Styrylbenzimidazoles. Synthesis and biological activity - part 3

Article abstract of DOI:10.2174/157340610791321514

As a follow up of an anti-Flaviviridae project, a new series of variously substituted 2-styryl-benzimidazoles were synthesized and tested in vitro for biological activity. Compounds were tested in cell-based assays against viruses representative of: i) two of the three genera of the Flaviviridae family, i.e. Pestiviruses and Flaviviruses; ii) other RNA virus families, such as Retroviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae; iii) two DNA virus families (Herpesviridae and Poxviridae) as well as for cytotoxicity tests, run in parallel with antiviral assays,against MDBK, BHK and Vero 76 cells. In the series examined, new leads emerged against BVDV, CVB-2 and RSV. Compounds 11, 12, 17, 18, 24, 31 exhibited anti-BVDV activity in the concentration range 1.7-16 μM; among them, compound 17 was the most active, with an EC₅₀ = 1.7 μM. Compounds 18 and 21 were equally active against CVB-2, with EC₅₀ values of 7 - 8 μM, while the derivative 30 was active against RSV with EC₅₀= 1 μM and represents a new lead compound.

Full text of DOI:10.2174/157340610791321514

70
Medicinal Chemistry, 2010, 6, 70-78
Styrylbenzimidazoles. Synthesis and Biological Activity - Part 3
Gabriella Vitalea, Paola Coronaa, Mario Lorigaa, Antonio Cartaa, Giuseppe Pagliettia,*,
Cristina Ibbab, Gabriele Gilibertib, Roberta Loddob, Esther Marongiub and Paolo La Collab,*
b
aDipartimento Farmaco Chimico Tossicologico, University of Sassari, Via Muroni,23-07100 Sassari, Italy; Diparti-
mento di Scienze e Tecnologie Biomediche, Sez. di Microbiologia e Virologia Generale e Biotecnologie Microbiche,
Università di Cagliari, Cittadella Universitaria, SS 554 Km 4,500, 09042 Monserrato (CA), Italy
Abstract: As a follow up of an anti-Flaviviridae project, a new series of variously substituted 2-styryl-benzimidazoles
were synthesized and tested in vitro for biological activity.
Compounds were tested in cell-based assays against viruses representative of: i) two of the three genera of the Flaviviri-
dae family, i.e. Pestiviruses and Flaviviruses; ii) other RNA virus families, such as Retroviridae, Picornaviridae, Pa-
ramyxoviridae, Rhabdoviridae and Reoviridae; iii) two DNA virus families (Herpesviridae and Poxviridae) as well as for
cytotoxicity tests, run in parallel with antiviral assays,against MDBK, BHK and Vero 76 cells.
In the series examined, new leads emerged against BVDV, CVB-2 and RSV. Compounds 11, 12, 17, 18, 24, 31 exhibited
anti-BVDV activity in the concentration range 1.7-16 μM; among them, compound 17 was the most active, with an EC50
=
1.7μM. Compounds 18 and 21 were equally active against CVB-2, with EC50 values of 7 – 8μM, while the derivative 30
was active against RSV with EC50= 1 μM and represents a new lead compound.
Keywords: Antiviral activity, flaviridae, benzimidazoles.
INTRODUCTION
R=5,6-diMe and R1=NH2 resulted the most potent against
both human haematologic and solid tumor cell lines; interest-
ingly, this compound turned out to be as potent as Etoposide
and more potent than 6-mercaptopurine (6-MP), which were
used as reference antiproliferative agents. Analogously, in
the series 2, compounds bearing (R=Me; R1=NH2) and
(R=Cl; R1=NH2) showed a potency similar to that of both 6-
MP and etoposide. In addition, in some other compounds of
both series an antiviral activity emerged at concentrations
not cytotoxic for uninfected cells. In particular, compounds
1a-c were active against YFV, with EC50s in the range 6-27
μM [1], whereas compound 2d was the most active against
the YFV (EC50 = 0.5 μM), and also exhibited good activity
against CVB-2 (EC50 = 1 μM) (Fig. 1) [2].
In our ongoing research program on benzimidazole de-
rivatives, some of which were designed as antiproliferative
and some as antiflaviviridae agents, we described so far two
distinct series of compounds related to structures 1[1] and 2
[2].
N
R
R1
N
H
1
N
R
R1
N
N
H
R1
R
N
H
2
R=H; 5-Me; 5,6-diMe, 5-Cl; 5,6-
diCl; 5-CF3; 5-COCH3;
R1= H; NO2; NH2; NHCOCH3;
YFV (EC50 μM)
1a, 5,6-diMe; R1=H
1b, 5-Cl; R1= NH2
1c, 5,6-diCl; R1=NH2
6
27
16
In this context we observed, whatever substituent was
considered, an increase of antiproliferative activity in some
compounds of the series 1, among which the derivative with
N
R
R1
N
H
*Address correspondence to these authors at the Dipartimento Farmaco
Chimico Tossicologico, University of Sassari, Via Muroni,23-07100
Sassari, Italy; Tel: +39 079 228704; Fax: +39 079 228720;
2d, R=COCH3; R1=NO2
E-mail paglietti@uniss.it; Dipartimento di Scienze
e
Tecnologie
YFV (EC50 =0.5μM); CVB-2(EC50 =1 μM)
Biomediche, Sez. di Microbiologia e Virologia Generale e Biotecnologie
Microbiche, Università di Cagliari, Cittadella Universitaria, SS 554 Km
4,500, 09042 Monserrato (CA), Italy; Tel: +39 070 6754147;
Fax: +39 070 6754210; E-mail placolla@unica.it.
Fig. (1). Reported antiviral activity for previously described ben-
zimidazoles.
1573-4064/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

Styrylbenzimidazoles. Synthesis and Biological Activity - Part 3
Medicinal Chemistry, 2010, Vol. 6, No. 2 71
These findings were considered most encouraging to af-
ford the screening of new benzimidazole series that were
designed to lead to lower cytotoxicity and/or more potent
antiviral activity.
CHEMISTRY
The preparation of (E)-5- or 5,6-substituted -2-(4-R1-
styryl)-1H-benzimidazoles (11-31) has been accomplished
according to Scheme 1.
To pursue this objective, we designed the molecules 11-
31 in Fig. (2), where the phenyl ring is separated from the
benzimidazole moiety by an ethylene spacer that was ob-
tained only in trans conformation. Noteworthy, this spacer is
certainly shorter than a phenyl placed in 2-diphenyl-
benzimidazoles and less bulky than a naphtyl benzimidazole
previously described.
The diamines 3-9 were condensed with bisulphite com-
pound 10, according to the procedure described by Ridley et
al. [9], to give the desired derivatives 11-17 in good yield.
The nitro derivatives 11-17 were in turn converted into the
amines 18-24, by reduction with SnCl2 in ethanol at 80°C in
1:3 molar ratio, with 37% hydrochloric acid aqueous solu-
tion. The primary amines 18-24 were then acetylated with
acetic anhydride to give the acetyl derivatives 25-31. The
yields recorded in the last steps varied from very high to
moderate. Elucidation of the compounds’ structures was
achieved by the whole elemental analyses and spectroscopic
data.
It has to be noted that some compounds listed and refer-
enced in Fig. (2) have been previuosly reported as antimi-
crobial derivatives but no one was tested as antiviral or anti-
proliferative agents.
R
N
R2
N
H
R1
11-31
R1
Compds
R
R2
Lit
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
H
CH3
CH3
Cl
H
H
NO2
NO2
[3,4]
[5]
CH3
H
NO2
NO2
[6,7]
Cl
Cl
H
NO2
CF3
COCH3
H
NO2
H
NO2
H
NH2
[8]
CH3
CH3
Cl
H
NH2
CH3
H
NH2
[8]
[8]
NH2
Cl
Cl
H
NH2
CF3
COCH3
H
NH2
H
NH2
H
NH COCH3
NH COCH3
NH COCH3
NH COCH3
NH COCH3
NH COCH3
NH COCH3
CH3
CH3
Cl
H
CH3
H
Cl
Cl
H
CF3
COCH3
H
Fig. (2). General structure of the newly described compounds.

72 Medicinal Chemistry, 2010, Vol. 6, No. 2
Vitale et al.
R
NH2
NaO3S
HO
NO2
i
R
NO2
N
+
NH2
R1
N
H
R1
10
3, R =R1=H
11-17
4, R=CH3 ; R1=H
5, R=R1=CH3
6, R=Cl; R1=H
7, R=R1=Cl
ii
8, R=CF3 ; R1=H
9, R=COCH3 ; R1=H
R
R1
N
iii
R
NH2
NHCOCH3
N
N
N
H
R1
H
25-31
18-24
Scheme 1. HCl (1:3 molar ratio), EtOH, 75°C for 25 to 60min; iii, acetic anhydride at 100°C,15 min.
RESULTS AND DISCUSSION
Antiviral Assays
position 5 of the hetero-ring (compound 17). In one case,
namely when the acetyl group was replaced by hydrogen
(11), the antiviral activity was retained, although at a concen-
tration 4-fold higher (7μM); however, this compound
showed a lower cytotoxicity for MT-4 cells, and a moderate
activity against CVB-2 (14 μM). In the group of the amino-
derivatives (18-24), the best result was recorded for com-
pound 18, that exhibited both anti-BVDV (EC50 = 16 μM)
and anti-CVB-2 activity (EC50 = 7 μM), while compound 21
showed only anti-CVB-2 activity. When the amino group
was acetylated, as in compounds 25-31, anti-BVDV activity
appeared in 31 (EC50 = 12 μM). Interestingly, compound 30
showed anti-CVB-2 activity with an EC50 value of 11μM
and a remarkable anti-RSV activity at 1 μM. A comparison
of the antiviral activities of both compounds 11 and 18, that
do not bear substituents at position 5, shows that the change
of the substituent in the para-styryl moiety from nitro (11) to
amino (18) allows to shift the activity from BVDV to CVB-
2. The reverse is true in the case of 18. It is interesting to
note that the variation of the nitro group in compound 16 into
the acetylamino of compound 30 resulted in a increase of
RSV activity from 11 to 1μM and a strong reduction of the
cytoxicity (Vero-76 14 against 80 μM) (Table 1).
As reported in Tables 1 and 2, title compounds were
evaluated in cell-based assays for both cytotoxicity and anti-
viral activity against viruses representative of two of the
three genera of the Flaviviridae family, i.e. Pestiviruses (Bo-
vine Viral Diarrhoea Virus, BVDV) and Flaviviruses (Yel-
low Fever Virus, YFV), as Hepaciviruses can hardly be used
in routine cell-based assays. Title compounds were also
tested against representatives of other virus families. Among
ssRNA+ were a retrovirus [Human Immunodeficiency Virus
Type 1 (HIV-1)] and two Picornaviruses [Coxsackie Virus
Type B2 (CVB-2) and Poliovirus Type-1, Sabin strain (Sb-
1)]; among ssRNA- were a Paramixovirus [Respiratory Syn-
cytial Virus (RSV)] and a Rhabdovirus [Vesicular Stomatitis
Virus (VSV)]. Among double-stranded RNA (dsRNA) vi-
ruses was a Reoviridae representative (Reo-1). Two repre-
sentatives of DNA virus families (Herpesviridae and Pox-
viridae) were also included: Herpes Simplex Type-1 (HSV-
1) and Vaccinia Virus (VV).
AZT (3’-azido-thymidine), NM 108 (2’-ß-methyl-
guanosine), NM 176 (2’-ethynyl-D-citidine), NM 299 (6-
azauridine), M 5255 (Mycophenolic Acid) and ACG (acy-
cloGuanosine) were used as reference inhibitors of ssRNA+,
ssRNA- and DNA viruses, respectively.
Conclusions
The results obtained in this paper allow us to make some
considerations about the antiviral and antiproliferative activi-
ties of this class of benzimidazoles. Their antiviral activity
seems rather limited in comparison with the broader citotox-
icity recorded. However, from the whole of the results we
can observe that, on maintaining an identical pattern of sub-
stituents in the hetero-ring and varying the substituents at
position 2 as in the case of 2-styrylbenzimidazoles, a signifi-
cant decrease , up to complete loss, of both the cytotoxicity
and the antiproliferative activity recorded in the previous
series of naphtyl- and diphenylbenzimidazoles can be
achieved . As far as the antiviral activity against Flaviridae is
concerned, the two abovementioned series exhibited low
potency, although, in the case of diphenylbenzimidazoles an
interesting activity against YFV and CVB-2 was evidentiated
(Fig. 1). In the present series, the antiviral activity is mainly
directed against BVDV in those compounds bearing an ace-
tyl group, and against CVB-2 in the un-substituted ones.
As reported in Table 1, compounds 11-31 with a few
exceptions ( 11, 18, 28, 29, 31) show a general cytotoxicity
in MT-4 and Vero 76 cells test, which was lower than that
recorded for the previously reported naphtyl- and diphenyl-
benzimidazole derivatives [1,2]. As far as the antiviral activ-
ity is concerned, this was limited to a few virus of the
Flaviridae family (Table 2). Compounds 11, 12, 17, 18, 24,
31 were poorly cytotoxic in MT-4 and Vero 76 cells and
exhibited anti-BVDV activity in the concentration range 1.7-
16 μM, while some of them (11,18) were also displaying
activity against CBV-2 in the same range of concentration.
Compound 17 was the most active (EC50 = 1.7μM) against
BVDV followed, in decreasing order of activity, by 15, 24,
11, 19, 31, 18. A simple SAR survey suggests that, in the
series of the para-nitro-styryl derivatives, the preferred sub-
stitution conferring anti BVDV activity is an acetyl group at

Styrylbenzimidazoles. Synthesis and Biological Activity - Part 3
Medicinal Chemistry, 2010, Vol. 6, No. 2 73
Table 1. Cytotoxicity of Title and Reference Compounds
Cell lines
bMDBK
Compds
aMT-4
cBHK-21
dVERO-76
CC50
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
83
14
>100
78
ꢀ100
>100
>100
>100
7.6
>100
45
45
5
86
60
44
>100
>100
ꢀ100
14
15
>100
16
9
29
>100
18
>100
>100
75
>100
53
>100
86
55
35
69
>100
52
90
81
>100
61
90
9
8
16
50
17
9
21
70
44
55
33
80
25
>100
>100
23
90
10
>100
>100
70
7
18
17
>100
>100
38
>100
>100
>100
33
>100
100
80
>100
100
>100
>100
>100
>100
Reference Compounds
AZT
NM-108
NM-176
6-azaUdR
M-5255
ACG
50
>100
ꢀ100
0.2
>100
>100
>100
42
90
>100
>100
20
>100
>100
>100
>100
0.2
ꢀ13
>100
>100
aCompound concentration (μꢀ) required to reduce the viability of mock-infected MT-4 (CD4+ human T cells containing an integrated HTLV-1 genome) cells by 50%, as determined
by the MTT method.
bCompound concentration (μꢀ) required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as determined by the MTT method.
cCompound concentration (μꢀ) required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as determined by the MTT method.
dCompound concentration (μꢀ) required to reduce the viability of mock-infected VERO-76 (Monkey normal kidney) monolayers by 50%, as determined by staining with crystal
The appearance of a novel activity against RSV strongly
suggests that a new lead has been discovered. Therefore, we
conclude that the benzimidazole ring is an interesting chemi-
cal feature that, according to the substituents, can be modu-
lated in both cytotoxic/antiproliferative and antiviral activi-
ties. In particular, the styryl moiety brings about a few novel
interesting activities that can be further implemented. The
next step will lead us to consider a modification of the acetyl
group to investigate whether it can positively affect the re-
corded antiviral activities.

74 Medicinal Chemistry, 2010, Vol. 6, No. 2
Vitale et al.
Table 2. Antiviral Activity of Title and Reference Compounds Against Viruses Containing an RNA and DNA Genome
Genome
Genus
ssRNA+
Flavi
dsRNA
Reo
ssRNA-
Paramyxo
DNA
Retro
aHIV-1
MT-4
Pesti
Entero
dCVB-2
Rhabdo
dVSV
Simplex
dHSV-1
Ortopox
dVV
Virus
bBVDV
MDBK
cYFV
dSb-1
cReo-1
BHK
dRSV
Cell line
BBHK-21
Vero 76
Vero-76
Compd
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
EC50
>83
>14
>5
7
9
>78
>100
>100
>100
>100
>7.6
>18
14
2.5
>100
>45
>100
>100
>100
>100
>100
>23
55
>45
>60
>100
>100
11
>100
>45
>100
>45
>30
>45
>86
100
3
>60
>100
>100
>14
>100
7
>60
>60
>60
>60
>44
>15
>16
>29
>100
>53
>69
>81
>9
>100
>100
>14
>100
>100
>14
>100
>100
>14
>100
>100
>14
ꢀ3.7
1.7
>100
>10
>100
>60
>100
>100
>75
>90
>80
>50
>70
>80
>90
>100
>70
>100
>100
1
>100
>100
>75
>100
>100
>75
>100
>100
>75
16
>86
12
>35
>75
>90
8
>75
>35
>100
65
>52
>90
>43
>90
>90
>90
>61
>80
>33
>80
>90
>80
>8
>16
>50
>70
>80
20
>50
>23
>50
>50
>50
>17
>44
>25
>10
>7
>9
>21
>70
>23
>70
>70
>70
6
>33
>80
>48
>80
>80
>80
>100
>100
>18
>100
>100
>100
12
>100
>23
>90
>100
>100
>19
>90
>90
>90
29
>100
>70
>100
>70
>100
>70
>100
>70
>17
>70
>100
>100
11
>100
>100
>38
86
>100
>100
>33
>100
>100
>80
>100
>100
>33
>100
>100
>80
>100
>100
>80
>100
>100
>80
>100
>100
>100
>100
>100
>100
>100
>100
Reference Compounds
AZT
NM-108
NM-176
6-azaUdR
M-5255
ACG
0.01
>100
>100
>0.2
>0.2
1.7
38
1.8
>100
26
20
24
>100
2.4
>100
>100
1.2
>100
>100
>20
>100
>100
>20
>13
3
>100
>100
11
20
>20
>13
>100
>100
>100
>100
>100
>100
>42
>100
>20
>13
>100
>100
>100
0.6
>13
1.8
>100
>100
>100
aCompound concentration (ꢁM) required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity, as determined by the MTT method.
bCompound concentration (μꢀ) required to achieve 50% protection of MDBK cells from the BVDV- (Bovine Viral Diarrhoea Virus) induced cytopathogenicity, as determined by the
MTT method.
cCompound concentration (μꢀ) required to achieve 50% protection of BHK (Kidney fibroblast) cells from the YFV- (Yellow Fever Virus) and Reo- (Reovirus 1) induced cytopatho-
genicity, as determined by the MTT method.
dCompound concentration (μM) required to reduce the plaque number of CVB-2 (Coxsackie virus B2), Sb-1 (Polio virus 1), RSV (Respiratory syncytial virus), VSV (Vesicular
stomatitis virus), HSV-1 (Herpes simplex type 1) and VV (Vaccinia) by 50% in VERO-76 monolayers, as determined by staining with crystal violet dye.
eReference Compounds: AZT (3’-azido-thymidine), NM-108 (2’-C-methylguanosine), NM-176 (2’-C-ethynil-citidine), 6-azaUdR (6-azauridine), M-5255 (mycophenolic acid), ACG
(acycloguanosine).

Styrylbenzimidazoles. Synthesis and Biological Activity - Part 3
Medicinal Chemistry, 2010, Vol. 6, No. 2 75
1
EXPERIMENTAL SECTION
General Remarks
1383; 1340; 1109. ꢂmax nm: 376; 205. H-NMR (DMSO-d6)
ꢁ: 8.25 (2H, d, J = 8.8 Hz, H-3’,5’); 7.83 (2H, d, J = 8.6 Hz,
H-2’,6’); 7.74 (1H, d, J = 16.0 Hz, CH=CH-C6H4-NO2);
7.45 (1H, d, J = 8.2 Hz, H-7); 7.42 (1H, d, J = 2.2 Hz, H-4);
7.37 (1H, d, J = 16.0 Hz, CH=CH-C6H4-NO2); 7.04 (1H, d,
J = 8.0 Hz, H-6); 2.46 (3H, s, CH3).MS m/z 280.10 (M+H)+.
Melting points were carried out with a Köfler hot stage melt-
ing point apparatus and are uncorrected. Infrared spectra
were recorded as nujol mulls on NaCl plates with a Perkin-
Elmer 781IRspectrophotometerand areexpressed in ꢀ (cm-1).
UV spectra are qualitative and were recorded in nm for solu-
tions in EtOH with a Perkin-Elmer Lamba 5 spectropho-
tometer. Nuclear magnetic resonance (1H-NMR) spectra
were determined in CDCl3, DMSO-d6, DMSO-d6 / CDCl3 (in
the ratio 3:1) and were recorded in a Varian XL-200
(200MHz). Chemical shifts (ꢁ scale) are reported in parts per
million (ppm) downfield from tetramethylsilane (TMS) as
internal standard. Splitting patterns are designated, as fol-
lows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, mul-
tiplet; br s, broad singlet; dd, double doublet. Low-resolution
mass spectra (MS) analyses were performed using an Agilent
1100 series LC/MSD mass spectrometer in ESI ionization
mode.
5,6-Dimethyl-2-(4’-nitrostyryl-4-yl)-1H-benzimidazole
(13) Reaction time: 2h. Yield: 44%. M.p. 179-180°C. Rf :
0.42 (petroleum ether 40-60°C/ethyl acetate 6:4). Analysis
for C17H15N3O2; ꢀmax cm-1: 1660; 1593; 1515; 1383; 1337;
1
1109. ꢂmax nm: 385; 205. H-NMR (DMSO-d6) ꢁ: 8.24 (2H,
d, J = 8.0 Hz, H-3’,5’); 7.85 (2H, d, J = 8.0 Hz, H-2’,6’);
7.70 (1H, d, J = 16.0 Hz, CH=CH-C6H4-NO2); 7.37 (1H, d,
J = 16.0 Hz, CH=CH-C6H4-NO2); 7.33 (2H, s, H-4,7); 2.34
(6H, s, 2xCH3).
5-Chloro-2-(4’-nitrostyryl-4-yl)-1H-benzimidazole (14)
Reaction time: 3h. Yield: 83%. M.p. 238-240°C ( 242-244°C
[6,7]). Rf : 0.43 (petroleum ether 40-60°C/ethyl acetate 6:4).
Analysis for C15H10ClN3O2; ꢀmax cm-1: 1659; 1593; 1504;
1
1338; 1109; 665. ꢂmax nm: 368 ; 206. H-NMR (DMSO-d6)
ꢁ: 8.26 (2H, d, J = 8.8 Hz, H-3’,5’); 7.89 (2H, d, J = 8.8 Hz,
H-2’,6’); 7.78 (1H, d, J = 16.4 Hz, CH=CH-C6H4-NO2);
7.59 (1H, d, J = 2.2 Hz, H-4); 7.56 (1H, d, J = 9.0 Hz, H-7);
7.41 (1H, d, J = 16.4 Hz, CH=CH-C6H4-NO2); 7.19 (1H,
dd, J = 8.8 and 2.0 Hz, H-6).
Chemistry
Intermediates
The diamines (3-6) were commercially available. The
diamine (8) was prepared according to Walley [10] starting
from the commercially available parent nitro compound
(Aldrich), while the diamine 9 was obtained according to the
procedure of W. Borsche and J. Barthenheier [11] . The bi-
sulphite compound (10) was obtained in high yields from E -
3-(4-nitrophenyl)prop-2-enal (Aldrich) with Na2S2O5 in
ethanol at room temperature. Compounds 11,12,14,18,20,21
were previously described as referenced in Fig. (1) using a
different synthetic route. In addition compounds 18,20,21 are
included in a patent and the m.p.’s were not given.
5,6-Dichloro-2-(4’-nitrostyryl-4-yl)-1H-benzimidazole
(15) Reaction time: 3h. Yield: 60%. M.p. >290°C. Rf : 0.45
(petroleum ether 40-60°C/ethyl acetate 1:1). Analysis for
C15H9 Cl2N3O2; ꢀmax cm-1: 1595; 1507; 1340; 1298; 665. ꢂmax
1
nm: 366; 203. H-NMR (DMSO-d6) ꢁ: 8.27 (2H, d, J = 8.4
Hz, H-3’,5’); 7.94 (2H, d, J = 8.8 Hz, H-2’,6’); 7.82 (2H, s,
H-4,7); 7.81 (1H, d, J = 16.6 Hz, CH=CH-C6H4-NO2); 7.44
(1H, d, J = 16.6 Hz, CH=CH-C6H4-NO2).
5-Trifluoromethyl-2-(4’-nitrostyryl-4-yl)-1H-benzimida-
zole (16) Reaction time: 3h. Yield: 95%. M.p. 144°C. Rf :
0.46 (petroleum ether 40-60°C/ethyl acetate 6:4). Analysis
for C16H10F3N3O2; ꢀmax cm-1: 1680; 1594; 1511; 1342; 1244;
General Procedure for the Preparation of the 2-(4’-
Nitrostyryl)Benzimidazoles 5 and 5,6 Substituted (11-17)
1
To a solution of 0.2 g of 1,2-diaminobenzenes (3-9) in 10
ml of ethanol was added 10 derivative in the molar ratio of
1:1.1 and the reaction mixture was refluxed from 2 to 4 h.
After cooling compounds 11-17, which precipitate as solid,
were collected by filtration and washed with water. Further
amounts of products were obtained by evaporation in vacuo
of the solvent followed by washing with water. Compounds
11-17 were purified by crystallization from MeOH/H2O.
1111. ꢂmax nm: 357; 207. H-NMR (DMSO-d6) ꢁ: 8.26 (2H,
d, J = 8.8 Hz, H-3’,5’); 7.76 (2H, d, J = 8.8 Hz, H-2’,6’);
7.70 (1H, d, J = 16.0 Hz, CH=CH-C6H4-NO2); 7.68 (1H, d,
J = 8.8 Hz, H-6); 7.53 (2H, s, H-4); 7.48 (1H, d, J = 8.4 Hz,
H-7); 7.34 (1H, d, J = 16.0 Hz, CH=CH-C6H4-NO2).
5-Acetyl-2-(4’-nitrostyryl-4-yl)-1H-benzimidazole (17) Reac-
tion time: 3h. Yield: 50%. M.p. >300oC. Rf : 0.19 (petroleum
ether 40-60°C/ethyl acetate 1:1). Analysis for C17H13N3O2;
ꢀmax cm-1: 1660; 1592; 1506; 1425; 1334; 1287; 1105. ꢂmax
nm: 366; 204. 1H-NMR (DMSO-d6 -CDCl3) ꢁ: 8.25 (2H, d, J
= 8.8 Hz, H-3’,5’); 8.23 (1H, s, H-4); 7.88 (2H, d, J = 9.0
Hz, H-2’,6’); 7.86 (1H, s, H-6); 7.81 (1H, d, J = 16.6 Hz,
CH=CH-C6H4-NO2); 7.63 (1H, d, J = 8.4 Hz, H-7); 7.42
(1H, d, J = 16.6 Hz, CH=CH-C6H4-NO2); 2.66 (3H, s,
CH3CO).
2-(4’-Nitrostyryl-4-yl)-1H-benzimidazole (11) Reaction
time: 4h. Yield 55%. M.p. 220-221°C. (265-266°C [3]; 260-
262°C from acetic acid [4]); Rf : 0.23 (petroleum ether 40-
60°C/ethyl acetate 6:4). Analysis for C15H11N3O2; ꢀmax cm-1:
1591; 1341; 1319; 1107. ꢂmax nm: 369; 203. 1H-NMR
(DMSO-d6) ꢁ 8.24 (2H, d, J = 8.8 Hz, H-3’,5’); 7.80 (2H, d,
J = 8.8 Hz, H-2’,6’); 7,78 (1H, d, J = 16.4 Hz, CH=CH-
C6H4-NO2); 7.62-7.58 (2H, m, H-4,7); 7.38 (1H, d, J = 16.4
Hz, CH=CH-C6H4-NO2); 7.26-7.21 (2H, m, H-5,6). MS m/z
266.10 (M+H)+.
General Procedure for the Preparation of the 2-(4’-
Aminostyryl)Benzimidazoles 5 and 5,6 Substituted (18-24)
5-Methyl-2-(4’-nitrostyryl-4-yl)-1H-benzimidazole (12)
Reaction time: 2.5h. Yield 67%. M.p. 225-230°C ( 205-
208°C [5]) ;Rf : 0.34 (petroleum ether 40-60°C/ethyl acetate
6:4). Analysis for C16H13N3O2; ꢀmax cm-1: 1640; 1594; 1518;
To a solution of 0.2 g of 2-(4’-nitrostyryl)benzimidazoles
(11-17) in 3.0 ml of ethanol was added a mixture of stannous
chloride dihydrate, in the molar ratio 1:3, in 3.0 ml of 37%
hydrochloric acid and the reaction was stirred at 75°C from

76 Medicinal Chemistry, 2010, Vol. 6, No. 2
Vitale et al.
25 to 60 minutes. After concentration in vacuo of the organic
solvent, the acidic residue was diluted with iced water and
made alkaline with 6N NaOH. The solid products that pre-
cipitated were collected by filtration.
1334; 1248; 1154; 1098. ꢁmax nm: 365; 203. 1H-NMR
(DMSO-d6-CDCl3) ꢂ: 8.08 (1H, s, NH2); 7.80 (1H, s, H-4);
7.73 (1H, s, H-7); 7.70 (1H, d, J = 16.4 Hz, CH=CH-C6H4-
NH2); 7.42 (1H, s, H-6); 7.37 (2H, d, J = 8.4 Hz, H-2’,6’);
6.90 (1H, d, J = 16.4 Hz, CH=CH-C6H4-NH2); 6.69 (2H, d,
J = 8.4 Hz, H-3’,5’).
2-(4’-Aminostyryl-4-yl)-1H-benzimidazole (18) Reaction
time: 60’. Yield: 91%. M.p. 202-205°C ( compound cited in
reference [8], but m.p.not given ); Rf : 0.14 (petroleum ether
40-60°C/ethyl acetate 1:1). Analysis for C15H13N3; ꢀmax cm-1:
3362; 3213; 1603; 1319; 1277; 1176. ꢁmax nm: 359; 202. 1H-
NMR. (DMSO-d6 -CDCl3) ꢂ: 8.07 (2H, s, NH2); 7.62 (1H, d,
J = 16.6 Hz, CH=CH-C6H4-NO2); 7.54-7.49 (2H, m, H-
4,7); 7.32 (2H, d, J = 8.4 Hz, H-2’,6’); 7.21-7.16 (2H, m, H-
5,6); 6.84 (1H, d, J = 16.6 Hz, CH=CH-C6H4-NO2); 6.65
(2H, d, J = 8.4 Hz, H-3’,5’).
5-Acetyl-2-(4’-aminostyryl-4-yl)-1H-benzimidazole (24)
Reaction time: 50’. Yield: 89%. M.p. 191-192°C. Rf : 0.05
(petroleum ether 40-60°C/ethyl acetate 1:1). Analysis for
C17H15N3; ꢀmax cm-1: 3340; 3213; 1600; 1292; 1170. ꢁmax nm:
374; 202. 1H-NMR (DMSO-d6 -CDCl3) ꢂ: 8.29 (1H, s, H-4);
7.79 (1H, d, J = 8.4 Hz, H-6); 7.57 (1H, d, J = 16.6 Hz,
CH=CH-C6H4-NH2); 7.54 (1H, s, H-7); 7.35 (2H, d, J = 8.0
Hz, H-2’,6’); 6.85 (1H, d, J = 16.6 Hz, CH=CH-C6H4-NH2);
6.61 (2H, d, J = 8.0 Hz, H-3’,5’); 2.62 (3H, s, CH3CO).
5-Methyl-2-(4’aminostyryl-4-yl)-1H-benzimidazole (19)
Reaction time: 40’. Yield: 89%. M.p. 113-115°C( compound
cited in reference [8], but m.p.not given ); Rf : 0.09 (petro-
leum ether 40-60°C/ethyl acetate 1:1). Analysis for
C16H15N3; ꢀmax cm-1: 3340; 3213; 1599; 1340; 1308; 1277;
General Procedure for the Preparation of the 2-(4’-
Acetylaminostyryl) Benzimidazoles 5 and 5,6 Substituted
(25-31)
1
0.1 g of 2-(4’-aminostyryl)benzimidazoles (18-24) were
suspended in 1 ml of acetic anhydride and stirred at 100°C
for 15 minutes. After cooling the solids formed were col-
lected by suction filtration and washed with ethyl ether.
1175. ꢁmax nm: 361; 203. H-NMR (DMSO-d6 -CDCl3) ꢂ:
8.25 (2H, s, NH2); 7.61 (1H, d, J = 16.0 Hz, CH=CH-C6H4-
NO2); 7.41 (1H, d, J = 8.2 Hz, H-7); 7.36 (2H, d, J = 8.8 Hz,
H-2’,6’); 7.31 (1H, s, H-4); 7.04 (1H, d, J = 8.4 Hz, H-6);
6.81 (1H, d, J = 16.0 Hz, CH=CH-C6H4-NO2); 6.63 (2H, d,
J = 8.0 Hz, H-3’,5’); 2.44 (3H, s, CH3).
2-(4’-Acetylaminostyryl-4-yl)-1H-benzimidazole
(25)
Yield: 85% M.p.: 281-282°C. Rf : 0.20 (petroleum ether 40-
60°C/ethyl acetate 1:1). Analysis for C17H15N3O; ꢀmax cm-1:
3279; 3170; 1723; 1665; 1595; 1307; 1284; 1179. ꢁmax nm:
350; 205. 1H-NMR (DMSO-d6) ꢂ: 10.20 (1H, s, HNCOCH3);
7.95-7.61 (6H, m, H-4,7,2’,3’,5’,6’); 7.39-7.25 (3H, m,
CH=CH-C6H4-NHCOCH3 + H-5‘,6’); 7.12 (1H, d, J = 16.6
Hz, CH=CH-C6H4-NHCOCH3); 2.08 (1H, s, CH3CONH).
5,6-Dimethyl-2-(4’aminostyryl-4-yl)-1H-benzimidazole
(20) Reaction time: 50’. Yield: 67%. M.p. >305°C ( com-
pound cited in reference [8], but m.p.not given ); Rf : 0.07
(petroleum ether 40-60°C/ethyl acetate 1:1). Analysis for
C17H17N3; ꢀmax cm-1: 3361; 3234; 1601; 1458; 1319; 1298;
1
1176. ꢁmax nm: 364; 203. H-NMR. (DMSO-d6 -CDCl3) ꢂ:
8.20 (2H, s, NH2); 8.00 (1H, d, J = 16.4 Hz, CH=CH-C6H4-
NO2); 7.44 (2H, s, H-4,7); 7.39 (1H, d, J = 8.4 Hz, H-2’,6’);
6.83 (1H, d, J = 16.4 Hz, CH=CH-C6H4-NO2); 6.66 (2H, d,
J = 8.6 Hz, H-3’,5’); 2.38 (6H, s, 2xCH3).
5-Methyl-2-(4’-acetylaminostyryl-4-yl)-1H-benzimida-
zole (26) Yield: 73%. M.p.: 290-293°C. Rf : 0.13 (petroleum
ether 40-60°C/ethyl acetate 1:1). Analysis for C18H17N3O;
ꢀmax cm-1: 3425; 3085; 1660; 1590; 1416; 1330; 1275; 1175.
1
ꢁmax nm: 354; 206. H-NMR (DMSO-d6) ꢂ: 10.19 (1H, s,
5-Chloro-2-(4’-aminostyryl-4-yl)-1H-benzimidazole (21)
Reaction time: 40’. Yield: 98%. M.p. 154°C ( compound
cited in reference [8], but m.p.not given ); Rf : 0.24 (petro-
leum ether 40-60°C/ethyl acetate 1:1). Analysis for
C15H12ClN3; ꢀmax cm-1: 3328; 3212; 1599; 1304; 1251; 1174;
650. ꢁmax nm: 365; 202. 1H-NMR (DMSO-d6 -CDCl3) ꢂ: 8.05
(1H, s, NH2); 7.87 (1H, s, H-4); 7.54 (1H, d, J = 16.4 Hz,
CH=CH-C6H4-NO2); 7.50 (1H, s, H-7); 7.36 (2H, d, J = 8.4
Hz, H-2’,6’); 7.11 (1H, dd, J = 8.4 and 1.8 Hz, H-6); 6.85
(1H, d, J = 16.4 Hz, CH=CH-C6H4-NO2); 6.68 (1H, d, J =
8.2 Hz, H-3’,5’).
HNCOCH3); 7.72-7.62 (5H, m, H-7,2’,3’,5’,6’); 7.48 (1H, d,
J = 8.2 Hz, H-6); 7.42 (1H, d, J = 16.0 Hz, CH=CH-C6H4-
NHCOCH3); 7.17-7.13 (2H, m, CH=CH-C6H4-NHCOCH3
+ H-4); 2.44 (3H, s, CH3); 2.08 (3H, s, HNCOCH3).
5,6-Dimethyl-2-(4’-acetylaminostyryl-4-yl)-1H-benzimid-
azole (27) Yield: 54%. M.p.: 278-280°C. Rf : 0.06 (petro-
leum ether 40-60°C/ethyl acetate 1:1). Analysis for
C19H19N3O; ꢀmax cm-1: 3404; 3170; 1596; 1465; 1333; 1277;
1
1242; 1178. ꢁmax nm: 358; 203. H-NMR (DMSO-d6) ꢂ:
10.36 (1H, s, HNCOCH3); 8.01 (1H, d, J = 16.8 Hz,
CH=CH-C6H4-NHCOCH3); 7.75 (2H, d, J = 8.8 Hz, H-
2’,6’); 7.64 (2H, d, J = 8.8 Hz, H-3’,5’); 7.50 (2H, s, H-4,7);
7.13 (1H, d, J = 16.8 Hz, CH=CH-C6H4-NHCOCH3); 2.37
(6H, s, 2xCH3); 2.09 (3H, s, CH3CONH).
5,6-Dichloro-2-(4’-aminostyryl-4-yl)-1H-benzimidazole
(22) Reaction time: 30’. Yield: 88%. M.p. 140°C. Rf : 0.35
(petroleum ether 40-60°C/ethyl acetate 1:1). Analysis for
C15H11ClN3; ꢀmax cm-1: 3404; 3319; 1604; 1291; 1277; 1177;
665. ꢁmax nm: 372; 205. 1H-NMR (DMSO-d6-CDCl3) ꢂ:
12.25 (1H, sa, NH2); 7.65 (1H, d, J = 16.2 Hz, CH=CH-
C6H4-NH2); 7.54 (2H, s, H-4,7); 7.34 (2H, d, J = 8.2 Hz, H-
2’,6’); 6.85 (1H, d, J = 16.2 Hz, CH=CH-C6H4-NH2); 6.68
(2H, d, J = 8.4 Hz, H-3’,5’).
5-Chloro-2-(4’-acetylaminostyryl-4-yl)-1H-benzimida-
zole (28) Yield: 94%. M.p.: >305°C. Rf : 0.14 (petroleum
ether 40-60°C/ethyl acetate 1:1). Analysis for C17H14 ClN3O;
ꢀmax cm-1: 3106; 3042; 1636; 1589; 1330; 1298; 1174; 659.
1
ꢁmax nm: 354; 205. H-NMR (DMSO-d6) ꢂ: 10.18 (1H, s,
HNCOCH3); 7.65-7.52 (7H, m, CH=CH-C6H4-NHCOCH3
+ H-4,7,2’,3’,5’,6’); 7.21 (1H, d, J = 8.4 Hz, H-6); 7.10 (1H,
d, J = 16.8 Hz, CH=CH-C6H4-NHCOCH3); 2.08 (3H, s,
CH3CONH).
5-Trifluoromethyl-2-(4’aminostyryl-4-yl)-1H-benzimida-
zole (23) Reaction time: 25’. Yield: 68%. M.p.: 100-101°C.
Rf : 0.23 (petroleum ether 40-60°C/ethyl acetate 1:1). Analy-
sis for C16H12 F3N3; ꢀmax cm-1: 3500; 3362; 1605; 1418;

Styrylbenzimidazoles. Synthesis and Biological Activity - Part 3
Medicinal Chemistry, 2010, Vol. 6, No. 2 77
5,6-Dichloro-2-(4’-acetylaminostyryl-4-yl)-1H-benzimid-
azole (29) Yield: 88%. M.p.: >305°C. Rf : 0.12 (petroleum
ether 40-60°C/ethyl acetate 1:1). Analysis for C17H13Cl2N3O;
ꢀmax cm-1: 3234; 3040; 1668; 1641; 1592; 1428; 1306; 1268;
1175. ꢁmax nm: 354; 205. 1H-NMR (DMSO-d6) ꢂ: 10.17 (1H,
s, HNCOCH3); 7.76-7.62 (7H, m, CH=CH-C6H4-
NHCOCH3 + H-4,7,2’,3’,5’,6’); 7.09 (1H, d, J = 16.2 Hz,
CH=CH-C6H4-NHCOCH3); 2.07 (3H, s, CH3CONH).
without or with serial dilutions of test compounds. Then, 20
μL of an HIV-1 suspension containing 100 CCID50 were
added. After a 4-day incubation, cell viability was deter-
mined by the MTT method.
Activity of compounds against Yellow fever virus (YFV)
and Reo virus type-1 (Reo-1) was based on inhibition of vi-
rus-induced cytopathogenicity in acutely infected BHK-21
cells. Activities against Bovine viral diarrhoea virus
(BVDV), in infected MDBK cells, were also based on inhibi-
tion of virus-induced cytopathogenicity.
5-Trifluoromethyl-2-(4’-acetylaminostyryl-4-yl)-1H-ben-
zimidazole (30) Yield: 89%. M.p.: 254-257°C. Rf : 0.08 (pe-
troleum ether 40-60°C/ethyl acetate 1:1). Analysis for
C18H14F3N3O; ꢀmax cm-1: 3319; 3106; 1667; 1599; 1411;
BHK and MDBK cells were seeded in 96-well plates at a
density of 5x104 and 3x104 cells/well, respectively, and were
allowed to form confluent monolayers by incubating over-
night in growth medium at 37 °C in a humidified CO2 (5%)
atmosphere. Cell monolayers were then infected with 50 ꢃL
of a proper virus dilution (in serum-free medium) to give an
m.o.i = 0.01. 1 hr later, 50 ꢃL of MEM Earle’s medium,
supplemented with inactivated foetal calf serum (FCS), 1%
final concentration, without or with serial dilutions of test
compounds, were added. After a 3-4 days incubation at 37
°C, cell viability was determined by the MTT method [12].
1
1334; 1298; 1243. ꢁmax nm: 337; 208. H-NMR (DMSO-d6)
ꢂ: 10.16 (1H, s, HNCOCH3); 7.72-7.63 (6H, m, CH=CH-
C6H4-NHCOCH3 + H-7, 2’,3’,5’,6’); 7.50 (1H, d, J = 8.7
Hz, H-6); 7.16 (1H, d, J = 16.5 Hz, CH=CH-C6H4-
NHCOCH3); 2.08 (3H, s, CH3CONH).
5-Acetyl-2-(4’-acetylaminostyryl-4-yl)-1H-benzimidazole
(31) Yield: 87%. M.p.: >305°C. Rf : 0.09 (petroleum ether
40-60°C/ethyl acetate 1:1). Analysis for C19H17N3O2; ꢀmax
cm-1: 3310; 3191; 1670; 1597; 1315; 1255. ꢁmax nm: 343;
204. 1H-NMR (DMSO-d6) ꢂ: 10.21 (1H, s, HNCOCH3); 8.24
(1H, s, H-4); 7.84 (1H, d, J = 8.0 Hz, H-6); 7.66 (5H, s, H-
7,2’,3’,5’,6’); 7.37 (1H, d, J = 15.9 Hz, CH=CH-C6H4-
NHCOCH3); 7.14 (1H, d, J = 15.9 Hz, CH=CH-C6H4-
NHCOCH3); 2.64 (3H, s, CH3CO); 2.08 (3H, s, CH3CONH).
Activity of compounds against Coxsackie virus, B-2
strain (CVB-2), Polio virus type-1 (Polio-1), Sabin strain,
Vesicular Stomatitis Virus (VSV), Vaccinia Virus (VV),
Herpes Virus 1 (HSV-1) and against Respiratory syncytial
virus (RSV), A-2 strain, in infected Vero 76 cells, was de-
termined by plaque reduction assays in Vero 76 cell mono-
layers. To this end, Vero 76 cells were seeded in 24-well
plates at a density of 2x105 cells/well and were allowed to
form confluent monolayers by incubating overnight in
growth medium at 37 °C in a humidified CO2 (5%) atmos-
phere. Then, monolayers were infected with 250 ꢃL of
proper virus dilutions to give 50-100 PFU/well. Following
removal of unadsorbed virus, 500 ꢃL of Dulbecco’s modi-
fied Eagle’s medium, supplemented with 1% inactivated
FCS and 0.75% methyl cellulose, without or with serial dilu-
tions of test compounds, were added. Cultures were incu-
bated at 37 °C for 2 (Sb-1 and VSV), 3 (CVB-2, 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. 50% effective concentrations
(EC50) were calculated by linear regression technique.
Biological Assays
Compounds
Compounds were dissolved in DMSO at 100 mM, and
then diluted in culture medium.
Cells and Viruses
Cell lines were purchased from American Type Culture
Collection (ATCC). The absence of mycoplasma contamina-
tion was checked periodically by the Hoechst staining
method. Cell lines supporting the multiplication of RNA
viruses were the following: CD4+ human T-cells containing
an integrated HTLV-1 genome (MT-4); Madin Darby Bo-
vine Kidney (MDBK); Baby Hamster Kidney (BHK-21) and
Monkey kidney (Vero 76) cells.
Cytotoxicity/Antiproliferative Assays
AZT (3’-azido-thymidine), NM 108 (2’-ß-methyl-
guanosine), NM 176 (2’-ethynyl-D-citidine), NM 299 (6-
azauridine), M 5255 (Mycophenolic Acid) and ACG (acy-
cloGuanosine) were used as reference inhibitors of ssRNA+,
ssRNA- and DNA viruses, respectively.
For cytotoxicity tests, run in parallel with antiviral as-
says, MDBK, BHK and Vero 76 cells were resuspended in
96 multiwell plates at an initial density of 6x105, 1x106 and
5x105 cells/mL, respectively, in maintenance medium, with-
out or with serial dilutions of test compounds. Cell viability
was determined after 48-96 hrs at 37 °C in a humidified CO2
(5%) atmosphere by the MTT method. The cell number of
Vero 76 monolayers was determined by staining with the
crystal violet dye.
Linear Regression Analysis
Viral and cell growth at each drug concentration was
expressed as percentage of untreated controls and concentra-
tions resulting in 50% (EC50 and CC50) growth inhibition
were determined by linear regression analysis.
Antiviral Assays
Activity of compounds against Human Immunodefi-
ciency virus type-1 (HIV-1) was based on inhibition of virus-
induced cytopathogenicity in MT-4 cells acutely infected
with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50
μL of RPMI containing 1x104 MT-4 were added to each well
of flat-bottom microtitre trays containing 50 μL of RPMI,
AKNOWLEDGEMENTS
This work was supported by grants from MIUR for Ital-
ian FIRB Project [(RBNE01J3SK 001)], “Cybersar” and
“Sviluppo di metodologie per la modellizzazione e lo studio
di farmaci e biofarmaci” Projects.

78 Medicinal Chemistry, 2010, Vol. 6, No. 2
Vitale et al.
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Received: January 16, 2010
Revised: April 08, 2010
Accepted: April 09, 2010