized and evaluated in cell-based assays for cytotoxicity and anti-
viral activity against viruses representatives of the three genera of the Flaviviridae family, i.e.: Pestivirus
(BVDV), Flavivirus (YFV) and Hepacivirus (HCV). Title compounds were also tested against RNA viruses
representative of other single-stranded, positive-sense (ssRNAþ) negative-sense (RNAꢀ), or double-
stranded (dsRNA) genomes, as well as against representatives of two DNA virus families.
Received 8 April 2011
Received in revised form
16 March 2012
Accepted 21 March 2012
Available online 30 March 2012
Nine compounds showed activity against BVDV (EC50 ¼ 0.8e8.0
potent (EC50 ¼ 0.80 M) and selective (SI ¼ CC50/EC50 ¼ >100). When tested in an HCV replicon assay,
compound 31 resulted again the most potent, displaying an EC50 value of 1.11 M and an SI of 100.
Besides inhibiting BVDV, two compounds (35 and 38) showed a moderate activity also against YFV
M).
mM), compound 31 being the most
Keywords:
m
5-Acetyl-2-arylbenzimidazoles
Semi and thiosemicarbazones
Ketoximes
m
(EC50 ¼ 13
m
M). Interestingly, 35 was moderately active also against RSV (EC50 ¼ 25
m
Antiflaviviridae virus activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
hepatitis G virus, although closely related to HCV, represent unas-
signed members of Flaviviridae]; the Flavivirus genus comprises
Dengue Fever, Yellow Fever (YFV), West Nile, Japanese Encephalitis
and Tick-Borne Encephalitis viruses; the Pestivirus genus comprises
Bovine Viral Diarrhoea (BVDV), Border Disease and Classical Swine
Fever viruses.
HCV is a major cause of human hepatitis [11]. The WHO esti-
mates that over 170 million people worldwide are presently
infected by this virus [12,13]. Most infections become persistent,
and about 60% progress towards chronic liver disease. Chronic HCV
infection can lead to development of cirrhosis, hepatocellular
carcinoma and liver failure [14,15]. Pegylated interferon, in
combination with ribavirin, is used in the clinic for HCV infections.
Unfortunately, this therapy has limited efficacy and is often asso-
ciated with severe, adverse events [16].
Our interest on the chemistry and biological properties of
benzimidazoles has led us to synthesize numerous derivatives
endowed with analgesiceantiinflammatory [1,2] and choleretic
[3,4] activities, as well as with a chloropromazine-like activity in
the conditioned avoidance response [5,6] and, more recently, with
antiproliferative and antiviral activities [7e10]
In this context, we have described the series of 2-[4-substituted
naphthyl]- (1) [7], 2-[4-substituted biphenyl]- (2) [8], 2-[4-
substituted styryl]-benzimidazoles (3) [9] and 1-substituted-2-
[(benzotriazol-1/2-yl)methyl benzimidazoles (4) and (5) [10],
endowed with selective activity, at micromolar level, against
Respiratory Syncytial virus (RSV) and some members of the Flavi-
viridae family (Fig. 1).
The latter includes single-stranded positive-sense RNA (ssRNAþ)
viruses, which cause significant diseases in humans and animals.
They are distributed into three genera: the Hepacivirus genus
includes, as sole representative, the Hepatitis C virus (HCV) [agents
such as GB virus-A and GB virus-A-like, GB virus-D and GBV-C or
Flaviviruses are human pathogens prevalent throughout the
world and cause a range of acute febrile illnesses, encephalitides
and hemorrhagic diseases. Although an effective vaccine against
YFV has been available since the late 1930s, its use is not systematic
in many areas [17].
Pestivirus infections of domesticated livestock cause significant
economic losses worldwide. They cause a range of clinical mani-
festations, including abortion, teratogenesis, respiratory problems,
chronic wasting disease, immune system dysfunction and predis-
position to secondary viral and bacterial infections. BVDV can also
* Corresponding author. Tel.: þ39 079 228704; fax: þ39 079 228720.
** Corresponding author. Tel.: þ39 070 6754037; fax: þ39 070 6754210.
E-mail addresses: paglietti@uniss.it (G. Paglietti), placolla@unica.it (P. La Colla).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.038

84
G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
Fig. 1. Chemical structure and antiviral activity of the series of 2-arylbenzimidazoles previously described [7,8,9,10].
establish a persistent infection (PI) in animals, that succumb or
remain viraemic throughout life and serve as continuous virus
reservoirs. Furthermore, BVDV also shows the ability to cross the
placenta of susceptible animals causing a variety of foetal infections
[18,19].
With the exception of YFV, no vaccines exist against Flaviviridae
pathogens, as well as no selective antiviral drugs are yet available in
the clinic to prevent and/or treat their infections. The only excep-
tions are two protease inhibitors, recently approved by FDA for the
treatment of HCV infections [20ꢀ23]. Hence the need to continue
studies aimed at identifying new lead compounds targeted at virus-
specifc steps of the Flaviviridae replication cycle.
As part of a pluriennal program in this area [7e10], very recently
we described new 2-phenylbenzimidazoles [compounds 6 in
Fig. 2], which have been evaluated in cell-based assays for
cytotoxicity and activity against a panel of RNA and DNA viruses
[24]. 39 compounds exhibited antiviral activity at concentrations
respectively. In enzyme assays, 6b and 6d inhibited at low mM
concentrations the RNA-dependent RNA polymerase (RdRp) of
BVDV and HCV, respectively. This suggested that 2-
phenylbenzimidazoles could be attractive leads for further devel-
opment of inhibitors of poxviruses, pestiviruses and even HCV.
Therefore, we decided to carry on our program to evaluate whether
this type of compounds, purposely modified, could be improved in
both potency and spectrum of antiviral activity.
Focussing on the derivatization of the 5-acetyl group on
the benzimidazole, by using typical reagents of carbonyl group
as hydroxylamine, semicarbazide and thiosemicarbazide, we
designed and synthesized derivatives 7e31 (Fig. 3) in order to
evaluate the influence of this modification on the antiviral activity.
In support of our hypothesis was the example of the many
benzimidazole derivatives reported in the literature, where the
presence of a carboxylic or tetrazolyl group at position 5 of the
benzimidazole ring [as in compounds 32e34 (Fig. 4)] allowed
inhibition of the HCV RdRp at very low IC50 values [25e27].
In our opinion, the presence of an acetyl group in compounds
7e12, owing to a comparable electronegativity with both carboxy
and tetrazolyl groups mentioned above, and its transformation into
the derivatives 13e31, could represent a good strategy to improve
the antiviral properties of this class of compounds in analogy with
what observed in the known cases of enviroxime [28]a,b, 2-
acetylpyridine thiosemicarbazones [29] and methisazone [30].
Thus, we decided to proceed in two stages. First, we afforded the
simple modification of the acetyl group, as in compounds 7e31.
Once verified that this type of substitution could lead to selective
BVDV inhibitors, we prepared benzimidazoles 35e61 (Fig. 5) in
form of both simple acetyl derivatives and corresponding thio-
semicarbazones and semicarbazones, characterized by an increased
comprised between 0.1 and 10
mM, and four of them were
outstanding for their potency against VV (6a, R ¼ 5,6-diCl; R1 ¼ 4-
NO2) or BVDV (6b, R ¼ 5-NO2; R1 ¼ H; 4c, R ¼ 5-NO2; R1 ¼ COCH3;
6d, R ¼ 5,6-diCl; R1 ¼ COCH3), with EC50 ¼ 0.1, 1.5, 0.8 and 1.0
mM,
R=H; 5-CF3; 5-COCH3; 5,6-diCl;
N
R'
R'=2-NO2; 2,4-diNO2; 4-OH; 4-OCH3; 3-
OCH3; 2,4-diOCH3; 3,5-diOCH3; 2,3,4-
triOCH3; 4-F;2,6-diF; 4-NO2; 2-NO2; 4-
OCH3;2-NH2; 4-NH2 ; 4-OCH3
R
N
H
6
Fig. 2. Examples of 5R- and 2-aryl substituted benzimidazoles endowed with recorded
antiflaviridae activities.

G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
85
respiratory syncytial virus, RSV) and a rhabdovirus (vesicular
stomatitis virus (VSV). Among double-stranded RNA (dsRNA)
viruses, we tested reovirus type-1 (Reo-1). Two representatives of
DNA viruses were also included: human herpesvirus 1 (herpes
simplex type-1 HSV-1) and vaccinia virus (VV). Efavirenz, 20-C-
methyl-guanosine, 20-C-ethynyl-cytidine, 6-azauridine, mycophe-
nolic acid and acyclovir were used as reference inhibitors. Cyto-
toxicity was evaluated in parallel with the antiviral activity.
As far as the activity against Flaviviridae is concerned,
nine compounds (26e28, 30, 31, 53, 56, 57 and 60) exhibited
selective activity against BVDV in the low micromolar range
X
N
H3C
R1
N
R
7-31
Compd
X
R
R1
Ph
Ref
76
86
9
C=O
C=O
C=O
C=O
C=O
C=O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4’-Cl-Ph
4’- CF3-Ph
(EC50 ¼ 0.8e8.0
the most potent anti-BVDV activity (EC50 ¼ 0.83 ꢁ 0.09 and
0.80 ꢁ 0.06 M, respectively; doseeresponse curve in Fig. 7A) and,
like compounds 26, 57 and 60, were non cytotoxic at concentra-
tions up to 100 M.
Other compounds (11, 20, 36e38, 42 and 61) exhibited activity
against BVDV at higher M).
M concentrations (EC50 ¼ 16e23
Notewhorthy, compound 38 exhibited a selective, although not-
very potent, activity also against YFV (CC50 100 M;
M), whereas compound 35 showed activity
mM). In particular, derivatives 28 and 31 showed
106
1110
1210
136
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
m
4’-OCH3-Ph
2’,4’-diOCH3-Ph
2’,3’,4’-triOCH3-Ph
4’-OCH3-Ph
2’,4’-diOCH3-Ph
2’,3’,4’-triOCH3-Ph
Ph
m
CH-OH
CH-OH
m
m
>
m
CH-OH
EC50 ¼ 13.4 ꢁ 1.1
m
C=N-OH
against YFV (CC50 > 100
mM; EC50 ¼ 13.2 ꢁ 1.5
mM), but not against
C=N-OH
4’-Cl-Ph
BVDV.
C=N-OH
4’- CF3-Ph
When tested against representatives of other virus families
(Tables 1and 2), 5-acetyl-2-arylbenzimidazoles resulted mainly
inactive. Exceptions were: compound 20 and 35 which, besides
C=N-OH
4’-OCH3-Ph
2’,4’-diOCH3-Ph
2’,3’,4’-triOCH3-Ph
Ph
C=N-OH
BVDV, also inhibited CVB-5 (CC50 > 100
(CC50 > 100 M), respectively; and compound 43,
M; EC50 ¼ 25
active only against CVB-5 (CC50 > 100 M).
M; EC50 ¼ 47
mM; EC50 ¼ 43
mM) and RSV
C=N-OH
m
m
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
m
m
4’-Cl-Ph
SAR studies allow to conclude that derivatization of the 5-acetyl
group led to a significant improvement in the anti-BVDV activity
(compounds 7e11); transformation into ketoximes allowed to
achieve higher anti-BVDV potency (20); the conversion into semi-
carbazones also allowed to achieve an increase in potency (26);
4’- CF3-Ph
4’-OCH3-Ph
2’,4’-diOCH3-Ph
Ph
finally, the replacement of
a semicarbazide with a thio-
4’-Cl-Ph
semicarbazide to obtain the thiosemicarbazones allowed to achieve
fairly low EC50 values (28 and 31). On the contrary, no significant
extension of the antiviral spectrum was obtained, the only excep-
tion being compounds 20, 35 and 37.
Introduction of a cyclohexyl substituent at position 1 (deriva-
tives 35e61) failed to prove useful in improving the anti-BVDV
potency. However, compounds 35e38, 42, 48, 53, 57, 59e61
resulted active against this virus, the most potent being, again,
the thiosemicarbazones (53, 57, 60, 61). Moreover, susbstitution
with cyclohexyl (derivatives 35e41) allowed the appearance
of antiviral activities against different viruses: YFV and RSV, in
the case of compounds 35 and 37, and YFV in the case of
compound 38.
Structureeactivity relationship studies indicate that the acetyl
group as such, or its corresponding hydroxyethyl derivatives, are
unable to promote antiviral activity in the series of N-1-
unsubstituted benzimidazoles. Only when the phenyl bears a 2,4-
dimethoxy group at position 2 (as in the above cited compound
11), the improvement in anti-BVDV activity resulted higher than
that obtained previously [24]. As a cycloexyl substitution at posi-
tion 1 of the benzimidazole takes place, some compounds (35e38,
42, 48, 53, 57, 59e61) exhibit anti-BVDV activity, the most active
being those bearing the most lipophilic susbstituents in the phenyl
ring (2,4di-OCH3 ¼ Cl > OCH3). Bioisosteric replacement of the
phenyl with a furanyl or pyridinyl ring was partially successful in
4’- CF3-Ph
4’-OCH3-Ph
2’,4’-diOCH3-Ph
Fig. 3. First series of the new designed compounds 7e31.
lipophilicity due to the cyclohexyl substitution at position 1, as for
the above mentioned compounds 32e34.
In addition, we tested the effect of the isosteric replacement, at
position 2, of unsubstituted and substituted phenyl rings with
different aromatic rings, such as furan and pyridine, which are
present in the potent HCV RdRp inhibitors 62e63 (Fig. 6) described
by other authors [31,32].
In this paper we present the design and synthesis of the above
described derivatives and their evaluation for cytotoxicity and
antiviral activity in cell-based assays.
2. Results and discussion
5-acetyl-2-arylbenzimidazole derivatives were tested in cell-
based assays for cytotoxicity and antiviral activity against repre-
sentative members of a number of virus families (Tables 1and 2). In
addition to the Flaviviridae BVDV and YFV, among ssRNAþ viruses
we tested a retrovirus (human immunodeficiency virus type-1,
HIV-1), two picornaviruses, human enterovirus B (coxsackie virus
type B5, CVB-5) and human enterovirus C (polio virus type-1, Sb-1).
the case of 59 (EC50 ¼ 26
mM) and fairly good in the case of
compound 60 (EC50 ¼ 8 M).
m
Due to its interesting activity against BVDV, compound 31 was
also tested against HCV in a subgenomic replication assay that
allows viral replication in a human hepatoma cell line (GS4.1). In
Among ssRNAꢀ viruses we tested
a paramyxovirus (human

86
G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
O
C
N
N
F
O
N
N
N
N
HO
OCH3
N
N
N
O
H
O
32
33
Japan Tobacco
Japan US Pat 2003 50320 [Ref.25]
Bristol-Meyers-Squibb
USA WO 03 26587[Ref.26]
Cl
Cl
O
C
O
CH3
S
HO
N
N
O
34 [Ref.27]
Fig. 4. Examples of benzimidazole compounds patented as HCV polymerase inhibitors.
Cl
this assay, 31 selectively inhibits the HCV replication with an EC50 of
M (Table 3, doseeresponse curve in Fig. 7B).
53e61, respectively, using in turn semicarbazide hydrochloride and
thiosemicarbazide in refluxing ethanol and in the presence of
sodium acetate or glacial acetic acid, respectively.
1.11 ꢁ 0.15
m
Studies on the inhibition of BVDV and HCV recombinant RNA
polymerases, in vitro selection of BVDV resistant mutants, defini-
tion of the molecular basis of resistance, as well as in silico studies
aimed at detailing drug-RdRps interactions and mode of action of
the most potent 5-acetyl-2-arylbenzimidazoles will be the object of
separate publications.
The elucidation of all novel compounds was supported by
elemental analyses and 1H NMR spectra, that are fully consistent
with the described structures. In addition, for certain compounds
(14, 17, 22, 24, 25, 29, 39, 48, 50, 53, 55, 61) 13C NMR spectra were
recorded to further ascertain the localization of the carbon atoms
external to the hetero-rings. Examination of these 13C NMR spectra
(vide infra) allowed us to confirm that a few variations are con-
cerned with the presence of substituents in the phenyl moiety at
position 2 of the benzimidazole ring, whereas the chemical shifts of
C-8, C-9, and of C]S and C]O in side chains, were almost coinci-
dent (Table 4).
2.1. Chemistry
The preparation of compounds 7e31 and 35e61 was achieved
according to the sequence of reactions of Scheme 1. From the
commercially available bromoacetophenone (a) (Janssen), we
prepared the necessary intermediates (b,c) following a known
procedure for (b) [6], whereas (c) was obtained for the first time in
DMSO at 70 ꢂC for 3 h. The diamine (d) was previously described by
W. Borsche and J. Barthenheier [30], while (e) is a new compound.
Ring closure to benzimidazoles was accomplished in two different
ways. As the N-unsubstitued diamine (d) was used, condensation
with the bisulfite salts of the aldehydes fem yielded the desired
compounds 7e12, 42e43. Inthe case of the N-cyclohexylsubstituted
diamine (e), we had to carry on the reactions with the aldehydes fen
in DMF/water (30:1), at room temperature for 1 h, in the presence of
oxone to obtain compounds 35e41. In both cases yields were very
satisfactory (ranging from 58 to 98%). The intermediates 7, 8, 10-12
have been already described by some of us (G.P., M.L) as referenced
in Fig. 3, while compounds 9, 42, 43 are described here for the first
time. Compounds 13e15 were obtained on reduction by means of
NaBH4 in aqueous ethanol of the parent 10e12. The ketoximes
16e21 were obtained from the ketones 7e12 by reaction with
hydroxylamine. The acetyl derivatives 7e12 and 35e43 were alter-
natively converted into the corresponding semicarbazones 22e26
and 44e52, respectively, and thiosemicarbazones 27e31 and
3. Experimental section
3.1. General remarks
Melting points were carried out with a Kofler hot stage melting
point apparatus and are uncorrected. Infrared spectra were recor-
ded as nujol mulls on NaCl plates with a PerkineElmer 781 IR
spectrophotometer and are expressed in
n
(cmꢀ1). UV spectra are
qualitative and were recorded in nm for solutions in EtOH with
a PerkineElmer Lamba 5 spectrophotometer. Nuclear magnetic
resonance (1H NMR) spectra were determined in CDCl3, DMSO-d6,
CDCl3/DMSO-d6 (in the ratio 1:3) and were recorded in a Varian XL-
200 (200 MHz). Chemical shifts (d scale) are reported in parts per
million (ppm), downfield from tetramethylsilane (TMS) as internal
standard. Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet; br s, broad singlet;
dd, doublet of doublet.
The assignment of exchangeable protons (OH and NH) was
confirmed by the addition of D2O. Mass spectra were performed on

G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
87
combined HP 5790-HP 5970 GC/MS apparatus or with a combined
Liquid Chromatograph-Agilent 1100 series Mass Selective Detector
(MSD). Analytical thin-layer chromatography (TLC) was performed
on Merck silica gel F-254. Pure compounds showed a single spot in
TLC. For flash chromatography, Merck silica gel 60 was used with
a particle size 0.040e0.063 mm (230e400 mesh ASTM). Elemental
analyses were performed on a PerkineElmer 2400 instrument at
Laboratorio di Microanalisi, Dipartimento di Chimica, Università di
Sassari, Italy, and results were within ꢁ0.4% of theoretical values.
X
N
H3C
R1
N
R
35-61
X
R
R1
Ph
Compd
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
C=O
Cyclohexyl
3.2. Chemistry
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
H
4’-Cl-Ph
4’- CF3-Ph
4’-OCH3-Ph
2’,4’-diOCH3-Ph
3’-Furyl
3.2.1. Intermediates
The diamine (d) was not commercially available and was
purposely prepared as described by W. Borsche and J. Barthenheier
[33]. The bisulphite compounds (fem) were obtained in high yields
from the commercially available corresponding aldehydes (Aldrich)
with Na2S2O5 in ethanol, according to the procedure used by
Shriner and Land [34].
2’-Pyridinyl
3’-Furyl
H
Cyclohexyl
Ph
3.2.1.1. 1-(4-(Cyclohexylamino)-3-nitrophenyl)ethanone (b). To
a
solution of a (1 g, 41mmole) in DMSO (4 ml), cyclohexylamine
(0.85 g, 87 mmol) was added dropwise. The brown-yellow mixture
was then heated at 70 ꢂC for 3 h under magnetic stirring. On
cooling, it was diluted with water and the formed precipitate was
collected and dried on air. Compound b (1.03 g, 96% yield) was
yellow orange solid [Rf ¼ 0,36 (EP-EA 9:1)]; m.p, 115e118 ꢂC. Anal.
calc. C14H18N2O3 %: C, 64.10; H, 6.92; N, 10.68; found: C, 63.98; H,
7.10; N,10.72. nmax (nujol) cmꢀ1: 3583, 3339,1671,1613; lmax (EtOH)
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CO-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
C=N-NH-CS-NH2
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
H
4’-Cl-Ph
4’- CF3-Ph
4’-OCH3-Ph
2’,4’-diOCH3-Ph
3’-Furyl
nm: 308, 269, 198. 1H NMR (CDCl3)
d: 8,79 (1H, d, J 1.6, H-3), 8.52
(1H, br s, J 8.0, NH), 8.04 (1H, dd, J 8.4 and 1.6, H-5), 6.92 (1H, d, J 8.4,
H-6), 3.66e3.50 (1H, m, CH cyclohexyl), 2.56 (3H, s, CH3CO),
2.18e1.30 (10H, m, cyclohexyl).
2’-Pyridinyl
3’-Furyl
H
Cyclohexyl
Ph
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
H
4’-Cl-Ph
3.2.1.2. 1-(3-Amino-4-(cyclohexylamino)phenyl)ethanone (e). A sol-
ution of (c) (2 g, 762 mmol) in ethanol (120 ml) was added of 0.2 g
of 10% palladised charcoal and hydrogenated under moderate
pressure at 20 ꢂC within 2 h. Then, the mixture was filtered off
through filter paper and the solvent removed under vacuum. A
glassy residue of (e) (1.77 g, 97% yield) was pure at TLC [Rf ¼ 0.30,
EP-EA 6:4]; m.p. 86e88 ꢂC; Anal. calc. for C14H20N2O: C, 72.38; H,
4’- CF3-Ph
4’-OCH3-Ph
2’,4’-diOCH3-Ph
3’-Furyl
2’-Pyridinyl
3’-Furyl
8.68; N, 12.06; found: C, 72.50; H, 8.51; N, 12.00. nmax (nujol) cmꢀ1
3583, 3370, 1642, 1587; lmax (EtOH) nm: 349, 258, 216; 1H NMR
(CDCl3) : 7.50 (1H, dd, J 8.6 and 2.0, H-5), 7.39 (1H, d, J 1.8, H-3),
:
H
Cyclohexyl
d
6.58 (1H, d, J 8.4, H-6), 3.45e3.30 (1H, m, CH cyclohexyl), 2.49 (3H,
s, CH3CO), 2.15e1.30 (10H, m, cyclohexyl).
Fig. 5. Second series of the new designed compounds 35e61.
O
C
N
N
X
X= OH HCV NS5B polymerase IC 50= 4.3 µM
[Ref.31]
N
62
O
C
N
X
X= OH HCV NS5B polymerase IC 50= 1.6 µM
[Ref.32]
N
O
63
Fig. 6. Further examples of benzimidazole compounds described as HCV polymerase inhibitors.

88
G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
Table 1
Cytotoxicity and antiviral activity of 2-phenylbenzimidazole derivatives (7e31) against representatives of ssRNAþ (HIV-1, BVDV, YFV, CBV-5, Sb-1), ssRNAꢀ (RSV, VSV), dsRNA
(Reo-1) and dsDNA (VV, HSV-1) viruses.
X
N
H3C
R1
N
R
Compds.
X
R
R1
MT-4 HIV-1 MDBK BVDV BHK YFV
Reo-1 VERO 76 CVB-5 Sb-1 RSV
VSV
VV
HSV-1
a
b
c
d
e
f
g
h
i
j
k
l
m
n
CC50 EC50
CC50
>100 100
>100 >100 >100 >100 >100 >100
>100 15 >100 >100 >100 >100
EC50
CC50 EC50 EC50
CC50
EC50
EC50 EC50 EC50 EC50
EC50
7
8
9
C]O
C]O
C]O
C]O
C]O
C]O
CHeOH
CHeOH
CHeOH
CNeOH
CNeOH
CNeOH
CNeOH
CNeOH
CNeOH
C]NeNHeCOeNH2
C]NeNHeCOeNH2
C]NeNHeCOeNH2
C]NeNHeCOeNH2
C]NeNHeCOeNH2
C]NeNHeCSeNH2
C]NeNHeCSeNH2
C]NeNHeCSeNH2
C]NeNHeCSeNH2
C]NeNHeCSeNH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
ND
ND
ND
24
ND
ND
ND
>24
>50
>100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 ND
>100 >100 ND
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
Ph(40-Cl)
Ph(40- CF3)
Ph(40-OCH3)
Ph(20,40-OCH3)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
58
73
>58
21
40
50
>40
>50
ND
ND
>100
>100
ND
ND
>100 ND
>100 ND
50
Ph(20,30,40-OCH3) >100 >100 >100 >100 >100 >100 >100 >100
Ph(40-OCH3)
Ph(20,40-OCH3)
ꢃ100 >100 >100 >100 >100 >100 >100 >100
ꢃ100 >100 >100 >100 >100 >100 >100 >100
Ph(20,30,40-OCH3) 79
>79
ND
>20
ND
60
>60
60
>60
>100 ꢃ100
>100 >100 >100 ND
>100 >100 >100 >100 >100 >100
>100 >100
Ph
Ph(40-Cl)
Ph(40- CF3)
Ph(40-OCH3)
Ph(20,40-OCH3)
ND
20
ND
ND
>100 >100 >100 >100 >100 >100
37
55
44
18
>55
>44
33
ND
44
>33
ND
>44
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>100 >100
>100 >100 ND
43
>100 >100 ND
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 ND
ꢃ100 >100 >100 17
>100 >100 >100 >100
>100 >100 >100 >100 >100
ND >100 >100
Ph(20,30,40-OCH3) 51
>51
ND
ND
ND
ND
>100 >100 >100 >100 >100 >100
Ph
Ph(40-Cl)
ND
ND
ND
ND
21
>100 50
>100 >100 >100 >100 >100 >100
>100 45 >100 >100 >100 >100
25 11 ND ND ND ND
>100 2.55
ꢃ100 >100 >100 80
3.80 32 >32 >32 >100
>100 0.85 >100 >100 >100 >100
>100 >100 >100 >100
Ph(40- CF3)
Ph(40-OCH3)
Ph(20,40-OCH3)
Ph
ND
>80
ND
>80
ND
ND
ND
>80
ND
80
ND
ND
ND
ꢃ21
ꢃ100 >100 32
>100 >100 >100 >100 ND
>100 >100 ND
>22 >22 ND
>100 >100 ND
>100 >100 >100 >100 ND
Ph(40-Cl)
2
>2
>100 >100 ND
ND ND ND
>100 >100 ND
Ph(40- CF3)
Ph(40-OCH3)
Ph(20,40-OCH3)
2
>2
5
>1.8
5
>5
ND
22
5
>5
13
1.85
13
28
>13
>28
>13
>28
>100
>100
72
>72
>100 0.80
>100
Reference compounds
Efavirenz
40
0.002
20-C-methyl-guanosine
20-C-methyl-cytidine
20-C-ethynyl-cytidine
6-aza-uridine
Mycophenolic acid
Acycloguanosine
>10
1.1
>10
>100
1.9
16
>100
ꢃ12.5
ꢃ12.5
>100
27
23
1.2
1.5
3
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%.
ND ¼ Not determined.
a
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
m
m
m
m
m
m
m
m
m
m
m
m
m
m
M) required to reduce the proliferation of mock-infected MT-4 cells by 50%, as determined by the MTT method.
b
c
M) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method.
M) required to achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected BHK cells by 50%, as determined by the MTT method.
M) required to achieve 50% protection of BHK cells from YFV-induced cytopathogenicity, as determined by the MTT method.
M) required to achieve 50% protection of BHK cells from Reo-1-induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected VERO-76 cells by 50% as determined by the MTT method.
M) required to reduce the plaque number of CVB-5 by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of Sb-1 by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of RSV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of VSV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of VV by 50% in VERO-76 monolayers.
d
e
f
g
h
i
j
k
l
m
n
M) required to reduce the plaque number of HSV-1 by 50% in VERO-76 monolayers.
3.3. General procedure for the preparation of the 2-[R-
substitutedphenyl]-5-acetyl-benzimidazoles (7e12) and 2-
[cycloakyl or heteroaryl]-5-acetyl-benzimidazoles (42e43)
ethanol. After cooling, an excess of the sodium salt was filtered off
through filter paper and the mother liquors were evaporated to
dryness under reduced pressure. The solid residues, coloured from
yellow to dark brown, were resuspended in dry ether and then
purified, if necessary, by recrystallization from EtOH/H2O, or by silica
gel column chromatography, using a mixture of the solvent indi-
cated under the single compounds described below. Compounds 7,
8, 10, 11, 12 have been described previously, as referenced in Fig. 3.
(A)-A mixture of 5-acetyl-1,2-diaminobenzene (d) (10 mmol)
and of either sodium hydroxy(R-substituted-phenyl-1-yl)meth-
anesulfonate (fek) or sodium hydroxy(cyclohexyl or furan-3-yl)
methanesulfonate (lem) (11 mmol) was refluxed for 4 h in 80 mL of

G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
89
Table 2
Cytotoxicity and antiviral activity of 2-phenylbenzimidazole derivatives (35e61) against representatives of ssRNAþ (HIV-1, BVDV, YFV, CBV-5, Sb-1), ssRNAꢀ (RSV, VSV), dsRNA
(Reo-1) and dsDNA (VV, HSV-1) viruses.
X
N
H3C
R1
N
R
Compds.
X
R
R1
MT-4 HIV-1 MDBK BVDV BHK YFV
Reo-1 VERO 76 CVB-5 Sb-1 RSV
VSV
VV
HSV-1
a
b
c
d
e
f
g
h
i
j
k
l
m
n
CC50 EC50
CC50
EC50 CC50 EC50 EC50
CC50
13.2 >100 80
>100 >100 >100 >100
33 >33 >33 35
>100 13.4 >100 30
EC50
EC50 EC50 EC50 EC50
EC50
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
C]O
C]O
C]O
C]O
C]O
C]O
C]O
C]O
C]O
c-hexyl Ph
37
58
41
27
>37
>58
>41
>27
>21
>63
>70
>100 60
>100 23
52
>100 21
21 >21
>100 >100 77
100
>80
>80 25 >80 >80
>80
c-hexyl Ph(40-Cl)
c-hexyl Ph(40-CF3)
c-hexyl Ph(40-OCH3)
>100 >100 >100 >100 >100 >100
23
>35
>30
>70
>80
>90
>35 20
>30 >30 >30 >30
>70 >70 >70 >70
>80 ND
>90 ND
>35 >35
>35
>30
>70
>80
>90
c-hexyl Ph(20,40-OCH3) 21
8.5
>8.5 >85
>77 ND
70
80
c-hexyl 30-Furyl
63
70
ND
>80
c-hexyl 20-Pyridinyl
>100 >100 ꢃ100 >100 ND
>90
>100
>90 >90
>100 >100 >100
>100 >100 >100
H
H
30-Furyl
c-hexyl
>100 >100 ꢃ100 22
98 >98 ND
>100 >100 ND
47 >100 ND
>100 >100 >100 >100 >100 >100 >100 >100
C]NeNHeCOeNH2 c-hexyl Ph
ND
ND
ND
ND
ND
ND
ND
ND
>23
ND
ND
ND
ND
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 ND
C]NeNHeCOeNH2 c-hexyl Ph(40-Cl)
C]NeNHeCOeNH2 c-hexyl Ph(40-CF3)
C]NeNHeCOeNH2 c-hexyl Ph(40-OCH3)
54
36
>54
22
ND
19
ND
ND
>19 ND
ND ND
ND
ND
20
ND
ND
>20
ND
ND
>20 ND
ND ND
ND
ND
ND
ND
ND
>20
ND
ND
>20
ND
C]NeNHeCOeNH2 c-hexyl Ph(20,40-OCH3) 23
C]NeNHeCOeNH2 c-hexyl 30-Furyl
C]NeNHeCOeNH2 c-hexyl 20-Pyridinyl
ND
ND
ND
ND
20
17
30
39
>100 100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
C]NeNHeCOeNH2
C]NeNHeCOeNH2
C]NeNHeCSeNH2 c-hexyl Ph
H
H
30-Furyl
c-hexyl
>100 60
>100 44
>100 >100 >100 >100
>100 >100 >100 >100
10
24
4.5
38
9
>38
20
>20
>20 >20 >20 >20
>20
C]NeNHeCSeNH2 c-hexyl Ph(40-Cl)
C]NeNHeCSeNH2 c-hexyl Ph(40-CF3)
C]NeNHeCSeNH2 c-hexyl Ph(40-OCH3)
ꢃ17
ꢃ30
>39
ꢃ17
>21
>23
>86
>48
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100
32
>100
7
4
ND
17
ND
>17 >17
ND
40
20
25
20
>40
>20
>25
>20
>40 >40 >40 >40
>20 >20 >20 >20
>40
>20
>25
>20
C]NeNHeCSeNH2 c-hexyl Ph(20,40-OCH3) 17
C]NeNHeCSeNH2 c-hexyl 30-Furyl
C]NeNHeCSeNH2 c-hexyl 20-Pyridinyl
21
23
86
48
>100 >100 >100 >100 ND
41
>100
>25 ND
>20 ND
>25 >25
>20 >20
26
8
24
>24 ND
C]NeNHeCSeNH2
C]NeNHeCSeNH2
H
H
30-Furyl
c-hexyl
>100 >100 >100 100
>100 100
>100 >100 >100 >100
>100 16
24
>24 ND
>100
>100 >100 >100 >100 >100 ND
Reference compounds
Efavirenz
40
0.002
20-C-methyl-guanosine
20-C-methyl-cytidine
20-C-ethynyl-cytidine
6-aza-uridine
Mycophenolic acid
Acycloguanosine
>10
1.1
>10 1.9
>100
16
>100
ꢃ12.5
ꢃ12.5
>100
27
23
1.2
1.5
3
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%.
ND ¼ Not determined.
a
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
m
m
m
m
m
m
m
m
m
m
m
m
m
m
M) required to reduce the proliferation of mock-infected MT-4 cells by 50%, as determined by the MTT method.
b
c
M) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method.
M) required to achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected BHK cells by 50%, as determined by the MTT method.
M) required to achieve 50% protection of BHK cells from YFV-induced cytopathogenicity, as determined by the MTT method.
M) required to achieve 50% protection of BHK cells from Reo-1-induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected VERO-76 cells by 50% as determined by the MTT method.
M) required to reduce the plaque number of CVB-5 by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of Sb-1 by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of RSV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of VSV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of VV by 50% in VERO-76 monolayers.
d
e
f
g
h
i
j
k
l
m
n
M) required to reduce the plaque number of HSV-1 by 50% in VERO-76 monolayers.
3.3.1. 2-(4-Trifluoromethylphenyl)-5-acetyl-1H-benzimidazole (9)
Yield: 98%. M.p. 230e232 ꢂC (EtOH/H2O). Anal. calc. for:
C16H11F3N2O: C, 63.16; H, 3.64; F, 18.73; N, 9.21; O, 5.26. Found %:
C, 63.00; H, 3.80; N, 9.10. nmax (Nujol) cmꢀ1: 3290, 1666; UV
7.87 (2H, d, J 8.6, H-20,60), 7.67 (1H, d, J 8.4, H-7), 2.66 (3H, s,
CH3CO).
3.3.2. 2-(3-Furanyl)-5-acetyl-1H-benzimidazole (42)
(EtOH) nm: 319, 261, 220; 1H NMR (CDCl3-DMSO-d6)
d
: 8.21 (2H,
Yield: 68%. Rf ¼ 0.18 (EP-EA 4:6); m.p. 215e218 ꢂC. Anal. calc. for
:C13H10N2O2: C, 69.02; H, 4.46; N, 12.38; found: C, 68.82; H, 4.58; N,
d, J 8.0, H-30,50), 8.27 (1H, s, H-4), 7.91 (1H, dd, J 8.4 and 1.6, H-6),

90
G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
Fig. 7. Compound 31. Doseeresponse curves from cell-based assays against BVDV (A) and HCV (B).
12.30; nmax (nujol) cmꢀ1: 3260, 1659; lmax (EtOH) nm: 308, 247,
228; 1H NMR (CDCl3-DMSO-d6)
: 8.36 (1H, dd, J 1.8 and 0.8, H-20),
8.19 (1H, s, H-4), 7.85 (1H, dd, J 8.6 and 1.6, H-6), 7.68 (1H,dd, J 1.8
and 1.6, H-40), 7.59 (1H, d, J 8.6, H-7), 7.11 (1H, dd, J 1.8 and 0.8, H-50),
2.65 (3H, s, CH3CO).
3.4.1. 1-Cyclohexyl-2-phenyl-5-acetyl-1 H-benzimidazole (35)
Yield: 69%. Rf ¼ 0.31 (PE-EA 6:4); m.p. 153e155 ꢂC; Anal. calc. for
C21H22N2O: C, 79.21; H, 6.96; N, 8.80; C, 78.96; H, 7.03; N, 8.70. nmax
d
(nujol) cmꢀ1: 1672. lmax (EtOH) nm: 242, 199. 1H NMR (CDCl3)
d:
8.42 (1H, s, H-4), 7.98 (1H,dd, J 8.0 and 1.6, H-6), 7.70 (1H, d, J 8.0, H-
7), 7.65e7.55 (3H, m, H-30,40,50), 7.54 (2H, m, H-20,60), 4.44e4.24
(1H, m, CH cyclohexyl), 2.64 (3H, s, CH3CO), 2.42e1.26 (10H, m,
cyclohexyl).
3.3.3. 2-(Cyclohexyl)-5-acetyl-1H-benzimidazole (43)
Yield: 58% after chromatography [Rf ¼ 0.23 (EP-EA 4:6)]; m.p.
45e48 ꢂC. Anal. calc. for: C15H18N2O: C, 74.35; H, 7.49; N, 11.56;
Found: C, 74.28; H, 7.70; N, 11.47. nmax (nujol) cmꢀ1: 1660; lmax
3.4.2. 1-Cyclohexyl-2-(4-chlorophenyl)-5-acetyl-1H-benzimidazole(36)
Yield: 76%. Rf ¼ 0.38 (PE-EA 7:3); m.p. 182e184 ꢂC; Anal. calc. for
C21H21ClN2O: C, 71.48; H, 6.00; Cl, 10.05; N, 7.94; found: C, 71.25; H,
6.40; Cl, 9.90; N, 7.85. nmax (nujol) cmꢀ1: 1674. lmax (EtOH) nm: 251,
(EtOH) nm: 268, 214; 1H NMR (CDCl3)
d: 8.22 (1H, s, H-4), 7.84 (1H,
dd, J 8.6 and 1.6, H-6), 7.58 (1H, d, J 8.6, H-7), 3.04e2.24 (1H, m, CH
cyclohexyl), 2.66 (3H, s, CH3CO), 2.24e1.18 (10H, m, cyclohexyl).
199. 1H NMR (CDCl3)
d: 8.41 (1H, s, H-4), 7.97 (1H,dd, J 8.0 and 1.6,
H-6), 7.69 (1H, d, J 8.6, H-7), 7.59 (2H, d, J 8.6, H-30,50), 7.53 (2H, d, J
8.6, H-20-60), 4.42e4.24 (1H, m, CH cyclohexyl), 2.69 (3H, s, CH3CO),
2.40e1.20 (10H, m, cyclohexyl).
3.4. General procedure for the preparation of the 2-[aryl,cycloalkyl
or heteroaryl]-5-acetyl-benzimidazoles (35e41)
(B) To a solution of (e) (258 mmol) in DMF-water in the ratio
30:1 (4 mL), a solution of the appropriate aldheyde (284 mmol) and
of oxone (2KHSO5eKHSO4eK2SO4) (167 mmol) were added. The
mixture was kept under stirring at rt and after 1 h was poured
dropwise into a stirred solution constituted by 1 M K2CO3 (1.80 mL)
and water (40 ml). The solid precipitates (35e41) were collected,
thoroughly washed with water and left to dry on air. In the case of
compounds 40e41, the aqueous phase was decanted and the
organic residue was taken up with chloroform. Then, the chloro-
form solution was dried over anhydrous sodium sulphate and
evaporated. Purification of all compounds was performed by silica
gel flash column chromatography, eluting with a mixture of petrol
ether (PE) ethylacetate (EA) in the ratio indicated under the Rf of
each compound, as described below.
3.4.3. 1-Cyclohexyl-2-(4-trifluoromethylphenyl)-5-acetyl-1H-
benzimidazole (37)
Yield: 78%. Rf ¼ 0.44 (PE-EA 7:3); m.p. 158e160 ꢂC; Anal. calc.%
for C22H21F3N2O: C, 68.38; H, 5.48; F, 14.75; N, 7.25; found C, 68.03;
H, 5.80; N, 7.11. nmax (nujol) cmꢀ1: 1678. lmax (EtOH) nm: 255, 228.
1H NMR (CDCl3)
d: 8.44 (1H, s, H-4), 7.99 (1H,dd, J 8.6 and 1.4, H-6),
7.85 (2H, d, J 8.4, H-30,50), 7.84 (2H, d, J 8.4, H-20-60), 7.72 (1H, d, J 8.4,
H-7), 4.38e4.26 (1H, m, CH cyclohexyl) 2.69 (3H, s, CH3CO),
2.45e1.25 (10H, m, cyclohexyl).
3.4.4. 1-Cyclohexyl -2-(4-methoxyphenyl)-5-acetyl-1H-
benzimidazole (38)
Yield: 82%. Rf ¼ 0.31 (PE-EA 1:1); m.p. 138e140 ꢂC; Anal. calc. for
C22H24N2O2: C, 75.83; H, 6.94; N, 8.04; found: C, 76.01; H, 6.68; N,
7.98. nmax (nujol) cmꢀ1: 3583, 1664. lmax (EtOH) nm: 257, 201. 1
H
Table 3
NMR (CDCl3) d: 8.40 (1H, s, H-4), 7.95 (1H,dd, J 8.8 and 1.6, H-6),
7.68 (1H, d, J 8.8, H-7), 7.59 (2H, d, J 8.0, H-30,50), 7.06 (2H, d, J 8.0, H-
20-60), 4.48e4.28 (1H, m, CH cyclohexyl), 3.90 (3H, s, OCH3), 2.68
(3H, s, CH3CO), 2.40e1.26 (10H, m, cyclohexyl). LC/MS: 349 (M þ 1).
Comparative activity of compound 31 against BVDV and HCV in cell-based assays.
Cell-baseda
Compd
BVDV
HCV-1b
bCC50
cEC50
dCC50
eEC50
3.4.5. 1-Cyclohexyl-2-(2,4-dimethoxyphenyl)-5-acetyl-1H-
benzimidazole (39)
31
>100
0.80 ꢁ 0.06
11.3 ꢁ 1.5
1.11 ꢁ 0.15
a
Data represent mean values for three independent determinations.
b
Yield: 90%.Rf ¼ 0.30 (PE-EA 1:1); m.p. 76e78 ꢂC; Anal. calc.% for
C23H26N2O3: C, 72.99; H, 6.92; N, 7.40; found: C, 73.10; H, 7.04; N,
7.20. nmax (nujol) cmꢀ1: 1672. lmax (EtOH) nm: 285, 239, 203. 1H
Compound concentration (
mM) required to reduce the viability of mock-infected
MDBK cells by 50%, as determined by the MTT method.
c
Compound concentration (
mM) required to achieve 50% protection of MDBK
cells from BVDV-induced cytopathogenicity, as determined by the MTT method.
NMR (CDCl3) d: 8.39 (1H, s, H-4), 7.94 (1H,dd, J 8.0 and 1.6, H-6),
d
Compound concentration (mM) required to reduce the viability of GS4.1 cells by
7.66 (1H, d, J 8.4, H-7), 7.43 (1H, d, J 8.4, H-60), 6.64 (1H, dd, J 8.4 and
1.2, H-50), 6.58 (1H, d, J 2.2, H-30), 4.06e3.90 (1H, m, CH cyclohexyl),
3.90 (3H, s, OCH3), 3.79 (3H, s, OCH3), 2.68 (3H, s, CH3CO), 2.30e1.20
50%, as described in the Experimental section.
e
Compound concentration (mM) required to achieve 50% protection of GS4.1 cells
from cytopathogenicity, as described in the Experimental section.

G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
91
HO
O
O
N
H3C
NO2
Br
H3C
H3C
H
R1
H2N-R
N
R
a
i,ii
13-15 (R=H)
NO2
v
from 10-12
NH
R
O
b, R=H
X
from 7-12
N
c, R=cycloexyl
N
H3C
H3C
HO
R1
H
R1
iii
iv
H2/Pd
O
vi
N
R
R1
N
C
NaO3S
NH2
H3C
R
16-21 (R=H)
+
7-12 , 42-43 (R=H)
f-m
NH
X=C=N-OH
O
35-41(R= cyclohexyl)
C
R1
R
H
d, R=H
e, R=cycloexyl
f-n
from 7-11, 35-43
vii
viii
R1
f=Ph
X
N
H3C
g=4'-Cl-Ph
h=4'-CF3-Ph
i=4'-OMe-Ph
j=2'.4'-diOMe-Ph
k=2',3,'4'-triOMe-Ph
l=cycloexyl
m= 3'-furyl
n=2'-pyridinyl
R1
N
R
22-26 X=C=N-NH-CO-NH2
27-31 X=C=N-NH-CS-NH2
44-52 X=C=N-NH-CO-NH2
53-61 X=C=N-NH-CS-NH2
Scheme 1. i, In ethanol in sealed tube at 100 ꢂC; ii, in DMSO at 70 ꢂC for 3 h; iii, in refluxing ethanol for 4e5 h; iv, in DMF at rt for 1 h in the presence of oxone; v, NaBH4 in ethanol/
water; vi, NH2OH in ethanol; vii, semicarbazide hydrochloride in ethanol and in the presence of sodium acetate; viii, thiosemicarbazide in ethanol, water and acetic acid.
(10H, m, cyclohexyl). 13C NMR (DMSO-d6)
d
:197.54 (C]O), 162.31
Then, the solvent was evaporated under vacuum. The gummy
residue was shaken with water and eventually taken up with dry
ether to give a solid that was recrystallized from ethanol or
ethanolewater.
(C-2), 158.32 (C-40), 153.30 (C-20), 143.13 (C-3a)), 136.67 (C-7a),
132.82 (C-6), 130.74 (C-5), 121.72 (C-4), 120.60 (C-7), 112.44 (C-10),
111.62 (C-60), 105.63 (C-50), 98.34 (C-30), 57.01 (C-100-cyclohexane),
55.51 (OCH3), 55.39 (OCH3), 30.53 (C-200,600), 26.78 (CH3), 25.59 (C-
300,500), 24.54 (C-400).
3.5.1. 1-(2-(4-Methoxyphenyl)-1H-benzimidazol-5-yl)ethanol (13)
Yield: 94%; m.p.222e224 ꢂC as described [6].
3.4.6. 1-Cyclohexyl -2-[furan-3-yl]-5-acetyl-1H-benzimidazole (40)
Yield: 62%. Rf ¼ 0.38 (PE-EA 1:1); m.p. 117e118 ꢂC; Anal. calc.%
for C19H20N2O2: C, 74.00; H, 6.54; N, 9.08; found: C, 73.69; H, 6.82;
N, 8.81. nmax (nujol) cmꢀ1: 1672. lmax (EtOH) nm: 245. 1H NMR
3.5.2. 1-(2-(2,4-Dimethoxyphenyl)-1H-benzimidazol-5-yl)ethanol
(14)
Yield:
C17H18N2O3 þ 0.25H2O: C, 67.42; H, 6.07; N, 9.25; found: C, 67.38; H,
6.11; N, 9.45. 13C NMR (DMSO-d6) : 161.87 (C-40), 158.00(C-20),
80%;
m.p.
217e218
ꢂC;
Anal.
calc
for
(CDCl3) d
: 8.38 (1H, dd, J 8.0 and 0.8, H-20), 7.96 (1H, dd, J 8.0 and 1.8,
H-6), 7.90 (1H, s, H-4), 7.65 (1H, d, J 8.0, H-7), 7.60 (1H, dd, J 1.8 and
1.6, H-40), 6.80 (1H, dd, J 1.4 and 0.6, H-50), 4.60e4.40 (1H, m, CH
cyclohexyl), 2.67 (3H, s, CH3CO), 2.39e1.37 (10H, m, cyclohexyl).
d
149.16 (C-2), 142.70 (C-3a), 141.50 (C-7a), 134.55 (C-5), 130.80 (C-
60), 117.35 (C-6), 112.91 (C-4), 111.19 (C-7), 108.24 (C-10), 106.19 (C-
50), 98.55 (C-30), 68.61 (C-8), 55.80 (20-OCH3), 55.48 (40-OCH3),
26.50 (CH3).
3.4.7. 1-Cyclohexyl -2-[pyridin-2-yl]-5-acetyl-1H-benzimidazole (41)
Yield: 66%. Rf ¼ 0.28 (PE-EA 1:1); m.p. 125e128 ꢂC; Anal. calc.%
for C20H21N3O: C, 75.21; H, 6.63; N, 13.16; found: C, 74.89; H, 7.02;
N,13.00. nmax (nujol) cmꢀ1: 1673. lmax (EtOH) nm: 295, 237. 1H NMR
3.5.3. 1-(2-(2,3,4-Trimethoxyphenyl)-1H-benzimidazol-5-yl)
ethanol (15)
(CDCl3)
d
: 8.73 (1H, d, J 4.0, H-30), 8.44 (1H, d, J 1.4, H-4), 8.24 (1H, d,
Yield:
78%;
m.p.
140e142
ꢂC;
Anal.
calc
for
J 8.0, H-60), 7.94 (1H, dd, J 8.6 and 1.6, H-6), 7.90e7.84 (1H, m, H-50),
7,75 (1H, d, J 8.8, H-7), 7.44e7.36 (1H, m, H-40), 5.30e5.20 (1H, m,
CH cyclohexyl), 2.69 (3H, s, CH3CO), 2.42e1.20 (10H, m, cyclohexyl).
C18H20N2O4 þ 0.5H2O: C, 64.00; H, 6.27; N, 8.30; found: C, 63.83; H,
6.08; N, 8.35.
3.6. General procedure for the preparation of the ketoximes 16e21
3.5. General procedure for the reduction of the 2-[R-
substitutedphenyl]-5-acetyl-benzimidazoles (10e12) into the
derivatives (13e15)
A mixture of the ketone (7e12) (1.68 mmol) in ethanol (10 mL)
and hydroxylamine hydrochloride (1.78 mmol) in water (1 mL)
was heated under reflux for 2 h in the presence of a sodium
hydroxide aqueous solution (3.56 mmol in 2 ml of water).
On cooling, the mixture was diluted with water to cause
A suspension of 10e12 (34 mmol) and sodium borohydride
(68 mmol) in ethanol (20 mL) was refluxed under stirring for 2 h.

Table 4
13C NMR spectra of compounds 14,17,22,24,25,29,39,48,50,53,55,61, representative of the list of Figs. 3 and 5.
X
R
R1
C
N
3'
4'
5'
4
2'
1'
6'
8
H3C
5
3
3a
9
R3
2
6
1
7a
7
N
R4
Compd.
C-2
C-3a
C-4
C-5
C-6
C-7
C-7a
C-8 X ¼ O X ¼ NOH
X ¼ NeNH
68.61
C-9
C-10*
C-20*
C-30*
98.55
C-40*
C-50*
C-60*
X; N]NHeCOe(S)NH2
Other chemical
shifts
55.80 (20-OCH3),
55.48 (40-OCH3),
14
149.16
142.70
112.91
134.55
117.35
111.19
141.50
26.50
108.24
158.00
161.87
106.19
130.80
17
22
24
25
29
39
154.91
152.11
150.50
152.13
151.71
162.31
129.12
129.05
133.36
136.20
137.78
143.13
128.19
130.04
127.10
120.58
112.97
121.72
120.37
126.50
130.05
132.62
128.18
130.74
128.87
120.87
129.42
122.34
114.27
132.82
128.19
112.92
112.93
114.45
113.60
120.60
128.87
130.04
133.87
139.50
138.14
136.67
153.37
145.10
144.96
145.02
148.50
197.54
11.99
13.85
13.83
13.82
14.50
26.78
131.64
132.88
133.36
112.90
133.04
112.44
129.12
126.50
127.10
128.11
126.93
153.30
129.12
129.05
126.01
114.35
129.25
98.34
134.67
129.05
133.87
160.76
122.29
158.32
129.12
129.05
126.01
114.35
129.25
105.63.
129.12
126.50
127.10
128.11
126.93
111.62
157.60 C]O
157.66 C]O
157.50 C]O
178.81 C]S
121.35 (CF3)
55.38 (OCH3),
131.00 (CF3)
57.01 (C-100)
,55.51 (OCH3),
55.39 (OCH3),
30.53 (C-200,600),
25.59 (C-300,500),
24.54 (C-400).
56.74 (C-100),
55.45 (20-OCH3),
55.32(40-OCH3),
30.66 (C-200,600),
25.64 (C-300,500),
24.68 (C-400),
56.45 (C-100),
30.73 (C-200,600),
25.78 (C-300,500),
24.72 (C-400),
56.61 (C-100),
30.68 (C-200,600),
25.53 (C-300,500),
24.50 (C-400)
48
151.77
133.60
120.13
132.78
131.92
117.15
143.60
145.08
13.80
105.51
157.51
111.98
158.27
98.30
112.13
162.07 C]O
50
53
55
144.80
153.99
160.30
142.95
143.31
148.83
121.22
118.21
112.90
134.76
129.78
120.82
113.10
121.05
117.96
117.68
112.55
112.45
137.55
134.45
143.25
148.96
148.76
153.99
13.76
14.42
14.42
N-1
150.85
128.76
130.77
125.53
129.30
114.20
132.74
131.69
131.56
124.40
129.30
114.20
150.32
128.76
130.77
157.45 C]O
178.69 C]S
178.67 C]S
130.60
134.46
122.64 (CF3)
55.33 (C-100),
30.66 (C-200,600),
25.55 (C-300,500),
24.53 (C-400)
61
149.20
138.74
113.53
131.21
120.42
112.98
138.74
160.19
14.50
37.69
31.18
25.53
21.13
25.53
31.18
178.65 C]S
*Superscript is referred to both aromatic and cyclohexane ring at position 2 of benzimidazole; Superscript is referred to cyclohexane ring at position 1 of benzimidazole.

G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
93
precipitation of the oximes as crude solids, which were
recrystallized from ethanol.
4.80; N, 20.94; nmax (nujol) cmꢀ1: 3469,1731,1584; lmax (EtOH) nm:
329, 263, 204; 1H NMR ( CDCl3-DMSO-d6)
: 12.76 (1H, br s, NH
d
benzimidazole), 9.06 (1H, br s, NH), 8.18 (2H, d, J 8.2, H-30,50), 7.84
(1H, dd, J 8.2 and 1.2, H-6), 7.73 (1H, s, H-4), 7.68 (1H, d, J 8.6, H-7),
7.49 (2H, d, J 8.2, H-20,60), 6.20 (2H, br s, NH2), 2.32 (3H, s, CH3C]
Ne).
3.6.1. 1-(2-Phenyl-1H-benzimidazol-5-yl)ethanone oxime (16)
Yield: 75%; m.p. 236-231 ꢂC; Anal. calc. for C15H13N3O þ 0.5H2O:
C, 69.21; H, 5.42; N, 16.14; found: C, 69.10; H, 5.47; N, 15.37.
3.6.2. 1-(2-(4-Chlorophenyl)-1H-benzimidazol-5-yl)ethanone
3.7.3. 2-(1-(2-(4-Trifluoromethylyphenyl)-1H-benzimidazol-5-yl)
ethylidene) hydrazine carboxamide (24)
oxime (17)
Yield:
65%;
m.p.
254e257
ꢂC;
Anal.
calc.
for
Yield: 64%; Rf ¼ 0.28 (CHCl3eMeOH 9,5:0,5); m.p. 238e240 ꢂC;
Anal. calc for C17H14 F3N5O: C, 56.51; H, 3.91; N, 19.38; found: C,
56.18; H, 4.12; N, 19.20; nmax (nujol) cmꢀ1: 3417, 1655, 1566; lmax
C15H12ClN3O þ 1C2H6O: C, 61.54; H, 5.47; N, 12.66; found: C, 61.43;
H, 5.37; N,12.81. 13C NMR (DMSO-d6)
d: 153.37 (C]NOH),151.01 (C-
2), 134.67 (C-40), 131.64 (C-10), 129.12 (C- 30,50, 20,60, 3a), 128.87 (C-
6,7a) 128.19 (C-4,7), 120.37 (C-5), 11.99 (CH3).
(EtOH) nm: 327, 264, 201; 1H NMR (CDCl3-DMSO-d6)
d: 13.05 (1H,
br s, NH benzimidazole), 9.25 (1H, br s, NH), 8.39 (2H, d, J 8.4, H-
30,50), 8.07 (1H, d, J 1.4, H-4), 7.81 (2H,d, J 8.4, H-20,60), 7.74e7.45 (2H,
3.6.3. 1-(2-(4-Trifluoromethylphenyl)-1H-benzimidazol-5-yl)
ethanone oxime(18)
m, H-6 and H-7), 6.35 (2H, br s, NH2), 2.32 (3H, s, CH3C]Ne). 13
C
NMR (DMSO-d6)
d: 157.66 (C]O), 150,50 (C-2), 144.96 (C]
Yield: 76%; m.p. 277e282 ꢂC; Anal. calc. for C16H12F3N3O: C,
60.19; H, 3.79; N, 13.16; found: C, 60.08; H, 4.19; N, 12.97.
NeNHe), 133.87 (C-7a, 40), 133.36 (C-3a, 10), 130.05 (C-5), 129.42
(C-6), 127.10 (C- 4, 20,60), 126.01 (C- 30,50), 121.35 (CF3), 112.93 (C-7),
13.83 (CH3).
3.6.4. 1-(2-(4-Methoxyphenyl)-1H-benzimidazol-5-yl)ethanone
oxime(19)
3.7.4. 2-(1-(2-(4-Methoxyphenyl)-1H-benzimidazol-5 yl)
ethylidene)hydrazinecarboxamide (25)
Yield: 86%; m.p. 275e280 ꢂC; Anal. calc. for C16H15N3O2: C,
68.31; H, 5.37; N, 14.94; found: C, 68.28; H, 5.47; N, 14.87.
Yield: 23%; Rf ¼ 0.25 (CHCl3eMeOH 9,5:0,5); m.p. 127e129 ꢂC;
Anal. calc for C17H17N5O2: C, 63.15; H, 5.30; N, 21.66; found: C,
63.00; H, 5.70; N, 21.48; nmax (nujol) cmꢀ1: 1677, 1572; lmax (EtOH)
3.6.5. 1-(2-(2,4-Dimethoxyphenyl)-1H-benzimidazol-5-yl)
ethanone oxime (20)
nm: 327, 320, 266, 209; 1H NMR (CDCl3,DMSO-d6)
d: 12.60 (1H, br s,
Yield:80%; m.p. 245e250 ꢂC. Anal. calc. For C17H17N3O3: C,
65.58; H, 5.50; N, 13.50; found: C, 65.45; H, 5.70; N, 13.27.
NH benzimidazole), 9.11 (1H, br s, NH), 8.12(2H, d, J 8.0, H-30,50),
7.97 (1H, s, H-4), 7.69 (1H, d, J 8.4, H-7), 7.60e7.45 (1H, dd, J 9.2 and
1.6, H-6), 7.03 (2H, d, J 8.0, H-20,60), 6.23 (2H, br s, NH2), 2.31 (3H, s,
3.6.6. 1-(2-(2,3,4-Trimethoxyphenyl)-1H-benzimidazol-5-yl)
ethanone oxime(21)
CH3C]Ne). 13C NMR (DMSO-d6) : 160.76 (C-40), 157,50 (C]O),
d
152.13 (C-2), 145.01 (C]NeNHe), 139.50 (C-7a), 136.20 (C-3a),
132.62 (C-5), 128.11 (C-20,60), 122.34 (C-6), 120.58 (C-4), 114.45 (C-
30,50,7), 112.90 (C-10), 55.38 (OCH3), 13.82 (CH3).
Yield: 69%; m.p. 256e260 ꢂC; Anal. calc. for C18H19N3O4: C,
63.33; H, 5.61; N, 12.31; found: C, 62.89; H, 5.71; N, 12.17.
3.7. General procedure for the preparation of the semicarbazones
(22e26) and (44e52)
3.7.5. 2-(1-(2-(2,4-Dimethoxyphenyl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarboxamide (26)
Yield: 58%; Rf ¼ 0.22 (CHCl3eMeOH 9,5:0,5); m.p.186e188 ꢂC;
Anal. calc. for C18H19N5O3: C, 61.18; H, 5.42; N, 19.82; found: C,
60.94; H, 5.80; N, 19.51; nmax (nujol) cmꢀ1: 3194, 1683, 1609; lmax
To a solution of the ketone (7e12) (35e43) (1 mmol) in ethanol
(3 mL), an excess of semicarbazide hydrochloride (3.4 mmol) dis-
solved in an aqueous solution of sodium acetate (10 mmol in 10 mL
of water) was added. The mixture was refluxed for 3 h (24e26), 4 h
(22), 12 h (23) and 5 h (35e43). On cooling, the precipitates were
collected and left to dry on air. The crude products were then
purified on flash chromatography on silica gel column eluting with
a mixture of chloroform/methanol, as indicated below to give rise
to the semicarbazones (22e26) and (44e52).
(EtOH) nm: 330, 262, 204; 1H NMR (CDCl3-DMSO-d6)
d: 9.36 (1H, br
s, NH), 8.25 (1H, d, J 9.2, H-60), 8.13 (1H, s, H-4), 7.88 (1H, dd, J 8.6
and 1.4, H-6), 7.65 (1H, d, J 8.6, H-7), 6.77 (2H, s, H-50), 6.40 (2H, br s,
NH2), 4.09 (3H, s, OCH3), 3.91 (3H, s, OCH3), 2.30 (3H, s, CH3C]Ne).
3.7.6. 2-(1-(1-Cyclohexyl-2-phenyl-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarboxamide (44)
Yield: 94%. Rf ¼ 0.18 (CHCl3eMeOH 9:1); m.p. 228e230 ꢂC; Anal.
calc. for C22H25N5O: C, 70.38; H, 6.71; N, 18.65; found: C, 70.10; H,
6.84; N, 18.56; nmax (nujol) cmꢀ1: 3457, 1672, 1583; lmax (EtOH) nm:
3.7.1. 2-(1-(2-Phenyl-1H-benzimidazol-5-yl)ethylidene)
hydrazinecarboxamide (22)
Yield: 41%; Rf ¼ 0.36 (CHCl3eMeOH 9:1); m.p. 153e155 ꢂC; Anal.
calc for C16H15N5O: C, 65.52; H, 5.15; N, 23.88; found: C, 65.83; H,
5.08; N, 24.00 nmax (nujol) cmꢀ1: 3471, 1666; lmax (EtOH) nm: 323,
255, 203; 1H NMR (CDCl3)
d: 8.40 (1H, dd, J 8.0 and 1.6, H-6), 8.10
(1H, s, H-4), 7.75 (1H, d, J 8.0, H-7), 7.72e7.60 (3H, m, H-30,40,50),
7.60e7.50 (2H, m, H-20,60), 4.50e4.23 (1H, m, CH cyclohexyl), 2.31
(3H, s, CH3C]Ne), 2.08e1.18 (10H, m, cyclohexyl).
258, 206; 1H NMR (CDCl3-DMSO-d6)
d: 12.80 (1H, br s, NH benz-
imidazole), 9.17 (1H, s, NH) 8.18 (1H, dd, J 8.0 and 1.6, H-6), 7.98 (1H,
s, H-4), 7.73 (1H, d, J 8.0, H-7), 7.60e7.40 (5H, m, H-20,30,40,50,60),
6.29 (1H, br s, NH), 2.32 (3H, s, CH3C]Ne).13C NMR (DMSO-d6)
3.7.7. 2-(1-(2-(4-Chlorophenyl)-1-cyclohexyl-1H-benzimidazol-5-
yl)ethylidene)hydrazinecarboxamide(45)
d
:157.60 (C]O, hydrazide), 152.11 (C-2), 145.10 (C]NHeNH),
Yield: 51%. Rf ¼ 0.35 (CHCl3eMeOH 9.5:0.5); m.p. 212e214 ꢂC;
Anal. calc. for C22H24ClN5O: C, 64.46; H, 5.90; Cl, 8.65; N, 17.09;
found: C, 64.16; H, 6.30; Cl, 8.15; N, 17.40. nmax (nujol) cmꢀ1: 3473,
132.88 (C-10), 130.04 (C-), 129.05 (C-), 126.50 (C-5, 20,60), 120.87
(C-6), 112.92 (C-7), 13.86 (CH3).
1681, 1585; lmax (EtOH) nm: 244; 1H NMR (DMSO-d6)
d: 9.26 (1H,
3.7.2. 2-(1-(2-(4-Chlorophenyl)-1H-benzimidazol-5-yl)ethylidene)
hydrazinecarboxamide (23)
br s, NH)), 8.62 (2H, d, J 8.4, H-30,50), 8.01 (1H, s, H-4), 7.81 (1H, dd, J
8.0 and 68.13; H, 6.71; N, 17.27 1,6, H-6), 7.72 (1H, d, J 8.6, H-7), 7.56
(2H, d, J 8.4, H-20-60), 6.34 (2H, br s, NH2), 4.39e4.18 (1H, m, CH
cyclohexyl), 2.29 (3H, s, CH3C]Ne), 2.04e1.10 (10H, m, cyclohexyl).
Yield: 29%; Rf ¼ 0.31 (CHCl3eMeOH 9:1); m.p. 162e164 ꢂC; Anal.
calc for C16H14ClN5O: C, 58.63; H, 4.31; N, 21.37; found: C, 58.39; H,

94
G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
3.7.8. 2-(1-(1-Cyclohexyl-2-(4-(trifluoromethyl)phenyl)-1H-
benzimidazol-5-yl)ethylidene)hydrazine carboxamide (46)
3.7.13. 2-(1-(1-Cyclohexyl-2-(furan-3-yl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (51)
Yield: 50%. Rf ¼ 0.36 (CHCl3eMeOH 9.5:0.5); m.p. 215e218 ꢂC;
Anal. calc. for C23H24F3N5O: C, 62.29; H, 5.45; N, 15.79; found: C,
62.40; H, 5.15; N, 15.95. nmax (nujol) cmꢀ1: 3643, 1681, 1570; lmax
Yield: 38%. Rf ¼ 0,23 (EP-EA 4:6); m.p. 205e206 ꢂC; Anal. calc.
for C20H23N5OS: C, 62.97; H, 6.08; N, 18.36; found: C, 62.48; H, 6.15;
N, 18.14. nmax (nujol) cmꢀ1: 3120, 1619; lmax (EtOH) nm: 303, 232;
(EtOH) nm: 266, 227, 203; 1H NMR (CDCl3-DMSO-d6)
d
: 8.59 (1H, br
1H NMR ( CDCl3)
d: 8.77 (1H, br s, NH), 8.09 (1H, dd, J 0,8 and 1.8, H-
s, NH), 8.07 (1H, s, H-4), 7.94e7.65 (6H, m, H-6,7,20,30,50,60), 5.80
(2H, br s, NH2), 4.44e4.20 (1H, m, CH cyclohexyl), 2.33 (3H,s,
CH3C]Ne), 2.12e1.18 (10H, m, cyclohexyl).
20), 7.89 (1H, s, H-4), 7.70 (1H, dd, J 8,6 and 1.6, H-6), 7.62 (1H, d, J
8.6, H-7), 7.60 ( 1H, dd, J 1.8 and 1.6, H-40), 7.40 (1H, br s, NH), 6.80
(1H, dd, J 1,8 and 0,8, H-50), 6.36 ( 2H, br s, NH2), 4.30e4.20 (1H, m,
CH cyclohexyl), 2.39 ( 3H, s, CH3C]Ne), 2.37e1.20 (10H, m,
cyclohexyl).
3.7.9. 2-(1-(1-Cyclohexyl-2-(4-methoxyphenyl)-1H-benzimidazol-
5-yl)ethylidene)hydrazine carboxamide (47)
Yield: 83%. Rf ¼ 0.35 (CHCl3eMeOH 9.5:0.5); m.p. 213e215 ꢂC;
Anal. calc. for C23H27N5O2: C, 68.13; H, 6.71; N, 17.27; found: C,
68.31; H, 6.98; N, 17.20. nmax (nujol) cmꢀ1: 3468, 1679, 1583; lmax
3.7.14. 2-(1-(1-Cyclohexyl-2-(pyridin-2-yl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (52)
Yield: 83%. Rf ¼ 0,27 (CHCl3eMeOH 9.8:0,2); m.p. 194e196 ꢂC;
Anal. calc. for C21H24N6S: C, 64.26; H, 6.16; N, 21.41; found: C,
64.06; H, 6.24; N, 21.18. nmax (nujol) cmꢀ1: 3140, 1611; lmax (EtOH)
(EtOH) nm: 258, 202; 1H NMR (CDCl3-DMSO-d6)
d: 9.23 (1H, br s,
NH), 7.98 (1H, s, H-4), 7.78 (1H, dd, J 8.0 and 1.6, H-6), 7.67 (1H, d, J
8.0, H-7), 7.58 ( 2H, d, J 8.2, H-30,50), 7.09 (2H, d, J 8.2, H-20-60), 6.34
(2H, br s, NH2), 4.45e4.20 (1H, m, CH cyclohexyl), 3.89 (3H, s,
OCH3), 2.30 (3H, s, CH3C]Ne), 2.02e1.20 (10H, m, cyclohexyl).
nm: 307; 1H NMR ( CDCl3)
d: 8.80 (1H, br s, NH), 8.72 (1H, dd, J 4,8
and 1.8, H-30), 8.22 (1H, dd, J 8.0 and 1.4, H-6), 8.14 (1H, s, H-4),
7.93e7.80 ( 1H, m, H-50), 7.80e7.68 (1H, m, H-7), 7.50e7.35 (2H, m,
H-40,60), 6.42 (1H, br s, NH), 5.60e5.40 (1H, m, CH cyclohexyl), 2.38
(3H, s, CH3C]Ne), 2.37e1.20 (10H, m, cyclohexyl).
3.7.10. 2-(1-(1-Cyclohexyl-2-(2,4-dimethoxyphenyl)-1H-
benzimidazol-5-yl)ethylidene)hydrazine carboxamide (48)
Yield: 79%.Rf ¼ 0.26 (CHCl3eMeOH 9,5:0,5); m.p. 224e226 ꢂC;
Anal. calc. for C24H29N5O3: C, 66.19; H, 6.71; N, 16.08; found: C,
65.91; H, 6.77; N, 16.02. nmax (nujol) cmꢀ1: 3483, 1683, 1591; lmax
3.8. General procedure for the preparation of the
thiosemicarbazones (27e31) and (53e61)
(EtOH) nm: 284, 253, 203; 1H NMR (CDCl3)
d
: 8.05 (1H, s, H-4),
To a stated amount of ketone (7e12) (35e43) (42 mmole) in
ethanol (10 mL), a slight excess of thiosemicarbazide (44 mmole)
dissolved in a mixture of water (12 mL) and glacial acetic acid
(0.8 mL) was added. The mixture was the refluxed for 1 h (27e30),
6 h (31, 40, 59), 9 h (58), 12 h (60, 61) under stirring. On cooling, the
precipitate formed was collected and thoroughly washed with
water. The crude products, as amorphous powder coloured from
cream-yellow to brown, were recrystallized from ethanolewater.
7.70 (1H,dd, J 8.0 and 1.6, H-6), 7.60 (1H, d, J 8.0, H-7), 7.42 (1H, dd,
J 8.4 and 2.4, H-40), 6.61 (1H, dd, J 8.2 and 2.0, H-50), 6.57 (1H, d, J
2.0, H-30), 3.90 (3H, s, OCH3), 3.78 (3H, s, OCH3), 2.30 (3H, s,
CH3C]Ne), 2.22e1.20 (10H, m, cyclohexyl). 13C NMR (DMSO-d6)
d
:162.07 (C]O), 158.27 (C-40), 157.51 (C-20), 151.77 (C-2), 145.04
(C]NeNH), 143.61 (C-7a), 133.60 (C-3a), 132.78 (C-5), 131.92 (C-
6), 120.13 (C-4), 117.15 (C-7), 112.13 (C-60), 111.98 (C-30), 105.51 (C-
10), 98.30 (C-50), 56.74 (C-100 cyclohexane), 55.45 (20-OCH3),
55.32(40-OCH3), 30.66 (C-200,600), 25.64 (C-300,500), 24.68 (C-400),
13.80 (CH3).
3.8.1. 2-(1-(2-Phenyl-1H-benzimidazol-5-yl)ethylidene)
hydrazinecarbothioamide (27)
Yield: 77%; Rf ¼ 0.35 (CHCl3eMeOH 9.5:0.5); m.p. 180e185 ꢂC;
Anal. calc. for C16H15N5S þ H2O: C 58.64; H, 5.19; N, 21.38; found:
C, 58.39; H, 4.91; N, 20.92. nmax (nujol) cmꢀ1: 3418, 1588; lmax
3.7.11. 2-(1-(1-Cyclohexyl-2-(furan-3-yl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarboamide (49)
Yield: 83%. Rf ¼ 0.54 (CHCl3eMeOH 9:1); m.p. 270e272 ꢂC; Anal.
calc. for C20H23N5O2: C, 65.73; H, 6.34; N, 19.16; found: C, 65.80; H,
6.21; N, 18.94. nmax (nujol) cmꢀ1: 3469, 1680, 1584; lmax (EtOH) nm:
(EtOH) nm: 331, 258, 204; 1H NMR (CDCl3-DMSO-d6)
d: 10.10 (1H,
br s, NH), 8.20 (1H, dd, J 8.0 and 1.6, H-6), 8.06 (1H, s, H-4),
7.90e7.82 (3H, m, H-30-40-50), 7.72 (2H, br s, NH2), 7.64 (1H, d, J 8.0,
H-7), 7.68e7.44 (2H, m, H-20-60), 2.43 (3H, s, CH3CO).
295, 252, 206; 1H NMR (CDCl3)
d: 8.30 (1H, br s, NH), 8.08 (1H, dd, J
1.8 and 0.8, H-20), 7.89 (1H, s, H-4), 7.69 (1H, dd, J 8.4 and 1.6, H-6),
7.63 (1H, dd, J 1.8 and 1.6, H-40), 7.60 (1H, d, J 8.6, H-7 partially
obscured), 6.80 (1H, dd, J 1.8 and 0.8, H-50), 6.19 (1H, br s, NH), 5.14
(2H, br s, NH2), 4.61e4.37 (1H, m, CH cyclohexyl), 2.31 (3H,s,
CH3C]Ne), 2.18e1.20 (10H, m, cyclohexyl).
3.8.2. 2-(1-(2-(4-Chlorophenyl)-1H-benzimidazol-5-yl)ethylidene)
hydrazinecarbothioamide (28)
Yield: 63%; Rf ¼ 0.35 (CHCl3eMeOH 9.5:0.5); m.p. 200e205 ꢂC;
Anal. calc. for C16H14ClN5S; C, 55.89; H, 4.10; N, 20.37; found: C,
55.63; H, 4.60; N, 19.82. nmax (nujol) cmꢀ1: 3421, 1589; lmax (EtOH)
3.7.12. 2-(1-(1-Cyclohexyl-2-(pyridin-2-yl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarboxamide (50)
nm: 332, 261, 202; 1H NMR (CDCl3-DMSO-d6)
d: 10.16 (1H, br s, NH),
8.20 (1H, d, J 8.6, H-7), 8.12 (1H, s, H-4), 7.96 (1H, dd, J 8.6 and 1,4, H-
6), 7.80 (2H, br s, NH2), 7.64 (2H, d, J 9. H-30,50), 7.60 (2H, d, J 8.8, H-
20,60), 2.43 (3H, s, CH3CO).
Yield: 83%. Rf ¼ 0.42 (CHCl3eMeOH 9,5:0,5); m.p. 185e188 ꢂC;
Anal. calc. for C21H24N6O: C, 67.00; H, 6.43; N, 22.32.Found: C,
67.15; H, 6.73; N, 22.11. nmax (nujol) cmꢀ1: 3848, 1681, 1580; lmax
(EtOH) nm: 271, 232, 201; 1H NMR (CDCl3)
d
: 8.72 (1H, dd, J 4.0 and
3.8.3. 2-(1-(2-(4-Trifluoromethylphenyl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (29)
1.8, H-30), 8,22 (1H, d0d, J 8.0 and 1.2, H-6), 8.12 (1H, d, J 8.0, H-7),
7.93e7.80 (1H, m, H-5 ), 7.72 (1H, s, H-4), 7.42e7.35 (1H, m, H-40-60),
5.60e5.40 (1H, m, CH cyclohexyl), 2.32 (3H, s, CH3C]Ne),
Yield: 64%; Rf ¼ 0.33 (CHCl3eMeOH 9.5:0.5); m.p.190e193 ꢂC;
Anal. calc. for C17H14F3N5S: C, 54.10; H, 3.74; N, 18.56; found; C,
54.19; H, 3.85; N, 18.70; nmax (nujol) cmꢀ1: 3387, 1619; lmax (EtOH)
2.18e1.20 (10H, m, cyclohexyl). 13C NMR (DMSO-d6)
d: 157.46
(C]O), 150.85 (C-20 pyridinyl), 150.32 (C-60), 148.96 (C]NeNH),
144.80 (C-2), 142.95 (C-3a), 137.55 (C-7a), 134.76 (C-5), 132.74 (C-
40), 125.35 (C-30), 124.40 (C-50), 121.22 (C-4), 117.68 (C-7), 113.10 (C-
6), 56.45 (C-100 cyclohexane), 30.73 (C-200,600), 25.78 (C-300,500), 24.72
(C-400), 13.76 (CH3).
nm: 327, 262, 204; 1H NMR (CDCl3)
d: 10.07 (1H, br s, NH), 8.40
(1H,dd, J 8.0 and 1.2, H-6), 8.30 (1H, s, H-4), 8.00 (1H, d, J 8.0, H-70),
7.85 (2H, d, J 8.6, H-30,50), 7.74 (2H, br s, NH2), 7.61 (2H, d, J 8.6, H-2 -
60), 2.42 (3H, s, CH3C]Ne). 13C NMR (DMSO-d6)
d
: 178.81 (C]S),
151.71 (C-2), 148.50 (C]NeNH), 138.14 (C-7a), 137.78 (C-3a), 133.04

G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
95
(C-10), 131.00 (CF3), 129.25 (C-30,50), 128.18 (C-5), 126.93 (C-20,60),
3.8.9. 1-Cyclohexyl-2-(1-(2-(4-methoxyphenyl)-1H-benzimidazol-
122.29 (C-40), 114.27 (C-6), 113.60 (C-7), 112.97 (C-4), 14.50 (CH3).
5-yl)ethylidene)hydrazine carbothioamide (56)
Yield: 86%. Rf ¼ 0.39 (PE-EA 3:7); m.p. 250e252 ꢂC; Anal. calc.
for C23H27N5OS: C, 65.53; H, 6.46; N, 16.61; found: C, 65.67; H, 6.36;
N, 16.80; nmax (nujol) cmꢀ1: 3413, 1610; lmax (EtOH) nm: 319, 259,
3.8.4. 2-(1-(2-(4-Methoxyphenyl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (30)
Yield: 55%; Rf ¼ 0.35 (CHCl3eMeOH 9.5:0.5); m.p.230e235 ꢂC;
Anal. calc. for C17H17N5OS: C, 60.16; H, 5.05; N, 20.63; found: C,
59.95; H, 4.82; N, 21.10; nmax (nujol) cmꢀ1: 3434, 1608; lmax (EtOH)
203; 1H NMR (CDCl3)
d: 8.76 (1H, br s, NH), 8.11 (1H, s, H-4), 7.74
(1H, dd, J 8.0 and 1.6, H-6), 7.65 (1H, d, J 8.6, H-7), 7.60 (2H, d, J 8.8,
H-30,50), 7.41 (1H, br s, NH), 7.05 (2H, d, J 8.8, H-20-60), 6.34 (2H, br s,
NH2), 4.42e4.24 (1H, m, CH cyclohexyl), 3.90 (3H, s, OCH3), 2.38
(3H, s, CH3C]Ne), 2.38e1.25 (10H, m, cyclohexyl). LC/MS: 422
(M þ 1).
nm: 333, 265, 202; 1H NMR (DMSO-d6)
d: 10.31 (1H, br s, NH), 8.29
(1H, s, H-4), 8.18 (1H, dd, J 8.0 and 1.6, H-6), 8.10 (1H, d, J 8.0, H-7),
7.99 (2H, br s, NH2), 7.80 (2H, br s, NH2), 7.68 (2H, d, J 8.0, H-30,50),
7.25 (2H, d, J 8.8, H-20,60), 3.89 (3H, s, OCH3), 2.40 (3H,s, CH3C]Ne).
3.8.10. 1-Cyclohexyl-2-(1-(2-(2,4-dimethoxyphenyl)-1H-
3.8.5. 2-(1-(2-(2,4-Dimethoxyphenyl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (31)
benzimidazol-5-yl)ethylidene)hydrazine carbothioamide (57)
Yield: 86%. Rf ¼ 0.25 (EP-EA 3:7); m.p. 134e136 ꢂC; Anal. calc. for
C24H29N5O2S: C, 63.83; H, 6.47; N, 15.51; found: C, 63.54; H, 6.77; N,
15.42. nmax (nujol) cmꢀ1: 3583, 1614; lmax (EtOH) nm: 319, 253,
Yield: 53%; Rf ¼ 0.35 (PE-EA 2:8); m.p. 175e178 ꢂC; Anal. calc. for
C18H19N5O2S: C, 58.52; H, 5.18; N, 18.96; found: C, 58.60; H, 5.32; N,
18.64; nmax (nujol) cmꢀ1: 3628, 1611; lmax (EtOH) nm: 338, 262,
203; 1H NMR (CDCl3)
d: 8.76 (1H, br s, NH), 8.11 (1H, s, H-4), 7.70
205; 1H NMR (DMSO-d6)
d: 10.19 (1H, br s, NH), 8.24 (1H, d, J 9.0, H-
(1H, dd, J 8.2 and 1.4, H-6), 7.62 (1H, d, J 8.2, H-7), 6.62 (1H, dd, J 8.6
and 2.4, H-50), 6.57 (1H, d, J 2,0, H-30), 6.36 (2H, br s, NH2), 4.10e3.90
(1H, m, CH cyclohexyl), 3.90 (3H, s, OCH3), 3.79 (3H, s, OCH3), 2.37
(3H, s, CH3C]Ne), 2.34e1.20 (10H, m, cyclohexyl).
60), 8.05 (1H, s, H-4), 7.89 (1H, dd, J 8.6 and 1.4, H-6), 7.56 (1H, d, J
8.0, H-7), 6.76 (1H, s, H-30), 6.72 (1H, dd, J 9.0 and 1.6, H-50), 4.02
(3H, s, OCH3), 3.86 (3H, s, OCH3), 2.37 (3H, s, CH3C]Ne).
3.8.6. 1-Cyclohexyl-2-(1-(2-phenyl-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (53)
3.8.11. 2-(1-(1-Cyclohexyl-2-(furan-3-yl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (58)
Yield: 81%. Rf ¼ 0.18 (EP-EA 1:1); m.p. 217e219 ꢂC; Anal. calc.
for C22H25N5S: C, 67.49; H, 6.44; N, 17.89; found: C, 67.24; H, 6.59;
N, 17.56. nmax (nujol) cmꢀ1: 3583, 1603; lmax (EtOH) nm: 319, 251,
Yield: 38%. Rf ¼ 0.23 (EP-EA 4:6); m.p. 205e206 ꢂC; Anal. calc.
for C20H23N5OS: C, 62.97; H, 6.08; N, 18.36; found: C, 62.48; H, 6.15;
N, 18.14. nmax (nujol) cmꢀ1: 3120, 1619; lmax (EtOH) nm: 303, 232;
201; 1H NMR (CDCl3)
d
: 8.75 (1H, br s, NH), 8.13 (1H, s, H-4), 7.73
1H NMR (CDCl3)
d: 8.77 (1H, br s, NH), 8.09 (1H, dd, J 0.8 and 1.8, H-
(1H, dd, J 8.0 and 1.6, H-6), 7.65 (1H, d, J 8.0, H-7), 7.60e7.50 (5H,
m, H-20,30,40,50,60), 7.41 (1H, br s, NH), 6.34 (2H, br s, NH2),
4.45e4.32 (1H, m, CH cyclohexyl), 2.38 (3H, s, CH3C]Ne),
20), 7.89 (1H, s, H-4), 7.70 (1H, dd, J 8.6 and 1.6, H-6), 7.62 (1H, d, J
8.6, H-7), 7.60 (1H, dd, J 1.8 and 1.6, H-40), 7.40 (1H, br s, NH), 6.80
(1H, dd, J 1.8 and 0.8, H-50), 6.36 (2H, br s, NH2), 4.30e4.20 (1H, m,
CH cyclohexyl), 2.39 (3H, s, CH3C]Ne), 2.37e1.20 (10H, m,
cyclohexyl).
2.35e1.34 (10H, m, cyclohexyl). 13C NMR (DMSO-d6)
d :178.69
(C]S), 153.99 (C-2), 148.76 (C]NeNH), 143.31 (C-3a), 134.45 (C-
7a), 131.69 (C-40), 130.60 (C-10), 129.78 (C-5), 129.30 (C-30,50),
128.76 (C-20,60), 121.05 (C-6), 118.21 (C-4), 112.55 (C-7), 56.61 (C-100
cyclohexane), 30.68 (C-200,600), 25.53 (C-300,500), 24.50 (C-400), 14.42
(CH3).
3.8.12. 2-(1-(1-Cyclohexyl-2-(pyridin-2-yl)-1H-benzimidazol-5-yl)
ethylidene)hydrazinecarbothioamide (59)
Yield: 83%. Rf ¼ 0.27 (CHCl3eMeOH 9.8:0.2); m.p. 194e196 ꢂC;
Anal. calc. for C21H24N6S: C, 64.26; H, 6.16; N, 21.41; found: C,
64.06; H, 6.24; N, 21.18. nmax (nujol) cmꢀ1: 3140, 1611; lmax (EtOH)
3.8.7. 1-Cyclohexyl-2-(1-(2-(4-chlorophenyl)-1H-benzimidazol-5-
yl)ethylidene)hydrazinecarbothio amide (54)
nm: 307; 1H NMR (CDCl3)
d: 8.80 (1H, br s, NH), 8.72 (1H, dd, J 4.8
Yield: 83%. Rf ¼ 0.29 (EP-EA 6:4); p.f. 228e230 ꢂC; Anal. calc. for
C22H24 ClN5S: C, 62.03; H, 5.68; Cl, 8.32; N,16.44; found: C, 61.85; H,
5.90; Cl, 8.20; N, 16.19. nmax (nujol) cmꢀ1: 3583, 1602; lmax (EtOH)
and 1.8, H-30), 8.22 (1H, dd, J 8.0 and 1.4, H-6), 8.14 (1H, s, H-4),
7.93e7.80 (1H, m, H-50), 7.80e7.68 (1H, m, H-7), 7.50e7.35 (2H, m,
H-40,60), 6.42 (1H, br s, NH), 5.60e5.40 (1H, m, CH cyclohexyl), 2.38
(3H, s, CH3C]Ne), 2.37e1.20 (10H, m, cyclohexyl).
nm: 319, 280, 254, 202; 1H NMR (CDCl3)
d: 8.73 (1H, br s, NH), 8.11
(1H, s, H-4), 7.74 (1H, dd, J 8.0 and 1.6, H-6), 7.65 (1H, d, J 8.6, H-7),
7.60 (2H, d, J 9.0, H-30,50), 7.52 (2H, d, J 8.6, H-20-60), 7.40 (1H, br s,
NH), 6.34 (2H, br s, NH2), 4.42e4.24 (1H, m, CH cyclohexyl), 2.38
(3H, s, CH3C]Ne), 2.38e1.25 (10H, m, cyclohexyl).
3.8.13. 2-(1-(2-(Furan-3-yl)-1H-benzimidazol-5-yl)ethylidene)
hydrazinecarbothioamide (60)
Yield: 80%; Rf ¼ 0.23 (PE-EA 3:7); m.p.159e162 ꢂC; Anal. calc. for
C14H13N5OS: C, 56.17; H, 4.38; N, 23.40; found: C, 56.41; H, 4.19; N,
23.10. nmax (nujol) cmꢀ1: 3433, 1613. lmax (EtOH) nm: 327, 319, 250,
3.8.8. 1-Cyclohexyl-2-(1-(2-(4-trifluoromethylphenyl)-1H-
benzimidazol-5-yl)ethylidene)hydrazine carbothioamide (55)
Yield: 86%. Rf ¼ 0.29 (EP-EA 6:4); m.p. 228e230 ꢂC; Anal. calc.
for C23H24F3N5S: C, 60.11; H, 5.26; N, 15.24; found: C, 60.34; H, 5.77;
N, 15.41. nmax (nujol) cmꢀ1: 3392, 1612; lmax (EtOH) nm: 327, 313,
205. 1H NMR (CDCl3-DMSO-d6)
d: 9.94 (1H, s, NH), 8.31 (1H, dd, J 1.8
and 0.8, H-20), 8.0 (2H, br s, NH2), 7.95 (1H, s, H-4), 7,76 (1H, dd, J 8.2
and 1.6, H-6), 7.61 (1H, dd, J 1.8 and 0.8, H-40), 7.52 (1H, d, J 8.2, H-7),
7.01 (1H, dd, J 1.8 and 0.8, H-50), 2.42 (3H, s, CH3C]Ne).
202; 1H NMR (CDCl3)
d: 8.80 (1H, br s, NH), 8.14 (1H, s, H-4), 7.85
(1H, dd, J 9.2 and 1.6, H-6), 7.80 (1H, d, J 9, H-7), 7.75 (2H, d, J 8.8, H-
30,50), 7,66 (2H, d, J 8.8, H-20-60), 7.41 (1H, br s, 1H, NH2), 6.46 (1H, br
s, 1H, NH2), 4.42e4.24 (1H, m, CH cyclohexyl), 2.38 (3H,s, CH3C]
Ne), 2.34e1.25 (10H, m, cyclohexyl). 13C NMR (DMSO-d6)
3.8.14. 2-(1-(2-Cyclohexyl-1H-benzimidazol-5-yl)ethylidene)
hydrazinecarbothioamide (61)
Yield: 75%. Rf ¼ 0.27 (EP-EA 3:7); m.p. 145e148 ꢂC. Anal. calc. for
C16H21N5S: C, 60.92; H, 6.71; N, 22.20; found: C, 61.06; H, 6.51; N,
22.02. nmax (nujol) cmꢀ1: 3370,1589. lmax (EtOH) nm: 317, 225, 204;
d
:178.67 (C]S), 160.30 (C-2), 153.99 (C]NeNH), 148.83 (C-3a),
143.25 (C-7a), 134.46 (C-10), 131.56 (C-40), 130.77 (C-20,60), 122.64 (
CF3), 120.82 (C-5), 117.96 (C-6), 114.20 (C-30,50), 114.90 (C-4), 112.45
(C-7), 55.33 (C-100), 30.66 (C-200,600), 25.55 (C-300,500), 24.53 (C-400),
14.42 (CH3).
1H NMR (CDCl3-DMSO-d6)
d: 10.0 (1H, br s, NH), 8.14 (1H, s, H-4),
8.06 (1H, br s, NH), 7.73 (1H, dd, J 8.6 and 1.6, H-6), 7.65 (2H, br s,
NH2), 7.44 (1H, d, J 8.6, H-7), 2.88 (1H, m, CH cyclohexyl), 2.39 (3H,
s, CH3C]Ne), 2.18e1.25 (10H, m, cyclohexyl). 13C NMR (DMSO-d6)

96
G. Vitale et al. / European Journal of Medicinal Chemistry 53 (2012) 83e97
d
:178.65 (C]S), 160.19 (C]N), 149.20 (C-2), 138.74 (C-3a,7a), 131.21
in Dulbecco’s Modified Eagle Medium (D-MEM) with L-glutamine
(C-5), 120 (C-6), 113.53 (C-4), 112.98 (C-7), 37.69 (C-100), 31.18 (C-
and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures
were then incubated at 37 ꢂC in a humidified, 5% CO2 atmosphere in
the absence or presence of serial dilutions of test compounds. Cell
viability was determined after 48e96 h at 37 ꢂC by the crystal violet
staining method.
200,600), 21.13 (C-400), 14.50 ( CH3).
4. Biology
4.1. Test compounds
4.4. Antiviral assays
Compounds were dissolved in DMSO at 100 mM, and then
diluted in culture medium.
Compound’s activity against HIV-1 was based on inhibition of
virus-induced cytopathogenicity in MT-4 cell acutely infected with
4.2. Cells and viruses
a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50
containing 1 ꢄ 10 [4] MT-4 cells were added to each well of flat-
bottom microtitre trays, containing 50 L of RPMI without or
with serial dilutions of test compounds. Then, 20 L of a HIV-1
mL of RPMI
Cell lines were purchased from American Type Culture Collec-
tion (ATCC). The absence of mycoplasma contamination was
checked periodically by the Hoechst staining method. Cell lines
supporting the multiplication of RNA and DNA viruses were the
following: CD4þ human T-cells containing an integrated HTLV-1
genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATCC CCL
22 (NBL-1) Bos taurus]; Baby Hamster Kidney (BHK-21) [ATCC CCL
10 (C-13) Mesocricetus auratus] and Monkey kidney (Vero 76) [ATCC
CRL 1587 Cercopithecus aethiops]. Viruses were purchased from
American Type Culture Collection (ATCC), with the exception of
Yellow Fever Virus (YFV), and Human Immunodeficiency Virus
type-1 (HIV-1). Viruses representative of positive-sense, single-
stranded RNAs (ssRNAþ) were: (i) Retroviridae: the IIIB laboratory
strain of HIV-1, obtained from the supernatant of the persistently
infected H9/IIIB cells (NIH 1983); (ii) Flaviviridae: yellow fever virus
(YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)] and bovine
viral diarrhoea virus (BVDV) [strain NADL (ATCC VR-534)]; (iii)
Picornaviridae: human enterovirus B [coxsackie type B5 (CVB-5),
strain Ohio-1 (ATCC VR-29)], and human enterovirus C [poliovirus
type-1 (Sb-1), Sabin strain Chat (ATCC VR-1562)]. Viruses repre-
sentative of negative-sense, single-stranded RNAs (ssRNA-) were:
(iv) Paramyxoviridae: human respiratory syncytial virus (RSV)
[strain A2 (ATCC VR-1540)]; (v) Rhabdoviridae: vesicular stomatitis
virus (VSV) [lab strain Indiana (ATCC VR 1540)]. The virus repre-
sentative of double-stranded RNAs (dsRNA) Reoviridae was
reovirus type-1 (Reo-1) [simian virus 12, strain 3651 (ATCC VR-
214)]. DNA virus representatives were: (vi) Poxviridae: vaccinia
virus (VV) [vaccine strain ElstreeeLister (ATCC VR-1549)]; (vii)
Herpesviridae: human herpes 1 (HSV-1) [strain KOS (ATCC VR-
1493)].
m
m
suspension containing 100 CCID50 were added. After a 4-day
incubation at 37 ꢂC, cell viability was determined by the MTT
method. Compound’s activity against YFV and Reo-1 was based on
inhibition of virus-induced cytopathogenicity in BHK-21 cells
acutely infected with a m.o.i. of 0.01. Compound’s activity against
BVDV was based on inhibition of virus-induced cytopathogenicity
in MDBK cells acutely infected with a m.o.i. of 0.01. Briefly, BHK and
MDBK cells were seeded in 96-well plates at a density of 5 ꢄ 10 [4]
and 3 ꢄ 10 [4] cells/well, respectively, and were allowed to form
confluent monolayers by incubating overnight in growth medium
at 37 ꢂC in a humidified CO2 (5%) atmosphere. Cell monolayers were
then infected with 50
medium [MEM-Earl with
0.025 g/L kanamycin, supplemented with 0.5% inactivated FBS] to
give an m.o.i of 0.01. After 1 h, 50 L of maintenance medium,
mL of a proper virus dilution in maintenance
L-glutamine, 1 mM sodium pyruvate and
m
without or with serial dilutions of test compounds, were added.
After a 3-/4-day incubation at 37 ꢂC, cell viability was determined
by the MTT method.
Compound’s activity against CVB-5, Sb-1, VV, HSV-1 and RSV was
determined by plaque reduction assays in infected cell monolayers.
To this end, Vero 76-cells were seeded in 24-well plates at a density
of 2 ꢄ 10 [5] cells/well and were allowed to form confluent mono-
layers by incubating overnight in growth medium [Dulbecco’s
Modified Eagle Medium (D-MEM) with
-glucose and 0.025 g/L kanamycin, supplemented with 10% FBS] at
37 ꢂC in a humidified CO2 (5%) atmosphere. Then, monolayers were
infected for 2 h with 250 L of proper virus dilutions to give 50e100
PFU/well. Following removal of unadsorbed virus, 500 L of main-
tenance medium [D-MEM with -glutamine and 4500 mg/L
L-glutamine and 4500 mg/L
D
m
m
L
D-
4.3. Cytotoxicity assays
glucose, supplemented with 1% inactivated FBS] containing 0.75%
methylecellulose, without or with serial dilutions of test
compounds, were added. Cultures were incubated at 37 ꢂC for 2 (Sb-
1 and VSV), 3 (CVB-5, VV and HSV-1) or 5 days (RSV) and then fixed
with PBS containing 50% ethanol and 0.8% crystal violet, washed
and air-dried. Plaques were then counted.
Cytotoxicity assays were run in parallel with antiviral assays.
Exponentially growing MT-4 cells were seeded at an initial density
of 1 ꢄ 10 [5] cells/ml in 96-well plates in RPMI-1640 medium,
supplemented with 10% foetal bovine serum (FBS), 100 units/mL
penicillin G and 100 mg/mL streptomycin. Cell cultures were then
incubated at 37 ꢂC in a humidified, 5% CO2 atmosphere, in the
absence or presence of serial dilutions of test compounds. Cell
viability was determined after 96 h at 37 ꢂC by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
method [35]. MDBK and BHK cells were seeded in 24-well plates at
an initial density of 6 ꢄ 10 [5] and 1 ꢄ 10 [6] cells/mL, respectively,
4.5. Linear regression analysis
The extent of cell growth/viability and viral multiplication, at
each drug concentration tested, were expressed as percentage of
untreated controls. Concentrations resulting in 50% inhibition (CC50
or EC50) were determined by linear regression analysis.
in Minimum Essential Medium with Earle’s salts (MEM-E), L-
glutamine, 1 mM sodium pyruvate and 25 mg/L kanamycin, sup-
plemented with 10% horse serum (MDBK) or 10% foetal bovine
serum (FBS) (BHK). Cell cultures were then incubated at 37 ꢂC in
a humidified, 5% CO2 atmosphere in the absence or presence of
serial dilutions of test compounds. Cell viability was determined
after 48e96 h at 37 ꢂC by the MTT method. Vero-76 cells were
seeded in 24-well plates at an initial density of 4 ꢄ 10 [5] cells/mL,
4.6. HCV replicon assays
A human hepatoma cell line (Huh-7) bearing the HCV genotype
1b replicon (GS4.1 cells), kindly provided by C. Seeger (Fox Chase
University, Philadelphia, PA, USA) through Idenix Pharmaceuticals,
was grown in D-MEM supplemented with 10% FBS, 2 mM
mine, 110 mg/L sodium pyruvate, 0.1 mM non-essential amino
L-gluta-

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acetoneemethanol, washed twice with PBS containing 0.1% Tween
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containing 10% FBS. After further washing as above, the reaction
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a sigmoidal non-linear regression analysis based on four parame-
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