Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents

Article abstract of DOI:10.1016/j.bmc.2014.06.043

A library of eighty-six assorted benzimidazole derivatives was screened for antiviral activity against a panel of ten RNA and DNA viruses. Fifty-two of them displayed different levels of activity against one or more viruses, among which CVB-5, RSV, BVDV a

Full text of DOI:10.1016/j.bmc.2014.06.043

Accepted Manuscript
Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-
RSV and anti-Sb-1 agents
Michele Tonelli, Federica Novelli, Bruno Tasso, Iana Vazzana, Anna Sparatore,
Vito Boido, Fabio Sparatore, Paolo La Colla, Giuseppina Sanna, Gabriele
Giliberti, Bernardetta Busonera, Pamela Farci, Cristina Ibba, Roberta Loddo
PII:
S0968-0896(14)00489-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.06.043
BMC 11680
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 March 2014
19 June 2014
22 June 2014
Please cite this article as: Tonelli, M., Novelli, F., Tasso, B., Vazzana, I., Sparatore, A., Boido, V., Sparatore, F.,
Colla, P.L., Sanna, G., Giliberti, G., Busonera, B., Farci, P., Ibba, C., Loddo, R., Antiviral activity of benzimidazole
derivatives. III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents, Bioorganic & Medicinal Chemistry (2014), doi:
http://dx.doi.org/10.1016/j.bmc.2014.06.043
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-RSV
and anti-Sb-1 agents.
a,*
a
a
a
b
a
Michele Tonelli, Federica Novelli, Bruno Tasso, Iana Vazzana, Anna Sparatore, Vito Boido,
a
c
c
c
c
Fabio Sparatore, Paolo La Colla, Giuseppina Sanna, Gabriele Giliberti, Bernardetta Busonera,
c
c
c
Pamela Farci, Cristina Ibba and Roberta Loddo.
a
Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
b
Dipartimento di Scienze Farmaceutiche, Università di Milano, Via Mangiagalli 25, 20133 Milano,
Italy
c
Dipartimento di Scienze Biomediche, Università di Cagliari, Cittadella Universitaria, 09042
Monserrato (CA), Italy
*
Corresponding authors. Tel.: +39 010 3538867; fax: +39 010 3538358 E-mail address:
michele.tonelli@unige.it
Abstract
A library of eighty-six assorted benzimidazole derivatives was screened for antiviral activity against
a panel of ten RNA and DNA viruses.
Fifty-two of them displayed different levels of activity against one or more viruses, among which
CVB-5, RSV, BVDV and Sb-1 were the most frequently affected. In particular, fourteen
compounds exhibited an EC50 in the range 9-17 µM (SI from 6 to > 11) versus CVB-5, and seven
compounds showed an EC50 in the range 5-15 µM (SI from 6.7 to ≥ 20) against RSV, thus resulting
comparable to or more potent than the respective reference drugs (NM108 and ribavirin). Most of
these compounds derive from 2-benzylbenzimidazole, but also other molecular scaffolds [as 1-
phenylbenzimidazole
(2),
2-trifluoromethylbenzimidazole
(69),

2
dihydropyrido[3’,2’:4,5]imidazo[1,2-a][1,4]benzodiazepin-5-one (3), dibenzo[c,e]benzimidazo[1,2-
a]azepine (22), and 2-(tetrahydropyran-2-yl)benzimidazole (81, 82 and 86)] are related to
interesting levels of activity against these or other viruses (BVDV, Sb-1). Thus, these scaffolds
(
some of which, so far unexplored), represent valid starting points to develop more efficient agents
against pathologies caused by CVB-5, RSV, BVDV and Sb-1 viruses.
Keywords: Benzimidazole derivatives. RNA and DNA viruses. Anti-CVB-5, anti-RSV, anti-Sb-1
activity.
1. Introduction
Because of its isosterism with indole and purine nuclei, that are present in many fundamental
cellular components and bioactive compounds, the benzimidazole ring represents a kind of
privileged substructure from which several important drugs used in different therapeutic areas have
been obtained (proton pump inhibitors, angiotensin receptor antagonist, antihistaminics and
1
antihelmintics). An even greater number of still investigational therapeutic agents, including
antitumorals and antivirals, contain a benzimidazole nucleus.
Among the antiviral benzimidazoles, an important position is held by compounds acting against
2a
2b
HIV-1, HCV and RSV, but several other viruses resulted affected by varied benzimidazole
derivatives even if in a more occasional way (influenza B virus, rhinovirus, poliovirus, coxsackie
B3, cytomegalovirus, etc.). In the active compounds, the fundamental heterocyclic ring is decorated
with a variety of substituents, from simple atoms or small groups to highly complicated (even
multicyclic) moieties, so that structure-activity relationships may be obtained only within
homogeneous subsets of compounds, affecting a given molecular/cellular target.
Since many years we are interested in the chemistry and biological properties of benzimidazole
derivatives and, pursuing varied pharmacological aims, we have deeply investigated this chemical
space.

3
a
A
number of 2-[(benzotriazol-1/2-yl)methyl]benzimidazoles, bearing at position
1
dialkylaminoalkyl or quinolizidin-1-ylalkyl moiety, exhibited a selective and potent activity against
3
RSV, with EC50 as low as 20-30 nM for the best compounds. Only moderate activity against a few
other viruses (BVDV, YFV and CVB-2) was also observed for some of these compounds.
In a set of seventy-six non-basic derivatives of 2-phenylbenzimidazole, thirty-nine were found to
exhibit high activity (EC50= 0.1-10 µM) against at least one virus of a panel of ten RNA and DNA
4
viruses. Five of these compounds were outstanding for their high and selective activity against
single viruses (VV: EC50= 0.1 µM, BVDV: EC50= 0.8-1.5 µM; CVB-2: EC50= 4 µM) and low
toxicity for the corresponding host cells (CC50> 100 µM) (see Figure 1).
Based on these findings we evaluated, in cell based assays, the antiviral activity of a library of 86
assorted benzimidazole derivatives in order to find out some additional hits to be developed
together with those previously described. In Figure 2 are highlighted all the chemical features
explored in the library.
Figure 1
Figure 2
2
. Results and discussion
.1. Chemistry
2
Structurally, the tested compounds may be grouped according to the nature of substituents on the
position 1 and 2 of the benzimidazole ring (1- or 2-phenyl, 2-benzyl and various 1- or 2- non-
aromatic substituents), and also to the presence, or less, of a linear or cyclic basic moiety, as it is
indicated in Figure 3. A detailed representation of all structures is found in Figures 4-6.
Figure 3
Figure 4

4
Figure 5
Figure 6
Many of the 86 tested compounds of Fig. 4, 5 and 6 have been already prepared by some of us for
5
6
7
8
9
10
11
other pharmacological aims: 3; 6-11; 12, 13, 16, 69-71; 15, 34, 35, 39; 20, 59; 22; 23; 29-
12
13
14
15
16
17
18a,b
19
20
21
22
3
1; 37, 38, 41; 47, 48; 68; 72-74; 79, 82-86. Compounds 21;
26; 45; 51; 75;
23
24
7
7; 78 were already described by others and reprepared according to cited references.
The novel compounds were prepared as follows.
Most commonly the benzimidazole ring was formed by heating (in acetic acid or chloroform
solution) the properly substituted 1,2-phenylenediamine with the hydrochloride of the iminoester,
previously obtained from the corresponding nitrile, absolute ethanol and dry HCl (27, 28, 32, 33,
40, 42-44, 52-54, 57, 58 and 60; Scheme 1).
Scheme 1
Scheme 2
Alternatively, the heterocyclic ring may be formed by refluxing with 4N HCl the previously formed
N-acyl-1,2-phenylenediamine (1, 2, 14, 61, 62; Scheme 2) or the mixture of the substituted 1,2-
phenylenediamine with 4-aminophenylacetic acid (49, 55) or with the diaminoacid dihydrochlorides
2
5
obtained by the hydrolytic cleavage of tetrahydrocytisine (64, 65) and d,l-lupanine (66, 67;
Scheme 3).
In a few cases the ring formation was achieved by heating at 180°C, without solvent, the mixture of
the suitable 1,2-phenylenediamine and the acid (76, 80, 81; Scheme 4).
Scheme 3
Scheme 4

5
For the preparation of 2-phenylbenzimidazole derivatives was valuable the condensation of the N-
substituted-1,2-phenylenediamine with the arylaldehyde sodium bisulfite adduct (4, 5, 17-19;
Scheme 5).
2
0
2
-(4-Aminobenzyl)benzimidazoles 45, 49 and 55 (Scheme 3) were acylated in benzene solution to
give the acetylamino derivatives 46, 50 and 56 (Scheme 6).
-(4-Bromobenzyl)-5,6-dichlorobenzimidazole 53 (Scheme 1) was converted to sodium salt with
2
2
6
NaNH
2
and then alkylated with chlorolupinane (Scheme 7).
Scheme 5
Scheme 6
Scheme 7
By reacting commercial 2-chlorobenzimidazole with (1R,9aR)-(octahydro-2H-quinolizin-1-
2
7
yl)methane thiole (thiolupinine) the 2-(S-lupinylthio)benzimidazole 63 was obtained (Scheme 8).
Finally the benzoimidazo[1,2-c][1,2,3]benzotriazine derivatives 24 and 25 were obtained by
4
diazotization of the 2-(2-amino-4-methoxy)phenylbenzimidazoles, previously described (Scheme
9
). While from the 5,6-dichlorobenzimidazole derivative the single compound 25 is obtained,
starting from the corresponding 5-trifluoromethyl-derivative two isomeric compounds can be
1
formed. Indeed, the isolated compound exhibits splitted signals in the H NMR with one isomer
largely prevailing on the other (10:1). Compound 24 was used as such for antiviral tests.
For the synthesis of compounds 17 and 42-44 (Scheme 5 and 1, respectively), the novel N-
[
(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-4-trifluoromethyl-1,2-phenylenediamine
was
prepared by reacting the [(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methan]amine (epi-
2
8
lupinylamine)
with
4-chloro-3-nitrobenzotrifluoride.
The
N-substituted
2-nitro-4-
trifluoromethylaniline obtained was reduced by catalytic hydrogenation. (Scheme 10).

6
Scheme 8
Scheme 9
Scheme 10
1
All new compounds were characterized by elemental analyses and H NMR spectra that are fully
1
consistent with the described structure. The purity of compounds (elemental analyses and H NMR
spectra) resulted in all cases ≥ 95%.
2.2. Biological activity – General considerations
The 86 benzimidazole derivatives considered in this work were evaluated for antiviral activity
against ten RNA and DNA viruses. Among single-stranded, positive RNA viruses (ssRNA+), we
considered a Retrovirus (Human Immunodeficiency Virus type 1, HIV-1) two Picornaviruses
(
Coxsackie Virus type-5, CVB-5 and Poliovirus type-1, Sabin strain, Sb-1), a Flavivirus (Yellow
Fever Virus, YFV) and a Pestivirus (Bovine Viral Diarrhea Virus, BVDV). Among single-stranded,
negative RNA viruses (ssRNA-) a Paramyxoviridae (Respiratory Syncytial Virus, RSV) and a
Rhabdoviridae (Vesicular Stomatitis Virus, VSV) were selected as representatives. Among double-
stranded RNA (dsRNA) viruses, a Reoviridae family member (Reo-1) was included. Finally, two
representatives of DNA virus families were also included: Herpes Simplex Virus type-1, HSV-1
(
Herpesviridae) and Vaccinia Virus, VV (Poxviridae).
Cytotoxicity was evaluated in parallel with the antiviral activity. As reference inhibitors were used:
EFV (efavirenz) for HIV-1 only, NM 108 (2’-C-methyl-guanosine), NM 176 (2’-C-ethynylcytidine)
and ribavirin for the other ssRNA+ and dsRNA viruses; NM 299 (6-azauridine), ribavirin and M
5255 (mycophenolic acid) for the ssRNA- viruses; M 5255 and ACG (acyclovir) for DNA viruses.
Fifty-two of the 86 tested compounds (34 with and 18 without basic side-chain) exhibited antiviral
activity (EC50< CC50) against one or more viruses; in particular, 28 compounds displayed a

7
selective activity against a single virus, while 16, 7 and 1 molecules were active against two, three,
and four viruses, respectively. None of the active compounds inhibited the replication of HIV-1, but
an increasing number of molecules exhibited antiviral activity against, in the order: VSV (1), VV
(
2), HSV-1 (2), Reo-1 (3), YFV (4), Sb-1 (9), BVDV (13), RSV (16), and CVB-5 (35) (see Table
).
1
Thirty-four, out of 52 active compounds, showed an EC50≤ 30 µM against at least one virus, and
seven of them exhibited even an EC50≤ 10 µM. Thirty-four compounds [14 bearing a basic side-
chain and 20 devoid of it] were not able to inhibit the replication of any virus at concentrations up
to the corresponding CC50 for their host cells, or up to the highest concentration tested (100 µM).
Cytotoxicity and antiviral activities of all the 86 considered benzimidazole derivatives and reference
compounds are reported in Tables 1S, 2S and 3S. In Table 2-4 only the results of the 52 active
benzimidazoles and reference compounds are illustrated.
Table 1
Table 2
Table 3
Table 4
2.2.1. Cytotoxicity on host cells.
The studied compounds showed only moderate degree of cytotoxicity against the confluent cell
monolayer (in stationary growth) used to support the replication of the different viruses.
The exponentially growing lymphoblastoid human cells (MT-4) used to growth HIV-1 were the
most susceptible to toxicity, while the non-human host cell lines exhibited a progressively reduced
sensitivity in the order BHK-21≥MDBK>>Vero-76. Indeed, only 46% of compounds showed no
toxicity (CC50> 100 µM) against MT-4 cells, versus 76% on Vero-76 cells. Vice versa 18.6% of

8
compounds exhibited a CC50≤ 30 µM for MT-4 cells, compared with only 1.2% in the case of Vero-
76 cells.
More importantly, it is observed that the sensitivity of each host cell line is largely different versus
the single structural group of benzimidazole derivatives, making very difficult the definition of
general structure-toxicities relationships. The 2-benzylbenzimidazoles devoid of basic side-chain
are the most toxic group of compounds for Vero-76 and, particularly, for MDBK cells, but, on the
contrary, they are the least toxic for the commonly more sensitive human MT-4 cells. The
introduction of basic side-chains of whatever kind, inverted the toxicity for the foresaid cell lines: it
was minimal for MDBK and Vero-76 cells, and maximal for MT-4 cells. In the case of 1-/2-(non-
aromatic substituted)benimidazoles, the presence of a basic side chain is always bond to a net
increase of toxicity on all the four host cell lines.
Some compounds (as 35, 38, 43, 51, 53, 71 and 82), independently from the inhibition of viral
replication, may still deserve interest for their high toxicity (CC50 ≤10 µM) against one or more host
cell lines, that warrants their evaluation as possible antiproliferative agents. Indeed a strong
antiproliferative activity has been recently described for some other analogous benzimidazole
8
derivatives against a panel of 60 human tumor cell lines.
Figure 7
2.2.2. Antiviral activity and structure-activity relationships.
As indicated in Figure 7, the antiviral activity was found in all the subsets of benzimidazole
derivatives, but it was not evenly distributed among them. The activity is the results of several
interacting structural features, which may address it versus specific viruses, modulating also the
potency. Considering the two more remarkable features, it is observed that compounds bearing a
basic side-chain were more frequently active (34/48; 70.8%) than those devoid of it (18/38; 47.4%).
On the other hand 1/2-phenyl and 2-benzylbenzimidazole derivatives (with and without side-chain)

9
were more frequently active (38/59; 64.4%) than benzimidazole derivatives devoid of any aromatic
moiety in position 1 or 2 (14/27; 51.8%). The simultaneous lack of an aromatic moiety and of a
basic side-chain did not abolish, but strongly reduced the incidence of activity (5/15; 33.3%). These
values may be underestimated, because some compounds of this subset were not tested against all
viruses and some active compounds could have been undetected.
Confining the comparison within the 34 compounds with the higher activities (EC50≤ 30 µM against
at least one virus), it is observed an increasing importance of the presence of an aromatic moiety, in
respect to that of a basic side-chain for the expression of antiviral activity (24/34 compounds;
7
0.6%), while the absence of both structural features reduced the incidence of activity from 33.3%
to 11.8%. All compounds with EC50≤ 10 µM are derivatives of phenyl- (2, 22) and benzyl- (35, 36,
7, 54 and 57) benzimidazole; four of them contain a basic side-chain and three are devoid of it.
3
Beyond the definition of a generic importance of the presence of a basic side-chain for the
expression of antiviral activity, it was difficult to analyze the significance of each particular basic
chain, because of the limited availability of terms of comparison. Nevertheless it was observed that
the 1-dimethylaminoethyl- (4) and the 1-diethylaminopropyl- (5) derivatives of 2-(4-
methoxyphenyl)-5-trifluoromethylbenzimidazole were inactive, while the corresponding 1-lupinyl-
and 1-epi-lupinyl- derivatives (16 and 17) displayed some activity versus, respectively, Reo and
CVB-5 viruses. Vice versa, in the case of 2-(3,4-dichlorobenzyl)-5-trifluoromethylbenzimidazole,
the linear basic chain derivatives 32 and 33 were active against CVB-5 and Sb-1 viruses, while the
lupinyl derivative 39 was inactive.
The comparison of the 1-unsubstituted benzimidazole derivatives 52, 53 and 47 with the
corresponding 1-lupinyl derivatives 35, 36 and 38 indicated that the latter were the only active or
more active; however in the couple 48 and 41 the inactive compound was the 1-lupinyl-2-(4-
methoxybenzyl)-5-trifluoromethylbenzimidazole 41 (it is worth noting that compound 41 was found
8
endowed with potent antiproliferative activity versus a large number of human cancer cell lines).

1
0
Of course, the expression of activity is also associated to the groups that decorate the aromatic
nuclei, but it is observed that a given group may play different roles, even opposite, in relation to
the specific molecular context. These considerations may have somewhat different relevance when
considering each specific virus. Thus, for compounds active against CVB-5, RSV and BVDV (the
most frequently affected viruses) the presence of an aromatic moiety and of a basic side-chain play
always an important role, fairly in line with the foregoing general considerations. However, an
aromatic moiety is more frequently present in compounds active against RSV (87.5%) and on CVB-
5
(82.8%) and less in those active against BVDV (69.2%). Vice versa the presence of a basic chain
is more frequent in compounds active against BVDV (84.6%) and less in those active against CVB-
(68.6%) and against RSV (62.5%). These structural features have almost the same relevance for
5
the activity against CVB-5 and RSV, and indeed 12 out of the 16 compounds active against RSV
were also active against CVB-5.
On the other hand, the activity on YFV rely on the presence of a basic side chain (65, 66, 68 and
69), and activity against Sb-1 does not require, in one third of the cases (81, 82 and 86), neither the
presence of an aromatic moiety, nor that of a basic side-chain.
As far as the viral drug-sensitivity is concerned, it is worth noting the widespread sensitivity of
CVB-5 versus 35 over 75 tested compounds, with a mean EC50 of 34.5 µM. More importantly, 14
of these compounds (2, 8, 13, 33, 35, 37, 38, 45, 49, 55, 57, 58, 69 and 70) exhibited EC50 in the
range 9-17 µM (mean EC50 12.9 µM), thus resulting more potent than the reference compounds NM
108 and NM 176 (EC50= 20 and 30 µM, respectively). Also the selectivity indexes (SI= CC50/EC50)
of these compounds (with the exception of 13) are better (from ~6 up to >11) than those of the
reference drugs (>5 and 3.33, respectively), so that they may represent interesting starting models to
obtain improved agents against coxsackie viruses.
Coxsackie viruses are associated with several human pathologies (as hand-foot and mouth disease,
pleurodynia, myopericarditis, aseptic meningitis, etc.) and with damage of pancreas beta-cells,
2
9
prodromal to type 1 diabetes, but effective drugs are not availables. Ribavirin, considered a broad

1
1
spectrum antiviral agent, has been tentatively used in the treatment of coxsackie virus infections,
but with poor results, and indeed, it is practically inactive in in vitro assays (EC50> 100 µM versus
3
0
CVB-5, and, accordingly to Cheng et al., EC50> 1800 µM versus CVB-3). Three (13, 49 and 70)
of the 14 best compounds inhibited the growth of only CVB-5, while the remaining exhibited
different levels of activity against other viruses; particularly, six compounds (2, 35, 38, 55, 57 and
69) showed good activity also versus RSV.
2
b
As mentioned in the introduction, activity against RSV is a peculiarity of several chemotypes
related to benzimidazole, and, indeed, has been observed also for sixteen of the presently
investigated compounds, with the EC50 in the range 5-75 µM (mean EC50= 23.8 µM). More
importantly, the seven best compounds (54, 2, 36, 57, 22, 35 and 38, in order of decreasing SI)
displayed EC50 values in the range 5-15 µM (SI from ≥ 20 to > 6.7), that are comparable with that
of the reference drug ribavirin (EC50= 7 µM; SI≥ 14.3 ). The latter is the only drug available to treat
RSV infections, but, as a consequence of its limited efficacy, the need of prolonged aerosol
administration and the risk of toxicity, its use is restricted to children considered at high risk.
Therefore, effective and safe drugs to treat the severe RSV-linked respiratory pathologies, affecting
3
1
also adults and the elderly, are strongly needed. Since the pioneering work of Dubovi et al.,
illustrating the strong activity of bis(5-amidinobenzimidazolyl)methane (BABIM), a large number
of benzimidazole derived anti-RSV agents have been described, whose activity has been attributed
to the inhibition of the viral fusion process. Some of them even reached nano- and pico-molar
potency in vitro but so far none of them has found clinical application, because of some negative
3
2a,b,c,d
properties.
Thus the novel chemotypes of the present RSV inhibitors, exhibiting good though
not outstanding potency, might be interesting for the development of improved anti-RSV agents.
The present 1-substituted-2-benzylbenzimidazoles 27, 28, 35, 36, 40 and 57, and, maybe, also the 1-
substituted-2-phenylbenzimidazoles 7, 8 and 21, may inhibit the RSV replication by blocking the
3
2a,b
viral fusion and entry processes, as it has been demonstrated
for the structurally similar 1-
substituted-2-[(benzotriazol-1/2-yl)methyl]benzimidazoles and other analogous compounds.

1
2
The good activity of 2-(epi-lupinyl)-1-phenylbenzimidazole 2 versus RSV (EC50= 7 µM) and the
lower ones versus BVDV, CVB-5 and Sb-1, were strongly influenced by the configuration of the
basic side-chain. In fact the corresponding lupinyl derivative 1 was completely devoid of activity
versus RSV and Sb-1 and exhibited reduced activity against BVDV and CVB-5. Among the other
couples of epimeric compounds (16 and 17; 37 and 42; 39 and 43; 41 and 44; 60 and 62), higher
activity was commonly observed in the lupinyl epimers (particularly versus CVB-5, the EC50 for 37
was 10 µM and for 42 was 75 µM) or no difference could be detected, being inactive both epimers
(
39 and 43; 41 and 44).
Another interesting chemotype is represented by the pentacyclic compound 22, that is a rigid analog
of 1-benzyl-2-phenylbenzimidazole 21. Both compounds were selective inhibitors of the replication
of RSV, but the quasi planar 22 is about three times more potent than 21, whose aromatic
substituents are free to twist, assuming variable orientations in the space. The ability to adopt
”
butterfly-like” orientation is considered determinant for the expression of high inhibitory activity
on HIV-1 reverse transcriptase and on the multiplication of that virus, as observed for 1-(2,6-
3
3a,b,c
difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole and its analogs
and also for 1-benzoyl-2-
2-nitrophenyl)benzimidazole. However 1-benzyl-2-phenylbenzimidazole (21) itself is devoid of
activity against HIV-1 and the other viruses presently considered, with the exception of RSV, but it
3
4
(
3
5
has been shown to possess antitumoral activity, by inhibiting, in vitro and in vivo, the human
chondrosarcoma. An extended comparison of the antiviral and antitumoral activities of novel
derivatives
of
the
flexible
1-benzyl-2-phenylbenzimidazole
and
the
rigid
dibenzo[c,e]benzimidazo[1,2-a]azepine (22) is surely worthwhile.
2
-Trifluoromethylbenzimidazoles are an important class of compounds endowed with various
3
6a,b,c
biological activities, and particularly, the antiparasitic one.
However, when some derivatives of
this scaffold were evaluated as antivirals, they resulted either inactive, or highly cytotoxic versus
3
7
the host cell lines. Notwithstanding, the only one compound of this type we have tested (69)
exhibited a non-negligible activity against CVB-5, RSV and YFV, with EC50= 11, 22 and 33 µM,

1
3
respectively, and corresponding SI= 8.2, 4.1, 2.4. The activity of compound 69 may be related to
the presence of the cumbersome lupinyl (quinolizidin-1-ylmethyl) residue on position 1 of
benzimidazole ring, but the importance of the trifluoromethyl group is confirmed by the narrower
spectrum of activity and lower potency of the 2-methyl analog 68. Thus 69 represents another hit
for developing better antiviral agents against either one of the above viruses.
Even if activity against BVDV was shown by thirteen over eighty-five tested compounds, only two
of them (3 and 61) exhibited a worthwhile potency (EC50= 17 µM) and were selective for that virus.
The former is the unique known derivative of an unusual tetracyclic ring system
(
pyrido[3’,2’:4,5]imidazo[1,2-a][1,4]benzodiazepine-5-one) and the latter is characterized by the
connection of a lupinyl residue to position 2 of the benzimidazole nucleus, instead of the usual
N(1). Both compounds represent interesting starting points for the preparation of better anti-BVDV
agents.
Finally, among the nine compounds inhibiting the replication of the poliovirus Sb-1, three (81, 82
and 86) were devoid of aromatic substituent and of basic side-chain, thus deserving some comment.
They derive from 2-(tetrahydropyran-2-yl)benzimidazole that represent an unexplored scaffold for
the preparation of antiviral agents. Some related compounds (in particular compound 82) were
1
7
found, by some of us, to inhibit the growth of 19 human tumor cell lines at near micromolar
concentration, while lacking any anti-HIV-1 activity (the only one sought-after by us at that time).
The tetrahydropyranyl moiety of these compounds (particularly when bearing a 6-hydroxymethyl
group, as in 86) resembles the 2-glucopyranosyl moiety of some benzimidazole derivatives, that
3
8a,b
39
have been shown to inhibit the glycogen phosphorylase
and also the α-glycosidase. These
activities, besides their importance for the development of new anti-diabetic drugs, were also shown
4
0
to impact on the multiplication of viruses.
Comparing compounds 82 and 86, it is observed a 5-fold increase of activity, related to the presence
of an alcoholic group in the latter. On the other hand, it is observed that the mono-chloro derivative
81 is three times more potent than the di-chloro derivative 82, thus the mono-chloro analog of 86

1
4
could be expected to display an improved activity against Sb-1 virus. Therefore the synthesis and
evaluation of antiviral activity (particularly against Sb-1) of other derivatives of 2-(tetrahydropyran-
2
-yl)benzimidazole [including 2-(glucopyranosyl)benzimidazole] may be worthwhile. Indeed,
poliomyelitis has been practically eradicated in developed countries by extensive vaccination, but
cases still occur in sub-Saharan Africa and southern Asia, and a specific antiviral therapy is still
4
1
unavailable.
In spite of the structural analogy with 2-(glucopyranosyl)benzimidazole, all the 2-
tetrahydropyranyl)- and 2-(tetrahydrofuranyl)benzimidazoles (79-86) exhibited, paradoxally,
(
variable degree (sometime very high) of hyperglycemic activity when injected i.p. in
normoglycemic rats (unpublished results). The existence of any relation between antiviral activity
and the derangement of release and/or the metabolism of glucose is worthy of investigation.
3. Conclusions
A library of eighty-six assorted derivatives of benzimidazole was screened for antiviral activity
against a panel of ten RNA and DNA viruses, pointing out a number of novel hit compounds
suitable for future developments. Fifty-two of them (60.5%) displayed different degrees of activity
versus one or more viruses; more importantly, nineteen (22%) of them exhibited SI≥ 5, thus
representing interesting hit compounds (Table 1). Among the sensitive viruses CVB-5, RSV,
BVDV and Sb-1 were (in decreasing order) the more frequently (and effectively) affected, while
YFV, Reo-1, HSV-1, VV and VSV viruses were only occasionally (and often modestly) affected;
HIV-1 replication was not inhibited by any of these compounds. It is interesting that in several
cases CVB-5 and RSV share sensitivity versus the same compounds.
Activity was mainly found in compounds bearing in position 1 or 2 of the benzimidazole ring an
aromatic moiety (substituted phenyl or benzyl) and a basic side chain (linear or cyclic) (48.1%) or,
at least, either one of them (42.3%); only 9.6% of the active compounds is devoid of both these
structural features.

1
5
Among the thirty-five compounds endowed with activity against CVB-5, fourteen exhibited EC50 in
the range 9-17 µM (mean EC50= 12.9 µM), and among the sixteen compounds active versus RSV
seven exhibited EC50 in the range 5-15 µM (with the mean EC50= 9.4 µM), thus resulting
comparable to or more potent than the respective reference drugs (NM 108 and ribavirin).
Among the above most active compounds, 49 and 70 were selective for CVB-5, while 22 and 54
were selective for RSV; compounds 2, 35, 38 and 57 shared good activity against both CVB-5 and
RSV. Most of the best compounds active against CVB-5 and RSV were 2-benzylbenzimidazole
derivatives, bearing or not a basic side-chain. Thus, through the introduction of a variety of
substituents on the two aromatic nuclei of 2-benzylbenzimidazole, and the variation of the eventual
basic chain, improved inhibitors of CVB-5 and or RSV replication might be expected.
Besides 2-benzylbenzimidazole, other molecular cores have been identified as hits for developing
better antiviral agents, by molecular manipulation. Thus, taking as models compounds 2 and 69,
from 1-phenylbenzimidazole and 2-trifluoromethylbenzimidazole large spectrum antivirals could be
achieved. On the other hand, starting from the unexplored, rigid scaffolds of compounds 3
(
pyrido[3’,2’:4,5]imidazo[1,2-a][1,4]benzodiazepine-5-one) and 22 (dibenzo[c,e]benzimidazo[1,2-
a]azepine), improved and selective inhibitors versus, respectively, BVDV and RSV could be
obtained. Finally, on the 2-(tetrahydropyran-2-yl)benzimidazole core of 81, 82 and 86, better
antiviral molecules, particularly versus Sb-1, could be constructed.
Effective therapeutic agents for the human pathologies related to CVB-5 and RSV viruses are
unavailable, and hence urgently needed, but, despite the extensive vaccination, also agents against
poliovirus Sb-1 may be important in the underdeveloped countries.
4. Experimental
4.1. General. Chemicals, solvents and reagents used for the syntheses were purchased from Sigma-
Aldrich, Fluka or Alfa Aesar, and were used without any further purification. Column
chromatography (CC): neutral alumina (Al ), activity 1 (Merck). Mps: Büchi apparatus,
2
O
3

1
6
1
uncorrected. H NMR spectra: Varian Gemini-200 spectrometer; CDCl
to Me Si as internal standard. J in Hz. Elemental analyses were performed on a Carlo Erba EA-
110 CHNS-O instrument in the Microanalysis Laboratory of the Department of Pharmacy of
Genoa University.
3
or DMSO-d6; δ in ppm rel.
4
1
4.2. Benzimidazoles from iminoesters. General method of Scheme 1.
Cooling at 0°C, a solution of 4-substituted- or 3,4-disubstituted-phenylacetonitrile or of
4
2
cyanolupinane (6 mmol) and 0.35 ml of abs. EtOH in 5 mL of CHCl
3
was saturated with
anhydrous HCl and left overnight at 0-5°C. After evaporation of the solvent the residue was reacted
1
1
1
with the proper 1,2-phenylenediamine or its N -alkyl- , N -dialkylaminoalkyl- or N -
(octahydroquinolizin-1yl)methyl]derivative (3 mmol) in glacial CH COOH (8 mL) and the mixture
was heated at 45°C for 12 h. Then the reaction mixture was concentrated and the residue was taken
up with dil. aq. HCl and extracted with Et O. The acidic solution was basified with 2N NH and
extracted with Et O. The dried organic layer (Na SO ) was evaporated to afford an oily residue that
crystallized from the suitable solvent or was purified by CC.
Alternatively, the residue of iminoester hydrochloride was reacted with the 3,4-dichloro-1,2-
phenylenediamine in anhydrous CHCl solution (10 mL). After refluxing the mixture for 10 h the
[
3
2
3
2
2
4
3
2 4
chloroform solution was washed with water, dried (Na SO ) and evaporated; the residue was
crystallized from the indicated solvent.
1
Most of the required N -substituted-1,2-phenylenediamine were commercially available or
1
9,13,43
previously described.
Section.
The preparation of the only novel diamine is described at the end of the
4
.2.1. 2-(4-Bromobenzyl)-1-[(2-diethylamino)ethyl]benzimidazole (27)
Yield: 28%. M.p. 85-87°C (Et O/pentane). H NMR (CDCl ): 1.08 (t, J = 7.2, 6H, N(CH
.47-2.76 (m, 6H, CH CH N(CH CH ); 4.10-4.28 (m, 2H, CH CH N(C )); 4.37 (s, 2H, CH -
2
3
2 3 2
CH ) );
2
2
2
2
3
)
2
2
2
2
H
5
)
2
2

1
7
Ar); 7.18 (d, J = 8.4, 2 arom. H); 7.23-7.41 (m, 3 arom. H); 7.46 (d, J = 8.4, 2 arom. H); 7.73-7.84
m, 1 arom. H). Anal. Calcd for C20 : C, 62.18; H, 6.26; N, 10.88. Found: C, 62.40; H, 6.34;
N, 10.62.
(
H24BrN
3
4.2.2. 1-[(2-Diethylamino)ethyl]-2-(4-iodobenzyl)benzimidazole (28)
1
Yield: 42%. M.p. 79-82°C (Et
2
O/pentane). H NMR (CDCl
3
): 1.05 (t, J = 7.2, 6H, N(CH
CH N(C ) and 4.38, s, 2H,
-Ar, superimposed); 7.09 (d, J = 8.2, 2 arom. H); 7.17-7.45 (m, 3 arom. H); 7.66 (d, J = 8.2, 2
2 3 2
CH ) );
2
.46-2.77 (m, 6H, CH
2
CH
2
N(CH
2
CH
3
)
2
); 4.10-4.43 (m, 2H, CH
2
2
2 5 2
H )
CH
2
arom. H); 7.73-7.83 (m, 1 arom. H). Anal. Calcd for C20
C, 55.22; H, 5.62; N, 9.59.
24 3
H N I: C, 55.43; H, 5.58; N, 9.70. Found:
4
.2.3.
32)
Yield: 30%. M.p. 90-91°C (Et
CH CH N(CH )); 4.22-4.55 (m, 2H, CH
m, 1 arom. H); 7.34-7.59 (m, 4 arom. H); 8.07 (s, 1 arom. H). Anal. Calcd for C19
2-[(3,4-Dichloro)benzyl]-1-[(2-dimethylamino)ethyl]-5-trifluoromethylbenzimidazole
(
1
2
O). H NMR (CDCl
3
): 2.42 (s, N(CH
-Ar, superimposed); 7.14-7.26
: C,
3 2
) ); 2.63 (t, J = 7.0, 2H,
2
2
3
)
2
2
-Q and 4.41, s, 2H, CH
2
(
H
18Cl
2
F
3
N
3
54.82; H, 4.36; N, 10.09. Found: C, 54.50; H, 4.62; N, 10.00.
4.2.4.
2-[(3,4-Dichloro)benzyl]-1-[(3-diethylamino)propyl]-5-trifluoromethylbenzimidazole
(
33)
1
Yield: 31%. M.p. 53-54°C (pentane). H NMR (CDCl
3
): 1.07 (t, J = 7.2, 6H, N(CH
CH N(C )); 2.59 (q, 4H,
); 4.39 (s, 2H, CH -Ar); 7.08 (dd, J = 8.6,
.2, 1 arom. H); 7.36-7.62 (m, 4 arom. H); 8.07 (s, 1 arom. H). Anal. Calcd for C22 : C,
7.65; H, 5.28; N, 9.17. Found: C, 58.27; H, 5.37; N, 9.17.
.2.5. 2-[(4-Bromo)benzyl]-1-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-
2 3 2
CH ) ); 1.81-
2
.00 (m, 2H, CH
2
CH
2
CH
2
N(C
2
5
H )
2
); 2.44 (t, J = 7.0, 2H, CH
2
2
2 5 2
H )
N(CH CH ); 4.18 (t, J = 7.3, 2H, CH
2
3
)
2
2
CH CH N(C
2
2
2
H
5
)
2
2
2
5
4
2 3 3
H24Cl F N
methylbenzimidazole (40)
Yield: 28%. M.p. 148-151°C (Et
near N of Q); 4.20-4.58 (m, 2H, CH
1
2
O). H NMR (CDCl
3
): 1.18-2.58 (m, 14H, Q); 2.84-3.30 (m, 2H
α
2
-Q and 4.34, s, 2H, CH
2
-Ar superimposed); 7.18 (d, J = 8.0, 2

1
8
3 3 2
arom. H); 7.34-7.57 (m, 4 arom. H); 8.05 (s, 1 arom. H). Anal. Calcd for C25H27BrF N +0.25H O:
C, 58.77; H, 5.42; N, 8.22. Found: C, 58.71; H, 5.72; N, 8.16.
4
.2.6.
methylbenzimidazole (42)
Yield: 35%. M.p. 99-101°C (Et
.90-3.18 (m, 2H near N of Q); 3.40-3.60 (m, 1H, CH
AB syst., 2H, CH -Ar.); 7.20-7.57 (m, 7 arom. H); 8.07 (s, 1 arom. H). Anal. Calcd for
: C, 70.24; H, 6.60; N, 9.83. Found: C, 70.22; H, 6.48; N, 9.91.
.2.7. 2-[3,4-(Dichloro)benzyl]-1-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-
methylbenzimidazole (43)
Yield: 38%. M.p. 125-126°C (Et
near N of Q); 3.60-3.82 (m, 1H, CH
superimposed); 7.13-7.62 (m, 5 arom. H); 8.07 (s, 1 arom. H). Anal. Calcd for C25
2-Benzyl-1-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-
1
2
O). CC (Al
2
O
3
/ Et
2
O). H NMR (CDCl
3
): 0.78-2.45 (m, 14H, Q);
-Q); 4.32-4.54
2
α
2
-Q); 4.16-4.29 (m, 1H, CH
2
(
2
25 28 3 3
C H F N
4
1
2
O). H NMR (CDCl
3
): 0.93-2.62 (m, 14H, Q); 3.10-3.38 (m, 2H
-Q and AB syst., 2H, CH -Ar.,
: C,
α
2
-Q); 4.18-4.51 (m, 1H, CH
2
2
H
2 3 3
26Cl F N
60.49; H, 5.28; N, 8.46. Found: C, 60.53; H, 5.33; N, 8.34.
4.2.8. 2-[4-(Methoxy)benzyl]-1-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-
methylbenzimidazole (44)
1
Yield: 35%. M.p. 116-117°C (pentane/Et
2
O 2:1). CC (Al
2
O
3
/ Et
2
O). H NMR (CDCl
-Q and 3.81, s, OCH
-Ar., superimposed); 6.88 (d, J =
.8, 2 arom. H); 7.20 (d, J = 8.8, 2 arom. H). 7.32-7.57 (m, 2 arom. H); 8.07 (s, 1 arom. H). Anal.
Calcd for C26 O: C, 68.25; H, 6.61; N, 9.18. Found: C, 68.59; H, 6.67; N, 9.26.
.2.9. 2-[(3,4-Dichloro)benzyl]-5,6-dichloro-1H-benzimidazole (52)
3
): 0.84-2.60
(
m, 14H, Q); 3.04-3.35 (m, 2H
α
near N of Q); 3.57-3.84 (m, 1H, CH
2
3
,
superimposed); 4.12-4.46 (m, 1H, CH
2
-Q and AB syst., 2H, CH
2
8
30 3 3
H F N
4
1
Yield: 32%. M.p. 204-207°C (Et
.0, 1 arom. H); 7.54-7.70 (m, 2 arom. H); 7.79 (s, 2 arom. H); 12.67 (br. s, NH benzim., collapses
with D O). Anal. Calcd for C14 Cl : C, 48.59; H, 2.33; N, 8.09. Found: C, 48.40; H, 2.27; N,
.06.
2
O). H NMR (DMSO-d
6
): 4.25 (s, 2H, CH
2
-Ar); 7.33 (dd, J = 8.0,
2
2
H
8
4 2
N
8

1
9
4.2.10. 2-[(4-Bromo)benzyl]-5,6-dichloro-1H-benzimidazole (53)
1
Yield: 31%. M.p. 224-227°C (CHCl
3
/ Et
2
O 1:1). H NMR (DMSO-d
6
): 4.19 (s, 2H, CH -Ar); 7.30
2
(
d, J = 8.4, 2 arom. H); 7.54 (d, J = 8.4, 2 arom. H); 7.78 (s, 2 arom. H); 12.65 (br. s, NH benzim.,
2 9 2 2 2
collapses with D O). Anal. Calcd for C14H BrCl N +0.25H O: C, 46.69; H, 2.66; N, 7.78. Found:
C, 46.75; H, 2.88; N, 7.70.
4.2.11. 5,6-Dichloro-2-[(4-methoxy)benzyl]-1H-benzimidazole (54)
1
Yield: 73%. M.p. 191-193°C (CHCl
3
/ Et
2
O 1:1). H NMR (DMSO-d
6
): 4.11 (s, 5H, CH -Ar and
2
OCH
3
, superimposed); 6.10 (d, J = 8.8, 2 arom. H); 6.45 (d, J = 8.8, 2 arom. H); 6.86 (s, 2 arom.
H); 12.58 (br. s, NH-Benz., collapses with D
N, 9.12. Found: C, 58.58; H, 4.11; N, 9.00.
2 2 2
O). Anal. Calcd for C15H12Cl N O: C, 58.65; H, 3.94;
4.2.12. 1-Butyl-2-(3,4-dichlorobenzyl)-5-trifluoromethylbenzimidazole (57)
1
Yield: 45%. M.p. 71-74°C (pentane/Et
2
O 2:1). H NMR (CDCl
CH CH CH ); 1.50-1.77 (m, 2H, CH
); 4.41 (s, 2H, CH -Ar); 7.12-7.64 (m, 5 arom. H); 8.12 (s, 1 arom.
2 3 2
17Cl F N : C, 56.87; H, 4.27; N, 6.98. Found: C, 56.60; H, 4.17; N, 6.87.
3
): 0.95 (t, J = 7.4, 3H,
CH
t, J = 7.6, 2H, CH
H). Anal. Calcd for C19
2
CH
2
CH
2
CH
3
); 1.26-1.46 (m, 2H, CH
2
2
2
3
2
CH CH CH ); 4.11
2
2
3
(
2
CH CH CH
2
2
3
2
H
4.2.13. 2-(3,4-Dichlorobenzyl)-1-[(2-methoxy)ethyl]-5-trifluoromethylbenzimidazole (58)
1
Yield: 47%. M.p. 104-105°C (pentane/Et
2
O 2:1). H NMR (CDCl
3
): 3.27 (s, 3H, CH
CH OCH ); 4.46 (s, 2H, CH
.12-7.63 (m, 5 arom. H); 8.11 (s, 1 arom. H). Anal. Calcd for C18 O: C, 53.62; H, 3.75;
2
CH
2
OCH
3
);
3
.63 (t, J = 5.2, 2H, CH
2
CH
2
OCH
3
); 4.30 (t, J = 5.4, 2H, CH
2
2
3
2
-Ar);
7
H
2 3 2
15Cl F N
N, 6.95. Found: C, 53.39; H, 3.54; N, 7.24.
4.2.14. 2-[(1S,9aR)-Octahydro-2H-quinolizin-1-yl]benzimidazole (60)
1
Yield: 41%. M.p. 232-234°C (CH
near N of Q and 2H, CH
NH-Benz., exchanges with D O). Anal. Calcd for C17
5.92; H, 8.66; N, 15.70.
2
Cl
2
). H NMR (CDCl
3
): 1.20-2.40 (m, 14H, Q); 2.56-3.42 (m,
2
H
α
2
-Q); 7.22-7.37 (m, 2 arom. H); 7.55-7.86 (m, 2 arom. H); 10.05 (s,
: C, 75.80; H, 8.61; N, 15.60. Found: C,
2
23 3
H N
7

2
0
4.3. Benzimidazoles from acyl chlorides. General method of Scheme 2.
To a solution of the suitable N-phenyl- or 4-R-1,2-phenylenediamine (2 mmol) in anhydrous
4
4
dioxane (4 mL), a solution of homolupinanoyl or epi-homolupinanoyl chloride hydrochloride (2
mmol) in anhydrous dioxane (4 mL) was added drop by drop, and the mixture was refluxed for 4 h.
1
Similarly, a solution of N -[(1S,9aR)-(octahydroquinolizin-1yl)methyl]-4-trifluoromethyl-1,2-
1
3
phenylenediamine (3 mmol) obtained as described and 2,6-difluorobenzoyl chloride (3 mmol) in
anhydrous dioxane (6 mL) was refluxed for 4 h. In both cases, the solvent was removed and the
residue was taken up in 4N HCl (15 mL) and refluxed for 3 h and 12 h, respectively. The acidic
2 2 4
solution was basified with 6N NaOH and extracted with Et O; the dried organic layer (Na SO ) was
evaporated and the residue purified by CC (Al /Et O).
2
O
3
2
4.3.1. 2-[(1S,9aR)-Octahydro-2H-quinolizin-1-yl]-1-phenylbenzimidazole (1)
1
Yield: 40%. M.p. 106-108°C (Et
near N of Q and 2H, CH
: C, 79.96; H, 7.88; N, 12.16. Found: C, 79.77; H, 7.91; N, 12.15.
.3.2. 2-[(1R,9aR)-Octahydro-2H-quinolizin-1-yl]-1-phenylbenzimidazole (2)
2
O). H NMR (CDCl
3
): 1.10-2.50 (m, 14H, Q); 2.68-3.30 (m, 4H,
2
H
α
2
-Q); 7.10-7.70 (m, 8 arom. H); 7.8 (d, J= 8.8, 1 arom. H). Anal. Calcd
27 3
for C23H N
4
1
Yield: 32%. M.p. 102-103°C (Et
CH -Q); 2.71-3.00 (m, 2H near N of Q); 3.06-3.18 (m, 1H, CH
.78 (d, J= 9.0, 1 arom. H). Anal. Calcd for C23 : C, 79.96; H, 7.88; N, 12.16. Found: C, 80.09;
H, 7.87; N, 12.23.
.3.3. 2-[(2,6-Difluoro)phenyl]-1-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-
methylbenzimidazole (14)
Yield: 39%. M.p. 118-121°C (pentane). H NMR (CDCl
2
O). H NMR (CDCl
3
): 0.86-2.20 (m, 14H, Q); 2.44-2.68 (m, 1H,
2
α
2
-Q); 7.10-7.67 (m, 8 arom. H);
7
27 3
H N
4
1
3
): 0.90-2.20 (m, 14H, Q); 2.63-3.18 (m,
-Q); 7.07-7.24 (m, 2 arom. H); 7.46-7.80 (m, 3 arom. H);
+0.5H O: C, 62.87; H, 5.50; N, 9.17. Found: C,
2
8
6
H
α
near N of Q); 4.27-4.56 (m, 2H, CH
2
.16 (s, 1 arom. H). Anal. Calcd for C24
3.18; H, 5.81; N, 9.29.
H
24
F
5
N
3
2

2
1
4.3.4. 5-Chloro-2-[(1S,9aR)-Octahydro-2H-quinolizin-1-yl]benzimidazole (61)
1
Yield: 45%. M.p. 172-173°C (toluene). H NMR (CDCl
3
): 1.15-2.42 (m, 14H, Q); 2.90-3.14 (m,
-Q); 7.18 (d, J= 8.6, 1 arom. H); 7.40-7.58 (m, 2 arom.
O). Anal. Calcd for C17 : C, 67.20; H, 7.30; N,
2
H
α
near N of Q); 3.24 (d, J= 6.0, 2H, CH
2
H); 9.98 (s, NH-Benz., exchanges with D
2
H22ClN
3
13.83. Found: C, 66.85; H, 6.95; N, 13.81.
4.3.5. 2-[(1R,9aR)-Octahydro-2H-quinolizin-1-yl]benzimidazole (62)
1
Yield: 42%. M.p. 231-233°C (CH
2
Cl
2
). H NMR (CDCl
3
): 1.10-2.40 (m, 14H, Q); 2.63-2.78 (m,
-Q); 7.17-7.30 (m, 2 arom. H);
O). Anal. Calcd for C17
O: C, 74.56; H, 8.65; N, 15.34. Found: C, 74.85; H, 8.69; N, 15.57.
1
7
0
H, CH
.48-7.65 (m, 2 arom. H); 10.06 (s, NH-Benz., exchanges with D
.25H
2
-Q); 2.88-3.13 (m, 2H
α
near N of Q); 3.18-3.37 (m, 1H, CH
2
2
23 3
H N +
2
4.4. Benzimidazoles from acids. General method of Scheme 3.
a) A mixture of 4-aminophenylacetic acid (5.3 mmol) and 4-trifluoromethyl- or 4,5-dichloro-1,2-
phenylenediamine (3.4 mmol) in 15 mL of 4N HCl was refluxed for 10 h. After cooling, the
solution was neutralized with 2N NaOH and extracted with Et
2
O. The dried organic layer (Na
2
SO
O.
or d,l-lupanine
extracted from Lupinus albus seeds) were dissolved, respectively in 6N HCl (1g in 4 mL) and
conc. HCl (1g in 3 mL). The former solution was refluxed for 6 h, while the latter required 72 h at
4
)
was evaporated to afford a spongy residue that was purified by CC and crystallized from dry Et
2
4
5a,b
b) Tetrahydrocytisine, obtained by catalytic hydrogenation of cytisine,
(
2
5
1
50°C (closed tube). The acid concentration was adjusted with water to about 4N, then the
equimolar amount of the suitable 1,2-phenylenediamine was added and the mixture refluxed for 10
h. After cooling the acidic solution was worked up as in point a).
4.4.1. 2-[4-(Amino)benzyl]-5-trifluoromethyl-1H-benzimidazole (49)
1
Yield: 58%. M.p. 170-171°C (Et
2
O). CC (Al
2
O
3
/ Et
2
O). H NMR (DMSO-d
6
): 4.04 (s, 2H, CH -
2
Ar); 4.99 (s, NH
2
-Ar); 6.52 (d, J = 8.6, 2 arom. H); 6.98 (d, J= 8.6, 2 arom. H); 7.44 (d, J = 8.2, 1

2
2
arom. H); 7.66 (d, J = 8.2, 1 arom. H); 7.83 (s, 1 arom. H); 12.58 (br. s, NH-Benz., exchanges with
O). Anal. Calcd for C15 : C, 61.85; H, 4.15; N, 14.43. Found: C, 61.90; H, 4.18; N, 14.56.
D
2
12 3 3
H F N
4.4.2. 2-[(4-Amino)benzyl]-5,6-dichloro-1H-benzimidazole (55)
1
Yield: 73%. M.p. 113-115°C (Et
2
O). H NMR (DMSO-d
6
): 3.37 (s, NH
2
-Ar); 3.99 (s, 2H, CH -Ar);
2
6.52 (d, J = 8.6, 2 arom. H); 6.98 (d, J = 8.4, 2 arom. H); 7.74 (s, 2 arom. H); 12.52 (br. s, NH-
2 2 3 2
Benz., collapses with D O). Anal. Calcd for C14H11Cl N +0.5H O: C, 55.83; H, 4.01; N, 13.95.
Found: C, 55.59; H, 4.17; N, 13.75.
4.4.3. 2-[3-(3,7-diazabicyclo[3.3.1]nonan-2-yl)propyl]-1H-benzimidazole (64)
1
Yield: 28%. Oil. H NMR (CDCl
3
): 1.12-2.40 (m, 8H and 1H, NH exchanges with D
O); 7.10-7.30 (m, 2 arom. H); 7.43-
.72 (m, 2 arom. H); 9.96 (s, NH-Benz., collapses with D O). Hydrochloride, M.p. 250°C
O). Anal. Calcd for C17 +3HCl: C, 51.85; H, 6.91; N, 14.23. Found: C, 51.64; H,
.25; N, 13.88.
2
O); 2.68-3.82
(
m, 8H); 3.95-4.40 (m, 1H); 6.00 (br. s, NH, exchanges with D
2
7
2
(
EtOH/Et
2
24 4
H N
7
4
.4.4. 5-Chloro-2-[3-(3,7-diazabicyclo[3.3.1]nonan-2-yl)propyl]-1H-benzimidazole (65)
1
Yield: 27%. Oil. H NMR (CDCl
3
): 1.10-2.40 (m, 8H and 1H, NH exchanges with D
O); 7.10 (dd, J = 8.6, 2.0, 1 arom.
H); 7.37-7.58 (m, 2 arom. H); 10.05 (s, NH-Benz., collapses with D O). Hydrochloride, M.p.
250°C (EtOH/Et O). Anal. Calcd for C17 +3HCl: C, 47.68; H, 6.12; N, 13.08. Found: C,
7.59; H, 6.30; N, 12.76.
.4.5. 4-[3-(1H-Benzimidazol-2-yl)propyl]decahydro-1H-1,5-methanopyrido[1,2-a]
2
O); 2.72-3.88
(
m, 8H); 4.00-4.42 (m, 1H); 5.60 (br. s, NH, exchanges with D
2
2
>
2
H23ClN
4
4
4
[
1,5]diazocine (66)
1
Yield: 13%. Oil. H NMR (CDCl
3
): 0.96-2.20 (m, 15H); 2.50-3.00 (m, 6H); 3.04-3.36 (m, 3H); 5.40
O); 7.06-7.26 (m, 2 arom. H); 7.36-7.67 (m, 2 arom. H); 9.97 (s, NH-
O). Hydrochloride, M.p. >270°C (EtOH/Et O). Anal. Calcd for
2
O: C, 54.14; H, 7.57; N, 12.03. Found: C, 54.00; H, 7.91; N, 11.46.
(
br. s, NH exchanges with D
Benz., collapses with D
+3HCl+ H
2
2
2
C
21
H
30
N
4

2
3
4
.4.6. 4-[3-(5-Chloro-1H-benzimidazol-2-yl)propyl]decahydro-1H-1,5-methanopyrido[1,2-a]
1,5] diazocine (67)
Yield: 15%. M.p. 113-116°C (Et
[
1
2
O). H NMR (CDCl
3
): 1.00-2.20 (m, 15H); 2.56-3.00 (m, 6H);
3
7
6
.06-3.40 (m, 3H); 5.50 (br. s, NH exchanges with D
2
O); 7.14 (dd, J = 8.4, 1.8, 1 arom. H); 7.40-
.60 (m, 2 arom. H); 10.00 (s, NH-Benz., collapses with D
7.63; H, 7.84; N, 15.02. Found: C, 67.53; H, 8.18; N, 15.09.
2 4
O). Anal. Calcd for C21H29ClN : C,
4.5. Benzimidazoles from acids. General method of Scheme 4.
A mixture of 1,2-pheylenediamine or 4-chloro-1,2-phenylendiamine (5 mmol) and 2-ethyl-2-
hexenoic acid or tetrahydropyran-2-carboxylic acid (5 mmol) was heated in a sealed tube at 180°C
for 2h. The residue was taken up with dil. aq. HCl and filtered; then the acidic solution was washed
with Et
2
O, made basic with 6N NaOH and extracted with Et
O.
2
O. The solvent was removed and the
residue crystallized from dry Et
2
4.5.1. 2-(Hept-3-en-3-yl)-1H-benzimidazole (76).
1
2 3
Yield: 51%. M.p. 182-184°C (Et O). H NMR (CDCl ): 0.90 (t, J = 6.4, 3H, C(7)); 1.02 (t, J = 6.4,
3
H, C(1)); 1.44 (q, 2H, C(6)); 2.00 (q, 2H, C(2)); 2.08-2.26 (m, 2H, C(5)); 5.80 (t, J = 7.0, 1H,
C(4)); 7.16-7.28 (m, 2 arom. H); 7.40-7.68 (m, 2 arom. H); 10.12 (s, NH-Benz., exchanges with
O). Anal. Calcd for C14 : C, 78.46; H, 8.47; N, 13.07. Found: C, 78.38; H, 8.60; N, 13.12.
D
2
18 2
H N
4.5.2. 2-(Tetrahydropyran-2-yl)-5-trifluoromethyl-1H-benzimidazole (80)
1
Yield: 38%. M.p. 213-215°C (Et
2
O). H NMR (CDCl
3
): 1.68-1.94 (m, 2H, C(5) and 1H, C(4),
pyrane); 2.08-2.30 (m, 1H, C(4) and 1H, C(3), pyrane); 2.52-2.73 (m, 1H, C(3), pyrane); 3.70-4.12
m, 2H, C(6)); 6.05 (dd, J= 8.4, 2.8, 1H, C(2)); 7.16 (d, J= 8.8, 1 arom. H); 7.40-7.56 (m, 2 arom.
H); 10.82 (s, NH-Benz., exchanges with D O). Anal. Calcd for C13 O: C, 57.78; H, 4.85; N,
(
2
13 3 2
H F N
10.37. Found: C, 58.00; H, 5.01; N, 10.42.
4.5.3. 5-Chloro-2-(tetrahydropyran-2-yl)-1H-benzimidazole (81)

2
4
1
Yield: 33%. M.p. 166-167°C (Et
pyrane); 2.05-2.32 (m, 1H, C(4) and 1H, C(3), pyrane); 2.46-2.69 (m, 1H, C(3), pyrane); 3.75-4.20
m, 2H, C(6)); 6.05 (dd, J= 8.6, 2.8, 1H, C(2)); 7.26 (d, J= 9.0, 1 arom. H); 7.46-7.67 (m, 2 arom.
H); 12.28 (s, NH-Benz., exchanges with D O). Anal. Calcd for C12 O: C, 60.89; H, 5.54; N,
2 3
O). H NMR (CDCl ): 1.62-1.95 (m, 2H, C(5) and 1H, C(4),
(
2
H13ClN
2
11.84. Found: C, 60.68; H, 5.60; N, 11.72.
4.6. Benzimidazoles from aldehyde-sodium bisulfite adducts. General method of Scheme 5.
1
1
13
1
To a solution of the proper N -alkyl- or N -(dialkylaminoalkyl)-
octahydroquinolizin-1-yl)methyl]- 4-trifluoromethyl-1,2-phenylenediamine (3.5 mmol) in 18 mL
of EtOH the 4-methoxybenzaldheyde sodium bisulphite adduct (3.5 mmol) (prepared according to
or N -[(1R,9aR)-
(
4
6
Shriner and Land) was added and the mixture was refluxed for 3 h with stirring. Afterwards, the
solvent was evaporated and the residue was taken up with water and filtered, or extracted with Et O.
O; the oily 17 was converted into the
2
The solid compounds were crystallized from dry Et
monohydrochloride with 1N ethanolic solution of HCl.
2
4.6.1. 1-[(2-Dimethylamino)ethyl]-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (4)
1
Yield: 64%. M.p. 112-113°C (Et
2
O). H NMR (CDCl
); 4.48 (t, J = 7.3, 2H, CH
H); 7.59 (s, 2 arom. H); 7.08 (d, J = 8.8, 2 arom. H); 8.09 (s, 1 arom. H). Anal. Calcd for
O: C, 62.80; H, 5.55; N, 11.56. Found: C, 63.10; H, 5.71; N, 11.72.
3
): 2.32 (s, 6H, N(CH
3 2
) ); 2.77 (t, J = 7.1, 2H,
CH
2
CH
2
N(CH
3
)
2
)); 3.92 (s, OCH
3
2
CH N(CH ); 7.08 (d, J = 8.8, 2 arom.
2
3 2
)
19 20 3 3
C H F N
4.6.2. 1-[(3-Diethylamino)propyl]-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (5)
1
Yield: 64%. M.p. 107-109°C (Et
2
O). H NMR (CDCl
3
): 1.05 (t, J = 7.2, 6H, N(CH
CH N(CH CH )); 3.93 (s, OCH
); 7.08 (d, J = 8.6, 2 arom. H); 7.57 (s, 2 arom. H); 7.72
O: C, 65.17; H, 6.46; N,
2
CH
3
)
2
); 1.94-
2
.16 (m, 2H, CH
2
CH
2
CH
2
N(C
2
H
5
)
2
); 2.41-2.77 (m, 6H, CH
2
2
2
3
)
2
3
);
4.14 (t, J = 7.3, 2H, CH
2
CH
2
CH
2
N(C
2 5 2
H )
(
d, J = 8.4, 2 arom. H); 8.09 (s, 1 arom. H). Anal. Calcd for C22
0.36. Found: C, 65.51; H, 6.70; N, 10.50.
26 3 3
H F N
1

2
5
4.6.3.
4-(Methoxyphenyl)-1-[(1R,9aR)-Octahydro-2H-quinolizin-1-yl]-5-trifluoromethyl-
benzimidazole (17)
1
Yield: 35%. Oil. H NMR (CDCl
3
): 0.86-2.23 (m, 14H, Q); 2.42-2.64 (m, 1H, CH
-Q); 3.87 (s, OCH ); 7.05 (d, J = 8.8, 2 arom. H); 7.62-
.94 (m, 4 arom. H); 8.08 (s, 1 arom. H). Hydrochloride, M.p.>178-202°C (EtOH/Et O). Anal.
Calcd for C25 O+HCl+H O: C, 60.30; H, 6.43; N, 8.44. Found: C, 60.26; H, 6.13; N, 8.36.
.6.4. 1-Butyl-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (18)
2
-Q); 2.67-3.00
(
m, 2H
α
near N of Q); 3.05-3.16 (m, 1H, CH
2
3
7
2
H
29ClF
3
N
3
2
4
1
Yield: 48%. M.p. 81-82°C (Et
CH CH CH CH ); 1.24-1.42 (m, 2H, CH
s, OCH ); 4.32 (t, J = 7.8, 2H, CH CH
2
O/pentane 1:1). H NMR (CDCl
CH CH CH ); 1.76-1.95 (m, 2H, CH
CH CH ); 7.10 (d, J = 9.0, 2 arom. H); 7.44-7.64 (m, 2
3
): 0.92 (t, J = 7.4, 3H,
2
2
2
3
2
2
2
3
2
CH CH CH ); 3.93
2
2
3
(
3
2
2
2
3
19 3 2
arom. H); 7.73 (d, J = 9.0, 2 arom. H); 8.16 (s, 1 arom. H). Anal. Calcd for C19H F N O: C, 65.50;
H, 5.50; N, 8.04. Found: C, 66.43; H, 5.42; N, 7.83.
4.6.5. 1-[(2-Methoxy)ethyl]-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (19)
1
Yield: 52%. M.p. 110-112°C (Et
2
O). H NMR (CDCl
); 4.49 (t, J = 5.4, 2H, CH
arom. H); 7.55-7.69 (m, 2 arom. H); 7.84 (d, J = 8.8, 2 arom. H); 8.16 (s, 1 arom. H). Anal. Calcd
: C, 61.71; H, 4.89; N, 8.00. Found: C, 61.49; H, 4.63; N, 8.00.
3
): 3.32 (s, 3H, CH
2
CH
2
OCH
3
); 3.82 (t, J =
5
.4, 2H, CH
2
CH
2
OCH
3
); 3.92 (s, OCH
3
2
CH OCH
2
3
); 7.09 (d, J = 9.0, 2
17 3 2 2
for C18H F N O
4.7. Acetylation of 2-(4-aminobenzyl)benzimidazoles. General method of Scheme 6.
2
0
A suspension of the 2-[(4-amino)benzyl]benzimidazole 45 or of the above 2-[(4-amino)benzyl]-5-
trifluoromethyl- or 5,6-dichlorobenzimidazoles (49, 55) (1.2 mmol) in 2 mL of anh. benzene and
2
3.6 mmol of Ac O was refluxed for 2 h. After removing the solvent, the residue was taken up with 5
mL of EtOH plus 1 mL of water and refluxed for 2 h, in order to hydrolyze any acetyl groups
attached to benzimidazole nitrogen. The solvent was evaporated under vacuum and the residue
taken up with water, filtered and washed with water. The compounds were crystallized from
2
EtOH/H O.

2
6
4.7.1. 2-[4-(Acetylamino)benzyl]-1H-benzimidazole (46)
1
Yield: 63%. M.p. 243-245°C (EtOH/H
2
O). H NMR (CDCl
3
): 2.19 (s, CH
3
CO); 4.56 (s, 2H, CH
O);
O: C, 72.43; H, 5.70; N,
2
-
Ar); 7.16-7.55 (m, 6 arom. H); 7.75-8.20 (m, 2 arom. H); 9.88 (s, NHCO, collapses with D
2
1
1
4
2.28 (br. s, NH-Benz., collapses with D
2
O). Anal. Calcd for C16
H
15
N
3
5.84. Found: C, 72.64; H, 5.94; N, 16.09.
.7.2. 2-[4-(Acetylamino)benzyl]-5-trifluoromethyl-1H-benzimidazole (50)
1
Yield: 82%. M.p. 240-241°C (EtOH/H
CH
s, 1 arom. H); 9.94 (s, NHCO, collapses with D
Anal. Calcd for C17 O+0.25H O: C, 60.45; H, 4.32; N, 12.43. Found: C, 60.09; H, 4.14; N,
2
O). H NMR (DMSO-d
6
3
): 2.09 (s, CH CO); 4.19 (s, 2H,
2
-Ar); 7.26 (d, J = 8.2, 2 arom. H); 7.41-7.60 (m, 2 arom. H); 7.67 (d, J = 8.2, 2 arom. H); 7.85
O); 12.68 (br. s, NH-Benz., collapses with D O).
(
2
2
H
14
F
3
N
3
2
12.50.
4.7.3. 2-[(4-Acetylamino)benzyl]-5,6-dichloro-1H-benzimidazole (56)
1
Yield: 75%. M.p. 270-271°C (EtOH/H
CH
NHCO, collapses with D
O+ 0.25H O: C, 56.74; H, 4.02; N, 12.41. Found: C, 56.64; H, 3.93; N, 12.26.
2
O). H NMR (DMSO-d
6
3
): 2.03 (s, CH CO); 4.14 (s, 2H,
2
-Ar); 7.24 (d, J = 8.4, 2 arom. H); 7.53 (d, J = 8.2, 2 arom. H); 7.80 (s, 2 arom. H); 9.93 (s,
O); 12.57 (br. s, NH-Benz., collapses with D O). Anal. Calcd for
2
2
C
16
H
13Cl
2
N
3
2
4.8.
2-[(4-Bromo)benzyl]-5,6-dichloro-1-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl)]-
benzimidazole (36). (Scheme 7)
A solution of 3.5 mmol of the above described 2-[4-(bromo)benzyl]-5,6-dichlorobenzimidazole (53)
in anhydrous DMF (6 mL) was treated with finely ground NaNH
at 45°C under a stream of dry N . Then a solution of chlorolupinane (3.8 mmol) in DMF (3 mL)
was added and the mixture was heated at 140°C for 3 h. The solvent was removed under vacuum
and the residue was taken up with diluted aqueous NaOH and extracted with Et O. The dried
Na SO ) organic solution was evaporated and the spongy residue was crystallized from dry Et O.
2
(200 mg) and stirred for 30 min.
2
6
2
2
(
2
4
2

2
7
1
Yield: 49%. M.p. 125-127°C (Et
2
O). H NMR (CDCl
3
): 1.20–2.44 (m, 14H, Q); 2.82-3.28 (m, 2H
-Ar, superimposed); 7.07-7.70 (m, 5
2 3
H26BrCl N : C, 56.82; H, 5.17; N, 8.28. Found:
α
near N of Q); 4.09-4.52 (m, 2H, CH
2
-Q and, 4.30, s, 2H, CH
2
arom. H); 7.84 (s, 1 arom. H). Anal. Calcd for C24
C, 56.76; H, 5.24; N, 8.23.
4
.9.
Scheme 8)
Commercially available 2-chloro-1H-benzimidazole (2.0 mmol) was dissolved in 5 ml of CH
2-{[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]thio}-1H-benzimidazole
(63).
(
3
CN in
an Aldrich pressure tube. A solution of (1R,9aR)-(octahydro-2H-quinolizin-1-yl)methanthiole
2
7
(
thiolupinine, 2.0 mmol) in 1 ml of CH
3 2
CN was rapidly added; the tube was flushed with dry N
and thoroughly closed. The mixture was heated at 100°C for 4 h. After evaporation of the solvent,
the residue was crystallized from a mixture of CH
3
CN/dry Et
2
O (2:1).
): 0.92-2.38 (m, 14H, Q); 2.71-
-S); 7.04-7.37 (m, 3 arom. H); 7.41-7.72 (m, 1
arom. H superimposed with NH, that collapses with D O). Anal. Calcd for C17 S: C, 67.74; H,
1
Yield: 67%. M.p. 172-173°C (CH
.06 (m, 2H
3
CN/Et
2
O 2:1). H NMR (CDCl
3
3
α
near N of Q); 3.26-3.82 (m, 2H, CH
2
2
23 3
H N
7.69; N, 13.94. Found: C, 67.50; H, 7.63; N, 13.91.
4.10. Benzoimidazo[1,2-c][1,2,3]benzotriazines. (Scheme 9)
To a suspension of 2-[(2-amino-4-methoxy)phenyl]-5-trifluoromethylbenzimidazole or 2-[(2-
4
amino-4-methoxy)phenyl]-5,6-dichlorobenzimidazole (2.5 mmol) in 7 mL of 2N HCl, cooled in an
2
ice bath, a solution of sodium nitrite (2.7 mmol) in 4 mL of H O was added. After 30 min of
stirring, the precipitate was collected and washed with water. The solid, dissolved in EtOH, was
decolorized with activated charcoal. Concentration of the ethanolic solution afforded the crystalline
compound.
4.10.1. 3-Methoxy-9/10-(trifluoromethyl)benzo[e]benzo[4,5]imidazo[1,2-c][1,2,3]triazine (24)

2
8
1
Yield: 58%. M.p. 171-173°C (EtOH). H NMR (CDCl
arom. H); 7.96-7.83 (m, 2 arom. H); 8.09 (d, J = 8.6, 1 arom. H); 8.61 (d, J = 8.8, 1 arom. H); 8.70
d, J = 2.4, 1 arom. H). The splotted signals indicate the presence of two unresolvable isomers in the
ratio 10:1. Anal. Calcd for C15 O: C, 56.61; H, 2.85; N, 17.60. Found: C, 56.46; H, 2.71; N,
3 3
): 4.10 (s, OCH ); 7.64 (dd, J = 8.8, 2.4, 1
(
9 3 4
H F N
17.59.
4.10.2. 9,10-Dichloro-3-methoxybenzo[e]benzo[4,5]imidazo[1,2-c][1,2,3]triazine (25)
1
Yield: 65%. M.p. 255-257°C (dioxane). H NMR (CDCl
3
): 4.10 (s, OCH
3
); 7.62 (dd, J = 9.0, 2.4, 1
arom. H); 7.88 (d, J = 2.4, 1 arom. H); 8.09 (s, 1 arom. H); 8.49 (s, 1 arom. H); 8.57 (d, J = 9.0, 1
arom. H). Anal. Calcd for C14 Cl O: C, 52.69; H, 2.53; N, 17.56. Found: C, 52.86; H, 2.77; N,
H
8
2 4
N
17.40.
4.11.
2-Nitro-N-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-4-trifluoromethyl-
benzeneamine monohydrochloride.
2
8
A solution of [(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methan]amine (epi-lupinylamine) (15
mmol) and 4-chloro-3-nitrobenzotrifluoride (15 mmol) in DMF (3 mL) was heated with stirring (90
min; 170°) in a pressure tube (Aldrich). At r.t. the residue was taken up with dry Et
crystalline solid (as monohydrochloride) that was collected by filtration and washed thoroughly
with dry Et O.
Yield: 88%. M.p. 220-224°C. H-NMR (CDCl
of Q); 2.97-3.13 (m, 1H, CH -Q); 3.25-3.40 (m, 1H of CH
dd, J=9.4, 2.4, 1 arom. H); 8.04 (s, NH, exchanges with D
Calcd for C17 +HCl: C, 51.84; H, 5.89; N, 10.67. Found: C 51.66, H 5.96, N 10.49.
.12. N-[(1R,9aR)-(Octahydro-2H-quinolizin-1-yl)methyl]-4-trifluoromethyl-1,2-
2
O affording a
2
1
3
): 1.04-2.08 (m, 14H, Q); 2.66-2.84 (m, 2H near N
α
2
2
-Q); 6.72 (d, J=9.2, 1 arom. H,); 7.29
O); 8.10 (d, J=2.6, 1 arom. H). Anal.
(
2
22 3 3 2
H F N O
4
phenylenediamine monohydrochloride.
A solution of the above nitrocompound (10 mmol) in EtOH (50 mL) was hydrogenated at r.t. and
athmospheric pressure in the presence of 10% Pd/C (0.4 g). After 1 h the calculated volume of H
2
was absorbed. The catalyst was removed and the solvent was evaporated under vacuum, leaving the

2
9
2
1,2-phenylenediamine monohydrochloride that was crystallized by absolute EtOH/dry Et O and
was used as such for the preparation of benzimidazole derivatives (17, 42-44).
1
Yield: 92%. M.p. 160-163°C (EtOH/Et
2
O). H-NMR (CDCl
3
): 1.04-2.06 (m, 14H, Q); 2.68-2.86
-Q); 3.30 (s, 3H, NH and NH, exchange with D O);
+HCl: C, 56.12; H, 6.93; N, 11.55. Found C
(
m, 2H near N of Q); 3.06-3.18 (m, 2H, CH
2
2
2
α
6
.40-6.72 (m, 3 arom. H). Anal. calc for C17
H
24
F
3
N
3
56.00, H 6.67, N 11.70.
4
.13. Cell-based assays
.13.1. Compounds
4
Compounds were dissolved in DMSO at 100 mM and then diluted in culture medium. The final
DMSO concentration did not exceed 1% (commonly 0.1%) which did not affect the biological
assay results.
4.13.2. Cells and viruses
Cell lines were purchased from American Type Culture Collection (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: CD4+ 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) except Yellow Fever
Virus (YFV), and Human Immunodeficiency Virus type-1 (HIV-1). Viruses representative of
positive-sense single-strand RNA (ssRNA+) group used were: i) Retroviridae family: the laboratory
strain HIV-1 IIIB wild-type, obtained from the supernatant of the persistently infected H9/IIIB cells
(
NIH 1983); ii) Flaviviridae family: YFV [strain 17-D vaccine (Stamaril Pasteur J07B01)] and
Bovine Viral Diarrhea Virus (BVDV) [strain NADL (ATCC VR-534)] (title compounds were
evaluated in vitro for antiviral activity against viruses representative of only two of the three genera