Design, synthesis, and antiviral evaluations of 1-(substituted benzyl)- 2-substituted-5,6-dichlorobenzimidazoles as nonnucleoside analogues of 2,5,6- trichloro-1-(β-D-ribofuranosyl)benzimidazole

Article abstract of DOI:10.1021/jm970559i

We have recently reported that certain ribosylated polyhalogenated benzimidazoles are potent and selective inhibitors of HCMV replication at noncytotoxic concentrations. To extend the structure-activity relationship beyond these first-generation compounds, we alkylated 5,6dichloro-2- substituted-benzimidazoles with either a series of substituted benzyl halides or (2bromoethyl)benzene to obtain five series of nonnucleoside analogues. Evaluation of these compounds for activity against herpes viruses revealed that the new compounds were less active than the benzimidazole ribonucleosides against human cytomegalovirus (HCMV) and inactive against herpes simplex virus type 1 (HSV-1). However, as part of our broader antiviral testing, we found that some of these compounds were active against HIV. Comparisons of the biological data revealed that a chloro or bromo group was required at the 2-position for the best separation of activity against HIV and cytotoxicity. Evaluation of the most active compounds against drug- resistant HIV suggested that they act by a mechanism other than inhibition of reverse transcriptase.

Full text of DOI:10.1021/jm970559i

1252
J . Med. Chem. 1998, 41, 1252-1262
Design , Syn th esis, a n d An tivir a l Eva lu a tion s of 1-(Su bstitu ted
ben zyl)-2-su bstitu ted -5,6-d ich lor oben zim id a zoles a s Non n u cleosid e An a logu es of
2,5,6-Tr ich lor o-1-(â-D-r ibofu r a n osyl)ben zim id a zole
Anthony R. Porcari, Rodrigo V. Devivar,† Louis S. Kucera,‡ J ohn C. Drach, and Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature,
Sciences and Arts, and Department of Biologic and Material Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan 48109-1065, and Department of Microbiology and Immunology, Wake Forest University
School of Medicine, Winston-Salem, North Carolina 27157-1064
Received August 20, 1997
We have recently reported that certain ribosylated polyhalogenated benzimidazoles are potent
and selective inhibitors of HCMV replication at noncytotoxic concentrations. To extend the
structure-activity relationship beyond these first-generation compounds, we alkylated 5,6-
dichloro-2-substituted-benzimidazoles with either a series of substituted benzyl halides or (2-
bromoethyl)benzene to obtain five series of nonnucleoside analogues. Evaluation of these
compounds for activity against herpes viruses revealed that the new compounds were less active
than the benzimidazole ribonucleosides against human cytomegalovirus (HCMV) and inactive
against herpes simplex virus type 1 (HSV-1). However, as part of our broader antiviral testing,
we found that some of these compounds were active against HIV. Comparisons of the biological
data revealed that a chloro or bromo group was required at the 2-position for the best separation
of activity against HIV and cytotoxicity. Evaluation of the most active compounds against
drug-resistant HIV suggested that they act by a mechanism other than inhibition of reverse
transcriptase.
In tr od u ction
quently, DRB has little potential as an antiviral drug.11,16
The early studies by Tamm and co-workers prompted
us to synthesize a series of 2-substituted benzimidazole
ribonucleosides as potential anticancer agents17 and
more recently to examine the 2-substituted analogues
of DRB for activity against HCMV. We found that 2,5,6-
trichloro-1-(â-D-ribofuranosyl)benzimidazole (TCRB) and
its 2-bromo analogue (BDCRB) are potent and selective
inhibitors of HCMV replication at noncytotoxic concen-
trations.18 Both compounds act by a unique mechanism
which does not involve inhibition of DNA synthesis19
but does involve inhibition of DNA processing.20 As part
of a structure-activity relationship study,21,22 acyclic
analogues of TCRB, BDCRB, and related nucleosides
have been synthesized.22 None of the compounds,
including the acyclovir and ganciclovir analogues of
TCRB were active.
Human cytomegalovirus (HCMV) has become a for-
midable opponent in the battle against infection in
humans. Although relatively nonpathogenic in an
intact immune system, HCMV is a leading cause of birth
defects in the United States and causes morbidity and
mortality in immunocompromised individuals, trans-
plant recipients, and AIDS patients.1-4 The two most
widely used agents for the treatment of HCMV disease
are ganciclovir and foscarnet.5-7 Cidofovir,8 a new agent
against HCMV, has recently been approved for use in
the United States. Unfortunately, toxicity associated
with these agents,5-8 poor oral bioavailability, high
relapse rates, and drug resistance9 have made their use
less than optimal.
As a part of our search for new antiviral agents, we
have been exploring benzimidazole nucleosides as po-
tential agents for the treatment of HCMV infections.
In 1954 Tamm, Folkers and co-workers first reported
the synthesis and antiviral activity of halogenated
benzimidazole nucleosides.10,11 They found that 5,6-
dichloro-1-(â-D-ribofuranosyl)benzimidazole (DRB) has
multiple biological activities including activity against
RNA11 and DNA12 viruses. DRB inhibits viral13 and
cellular14 RNA synthesis most likely as a consequence
of inhibiting cellular RNA polymerase II.15 Thus, DRB
affects multiple cellular processes, and its antiviral
activity is poorly separated from cytotoxicity. Conse-
To better understand the requirement of a sugar
moiety for the potent and selective activity of TCRB and
the lack of activity of the acyclic analogues, we decided
to initiate the preparation of other nonnucleoside ana-
logues in which the â-D-ribofuranosyl moiety has been
replaced with a benzyl or phenylethyl group. A benzyl
group at the N-1 nitrogen position also makes it
convenient to employ the rational drug design method
developed by Topliss.23 We concentrated our efforts on
2-amino-, 2-chloro-, and 2-bromo-5,6-dichlorobenzimid-
azoles based upon our prior observations that the
ribosides of these latter two heterocycles were potent
and selective inhibitors of HCMV and that the 2-amino
analogue was somewhat active, albeit more cytotoxic.18
In addition, several other derivatives were synthesized
using the same rationale. These compounds were
† Present address: FlowGenix Corp., 100 East Nasa Rd One, Suite
102, Webster, TX 77598.
‡ Present address: Wake Forest University, Bowman-Gray School
of Medicine, Winston-Salem, NC 27103.
S0022-2623(97)00559-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/15/1998

Synthesis of Substituted 5,6-Dichlorobenzimidazoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1253
Sch em e 1.a Synthesis of the
2-Amino-5,6-dichloro-1-(substituted
benzyl)benzimidazole Series
Sch em e 3.a Synthesis of the
2-Bromo-5,6-dichloro-1-(substituted
benzyl)benzimidazole Series
a
a
Reagents: (a) CuBr2, tert-butyl nitrite, acetone; (b) sodium
Reagents: (a) hydrogen gas at 40 psi, Raney nickel, ethanol;
hydroxide, acetonitrile.
(b) cyanogen bromide, water, methanol; (c) sodium hydroxide,
acetonitrile.
Sch em e 4.a Synthesis of
1-(Phenylethyl)-2,5,6-trichlorobenzimidazole and
2-Bromo-5,6-dichloro-1-(phenylethyl)benzimidazole
Sch em e 2.a Synthesis of the 1-(Substituted
benzyl)-2,5,6-trichlorobenzimidazole Series
a
Reagents: (a) (2-bromoethyl)benzene, sodium hydroxide, ace-
tonitrile.
Sch em e 5.a Synthesis of 1-Benzyl-5,6-dichloro-2-
(isopropylamino)benzimidazole and
1-Benzyl-2-(cyclopropylamino)-5,6-dichlorobenzimidazole
a
Reagents: (a) sodium hydroxide, acetonitrile.
evaluated for antiviral activity against two herpes-
viruses [HCMV and herpes simplex virus type 1 (HSV-
1)] and human immunodeficiency virus (HIV) in an
effort to establish structure-activity relationships
(SAR’s). We report herein that the compounds were
essentially inactive against the herpesviruses, but the
testing provided an interesting SAR against HIV.
Resu lts a n d Discu ssion
a
Reagents: (a) isopropylamine or cyclopropylamine at 60 °C
in a sealed vessel.
Ch em istr y. For the synthesis of the requisite target
compounds in the first series (Scheme 1), we used
commercially available 4,5-dichloro-2-nitroaniline (1) as
our starting material instead of the considerably more
expensive 4,5-dichloro-1,2-phenylenediamine. A reduc-
tion of the nitro group without concomitant removal of
the chloro groups restricted the choice of reducing
agents and conditions. We found that treatment of 1
with Raney nickel in the presence of hydrogen gas
effected a clean reduction of the nitro group without any
affect on the chloro group to provide a good yield of 4,5-
dichloro-1,2-phenylenediamine (2). The ring annulation
of 2 was accomplished with cyanogen bromide in aceto-
nitrile to afford 2-amino-5,6-dichlorobenzimidazole (3)
in good yield. A series (4a -o) of 2-amino-5,6-dichloro-
1-(substituted benzyl)benzimidazoles (4) were prepared
by alkylating compound 3 with the appropriately
substituted benzyl halide in the presence of sodium
hydroxide.
For the synthesis of the target compounds in the
second series (Scheme 2), we prepared compound 5 by
published methods.18 A series (6a -o) of 1-(substituted
benzyl)-2,5,6-trichlorobenzimidazoles (6) were prepared
by alkylating compound 5 with the appropriately sub-
stituted benzyl halide in the presence of sodium hy-
droxide.
For the synthesis of the target compounds in the third
series (Scheme 3), we elected to use compound 3 as our
starting material. A diazotization of compound 3 with
tert-butyl nitrite was followed by a displacement of the
diazo group by a bromo group to furnish 2-bromo-5,6-
dichlorobenzimidazole (7). A series of 2-bromo-5,6-

1254 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Porcari et al.
Ta ble 1. Activity against Herpesviruses and Cytotoxicity of 1-(Substituted benzyl)-2-substituted-5,6-dichlorobenzimidazoles
50% inhibitory concentration (µM)
antiviral activity
cytotoxicityc
HCMVa
plaque
HSV-1b
ELISA
no.
R
V
W
Y
Z
visual
growth
4a
4b
4c
4d
4e
4f
4g
4h
4i
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Cl
Cl
H
H
Cl
OCH3
CH3
Cl
H
C(CH3)3
CF3
H
H
H
H
NO2
H
F
H
H
37d
>100e
>100
30
152
>100
>100
80
58d
>100
>100
32
170d
45
45
50
35
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
40
50
32
>100
70
>100
65
22d
60d
32d
50d
H
32
32
CH3
CF3
H
CF3
H
NO2
H
H
H
H
H
Cl
Cl
H
>100
80
60
>100
65
45
4j
4k
4l
32
32
32
32
32
32
>100
60
4m
4n
4o
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
>100
>100
12d
100
>100
>100
37d
35
65
70
>100
70
>100d
>100
>100
>100
>100
>100
>100
>100
>100
>100
30
>100d
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100d
>100
>100
>100
>100
70
>100d
>100
>100
32
>100d
>100
>100
>100
>100
Cl
OCH3
CH3
Cl
100
H
100d
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100d
>100
>100
>100
>100
>100
>100
>100
>100d
>100d
>10
C(CH3)3
CF3
H
H
H
H
NO2
H
F
H
Cl
OCH3
CH3
Cl
H
C(CH3)3
CF3
H
H
H
H
NO2
H
>100d
>100
>100
>100
156d
H
CH3
CF3
H
CF3
H
NO2
H
H
H
H
H
Cl
Cl
H
>100
>100
>100
>100
>100d
>100
>100
>100
>100
>100
>100
>100
>100d
>100d
>10
>100d
183d
239d
>100d
>100d
>100
55
>100
>100
>100
H
>100
>100
CH3
CF3
H
CF3
H
Br
Br
Br
Br
>100d
78d
70
40
>100
>100
>100
>100d
65
>100
>100
>100
>100d
8m
8n
8o
NO2
H
150
Br
F
a
b
Plaque reduction assays were performed in duplicate as described in the text. Compounds were assayed by ELISA in quadruplicate
wells. c Visual cytotoxicity was scored on HFF cells at time of HCMV plaque enumeration. Inhibition of KB cell growth was determined
as described in the text in quadruplicate assays. Average of IC50’s from two to five separate experiments. e A greater than sign (>)
d
indicates IC50 not reached at the noted (highest) concentration tested.
dichloro-1-(substitutedbenzyl)benzimidazoles (8a-o) were
synthesized by the alkylation of compound 7 with the
appropriately substituted benzyl halide in the presence
of sodium hydroxide.
Compounds 9 and 10 were prepared by alkylating
compounds 5 and 7, respectively, with (2-bromoethyl)-
benzene in the presence of sodium hydroxide in aceto-
nitrile (Scheme 4).
cyclopropylamine, respectively, at 60 °C in a sealed
vessel (Scheme 5).
All new compounds reported in this research inves-
1
tigation were characterized using H-nuclear magnetic
resonance spectroscopy, 13C-nuclear magnetic resonance
spectroscopy, elemental analysis, and thin-layer chro-
matography. More detailed proton and carbon assign-
ments of 3 representative compounds (4a , 6b, and 8i)
were obtained using distortionless enhancement by
polarization transfer spectroscopy (DEPT), hetero-
Compounds 11 and 12 were prepared by displacing
the 2-chloro group of 6a with either isopropylamine or

Synthesis of Substituted 5,6-Dichlorobenzimidazoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1255
Ta ble 2. Activity against Herpesviruses and Cytotoxicity of 1-Phenylethyl- and 1-Benzyl-2-substituted-5,6-dichlorobenzimidazoles
50% inhibitory concentration (µM)
antiviral activity
cytotoxicityc
HCMVa
plaque
HSV-1b
ELISA
no.
R1
R2
visual
growth
9
Cl
Br
phenylethyl
phenylethyl
benzyl
benzyl
â-D-ribose
â-D-ribose
>100d
>100
22
>100
>100
22
33
238
118
10
11
12
>100
50
>100
102
>100
45
60
210
>100
isopropylamino
cyclopropylamino
Cl
Br
18
2.9
0.7
TCRBe
BDCRBe
130
d
a,b,c See footnotes to Table 1. A greater than sign (>) indicates IC50 not reached at the noted (highest) concentration tested. e Data
also reported in ref 18; average derived from three to six experiments.
nuclear shift correlation spectroscopy (HetCor), and
correlation spectroscopy via long-range coupling (COLOC)
on a 500 MHz Bruker Avance DRX 500.
compound (not shown) were identical in wt and TCRB-
resistant strains25 and in wt and in 1263W94-resistant
strains24 of HCMV. Together, these data suggest that
the compounds act by a mechanism different from either
TCRB or 1263W94 or that the observed effects were due
to cytotoxicity.
When screened for activity in the HSV-1 assay, no
compounds in any of the five series were active at
noncytotoxic concentrations (Tables 1 and 2).
Biology. Evaluation of 2-amino-1-benzyl-5,6-di-
chlorobenzamidazole (4a ) and its 2-chloro (6a ) and
2-bromo (8a ) analogues established that the unsubsti-
tuted benzyl analogues were either inactive (6a and 8a )
or weakly active and cytotoxic (4a ) against HCMV and
HSV-1 (Table 1). By incorporating substituents on the
benzyl moiety, suggested by the Topliss tree approach,
we attempted to increase antiviral activity and reduce
cytotoxicity in the three series of compounds (Table 1).
In the 2-amino-5,6-dichloro-1-(substituted benzyl)benz-
imidazole series (4), compounds 4d , 4e, 4g, 4h , 4j, 4k ,
4l, and 4o showed some activity against HCMV but with
similar cytotoxicity. Consequently, this antiviral activ-
ity most likely was a result of cytotoxicity. Compound
4o was the most active with a 50% inhibitory concen-
tration (IC50) of 12 µM vs HCMV. However, a 50%
cytotoxic concentration (CC50) of 37 µM (in vitro thera-
peutic index ) 3) excludes it from being a potential
antiviral agent.
The parent compound (6a ) of the 2-chloro series (6)
had no activity. Likewise, all of the compounds in this
series were inactive with the exception of the o-chloro-
substituted compound (6k ) which had an IC50 of 30 µM
against HCMV. In the 2-bromo series (8), the parent
compound (8a ) and all its derivatives were inactive
against HCMV.
Two analogues closely related to compounds 6a and
8a also were made and tested. Like compounds 6a and
8a , 1-(phenylethyl)-2,5,6-trichlorobenzimidazole (9) and
the 2-bromo homologue (10) were inactive against
HCMV (Table 2). In addition, two analogues similar
to those in the 2-amino series (4) were synthesized based
upon the activity of 5,6-dichloro-2-(isopropylamino)-â-
All the compounds in the five series also were evalu-
ated for activity against human immunodeficiency virus
type 1 (HIV-1) using a syncytial plaque assay. Although
many of the compounds in the 2-amino series (4)
inhibited plaque formation, the activity was not well
separated from cytotoxicity, and therefore specific an-
tiviral activity against HIV-1 is questionable (Table 3).
Cytotoxicity in CEM-SS cells measured by [3H]dThd
incorporation into total acid precipitable material (Table
3) was similar to cytotoxicity in HFF and KB cells
reported in Table 1. The most promising was compound
4h , with an IC50 of 1.8 µM and a CC50 of 20 µM (in vitro
therapeutic index ) 11). In the 2-chloro series (6), 6b,
6c, 6d , 6i, 6k , 6m , 6n , and 6o had modest activity
against HIV-1 in the syncytial plaque assay (Table 3).
Of these, compound 6b was the most promising, with
an IC50 of 9.1 µM and a CC50 of greater than 100 µM.
In the 2-bromo series (8), compounds 8a , 8c, 8i, 8j, 8l,
8m , 8n , and 8o also were active in the HIV-1 syncytial
plaque assay (Table 3). Of these, compound 8i was the
most promising, with an IC50 of 3.5 µM and a CC50 of
greater than 100 µM.
When comparing trends among the series of com-
pounds (Table 3), we found that the 2-chloro series (6)
and the 2-bromo series (8) are less toxic than the
2-amino series (4). Overall, in the HIV-1 assay the
2-chloro series (6) was less active than or equally active
to the 2-amino series (except 4m ) and the 2-bromo series
(except 8b, 8d ). A comparison between the 2-amino
series (4) and the 2-bromo series (8) showed no trend.
Compounds 4b, 4d , 4e, 4f, and 4g showed greater
activity in the HIV-1 assay in the 2-amino series than
the 2-bromo series where compounds 8c, 8i, 8j, 8m , 8n ,
and 8o showed greater or equal activity.
L-ribofuranosylbenzimidazole
(1263W94)
against
HCMV.24
Both 1-benzyl-5,6-dichloro-2-(isopropyl-
amino)benzimidazole (11) and the 2-cyclopropylamino
analogue (12) were active in the HCMV assay, but with
only a small separation from cytotoxic concentrations
(Table 2).
To better understand the activity of the three amine-
substituted compounds (4a , 11, 12), their activity
against HCMV was compared in wild-type (wt) and
drug-resistant HCMV. Dose-response curves for each
Selected compounds in the 2-chloro and 2-bromo
series were examined in greater detail by assaying them
against virus which was resistant to AZT or to the

1256 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Porcari et al.
Ta ble 3. Activity of 1-(Substituted benzyl)-2-substituted-5,6-dichlorobenzimidazoles against Human Immunodeficiency Virus
50% inhibitory concentration (µM)
HIV
cytotoxicity
[3H]dThdb
no.
R
V
W
Y
Z
syncytial plaquea
4b
4c
4d
4e
4f
4g
4h
4i
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
Cl
Cl
H
Cl
OCH3
CH3
Cl
H
10
12
20
32
27
40
30
37
27
20
48
34
36
17
32
47
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
>6c,d
>30
10d
H
C(CH3)3
CF3
H
H
H
H
NO2
H
F
Cl
OCH3
CH3
Cl
H
C(CH3)3
CF3
H
H
H
H
NO2
H
F
H
Cl
OCH3
CH3
Cl
H
1.8d
12
CH3
CF3
H
CF3
H
NO2
H
H
H
H
Cl
Cl
H
4j
4k
4l
>6
6
>10
4m
4n
4o
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
21
21d
32
30
9.1d
26d
>100
>100
>100
>100
>100
>100
>100
>100
>100
70
33d
>100d
100d
>100
>100
14d
H
CH3
CF3
H
CF3
H
NO2
H
H
H
H
H
Cl
Cl
H
>100d
15d
>100
7.8d
20d
>100
51
>100
>100
>100
>100
60
>100
>100
>100
>100
>100
>100
>100
74
37d
27
>100
6d
>100
>100
>100
100d
>100
3.5d
12d
H
C(CH3)3
CF3
H
H
H
H
NO2
H
H
CH3
CF3
H
CF3
H
15d
Br
Br
Br
Br
5.9d
14d
>100
>100
>100
>100
1.9
8m
8n
8o
NO2
H
5.3d
30
F
zidovudine (AZT)
0.0026e
a
b
Syncytial plaque assay was performed in duplicate using CEM-SS cells. Incorporation of [3H]dThd was used as a measure of
cytotoxicity. Average results from duplicate experiments are presented. c A greater than sign (>) indicates IC50 not reached at the noted
(highest) concentration tested. Average of duplicate or triplicate experiments. e Mean from nine separate experiments ( standard deviation
d
of 0.0024.
nonnucleoside reverse transcriptase (RT) inhibitor,
TIBO. Data in Table 4 establish that other than
compound 6b, all of the compounds were active against
HIV-1 that was resistant to either AZT or TIBO, thereby
suggesting that the compounds are not RT inhibitors.
In contrast, none of the compounds with the possible
exception of 8n were active against HIV-2.
To better understand the activity of the benzylbenz-
imidazoles against HIV-1, selected compounds were
assayed in a different manner by determining their
effect on the production of reverse transcriptase (RT)
in HIV-1-infected cultures. (This assay measures RT
activity in culture supernatants as a marker for infec-
tious virus multiplication, it does not measure the direct
effect of compounds on RT itself.) In contrast to the
activity observed in the syncytial plaque assay, little
activity was observed using the RT marker assay (Table
5). The 2-amino compounds 4b and 4h were active but
at concentrations near those which were cytotoxic to
CEM-SS cells (Table 3). Compounds in the 2-chloro and
2-bromo series were found to be inactive except for
compound 8isthe most active compound in the
seriessand it was nearly 20-fold less active in the RT
assay. The identical activity of AZT in both assays

Synthesis of Substituted 5,6-Dichlorobenzimidazoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1257
Ta ble 4. Activity of 5,6-Dichloro-1-(substituted benzyl)-2-substituted-benzimidazoles against Selected Strains of HIV-1 and HIV-2
50% inhibitory concentration (µM)a
HIV-1
C-691c or
H-112c
(wt)
G-762c or
G-910c
b
IIIB
A-17
no.
R1
R2
(wt)
(AZTr)
(TIBOr)
HIV-2
6b
6i
6k
6o
8i
p-ClBn
m-CH3Bn
o-ClBn
Cl
Cl
Cl
Cl
Br
Br
Br
9.1
14
30
37
255
13
19d
16d
13d
19
17
11
>30e
>30
>30
>30
143
66
15
37
3.5
14
5.0
0.0026
p-FBn
m-CH3Bn
p-NO2Bn
m-NO2Bn
8.7
11
8.7
9.0
6.9
1.1
8m
8n
1.0
28
zidovudine (AZT)
0.002
>10
a
b
Syncytial plaque assay was performed in duplicate sets using CEM-SS cells. Data from Table 3. c Matched pairs of HIV sensitive
(wt) and resistant to AZT (AZTr). C-691 and G762 were used with 6 series compounds and zidovudine; H-112 and G910 were used with
8 series compounds. Average of duplicate experiments. e A greater than sign (>) indicates IC50 not reached at the noted (highest)
d
concentration tested.
Ta ble 5. Comparison of the Activity of 5,6-Dichloro-1-(substituted benzyl)-2-substituted-benzimidazoles against Human
Immunodeficiency Virus in Different Assays
50% inhibitory concentration (µM)
HIV assay
no.
R1
R2
syncytial plaquea
RTb
4b
4h
6d
6m
8c
8e
8i
p-ClBn
NH2
NH2
Cl
Cl
Br
Br
Br
Br
Br
10
1.8
33
7.8
6
10
20
p-CF3Bn
p-CH3Bn
p-NO2Bn
p-OCH3Bn
m,p-Cl2Bn
m-CH3Bn
m-CF3Bn
m-NO2Bn
>100
>100
>100
>100
66
>100
>32
>100
3.5
5.9
5.3
8l
8n
zidovudine (AZT)
0.0026 ( 0.0024c
0.0031 ( 0.0039c
a
b
Syncytial plaque assay was performed in duplicate sets using CEM-SS cells; data from Table 3. Reverse transcriptase (RT) assay
was performed in triplicate, also in CEM-SS cells. c Means ( standard deviations from 9 and 31 separate experiments.
validates the assays and virtually proves that the
benzylbenzimidazoles are acting by a mechanism dif-
ferent than AZT. Furthermore, the lack of activity of
these compounds in the RT marker assay suggests that
the compounds are inhibiting a late step in virus
multiplication with the production of noninfectious virus
particles.
via 6d , 6f, and 6k . In some cases, activity against
HIV-1 was well separated from cytotoxicity (i.e., 6b and
8i). Using these as lead compounds, it is plausible that
other modifications could uncover agents which are
more active against HIV-1.
Exp er im en ta l Section
Evaporations were carried out under reduced pressure
(water aspirator) with the bath below 50 °C. Silica gel 60 230-
400 mesh (E. Merck, Darmstadt, West Germany) was used for
chromatography. Thin-layer chromatography was performed
on silica gel GHLF-254 plates (Merck Reagents) using a 9:1
chloroform:methanol solvent system. Melting points were
determined with a Thomas-Hoover capillary apparatus and
are uncorrected. Proton magnetic resonance (1H NMR) spectra
were obtained with a Bruker WP270SY spectrometer (solu-
tions in dimethyl-d6 sulfoxide), with chemical shift values
reported in δ, parts per million, relative to the internal
standard. Carbon magnetic resonance (13C NMR) spectra were
obtained at 90 MHz in DMSO-d6 with an IBM WM-360
spectrometer. Elemental analyses were performed by M-H-W
Laboratories, Phoenix, AZ.
In summary, N-1 benzyl and phenylethyl analogues
of TCRB were synthesized to expand our understanding
of the structure-activity relationship among nonnucleo-
side benzimidazole analogues. However, the compounds
which we prepared were inferior in activity to the
benzimidazole ribonucleosides as agents against HCMV.
Furthermore, the antiviral activity was poorly separated
from the cytotoxicity. As part of our broader antiviral
testing, we found these compounds were inactive against
HSV-1 but active against HIV-1. We found that the
Topliss tree provided some useful suggestions for sub-
stitutions on the 1-benzyl group; for example, if one
assumes that 6b is equally as active as 6a in the HIV
assay (Table 3), then the Topliss tree will lead to 6m
4,5-Dich lor o-1,2-p h en ylen ed ia m in e (2). 4,5-Dichloro-2-
nitroaniline (1) (51.7 g, 250 mmol) and Raney nickel (8 g) were

1258 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Porcari et al.
mixed in ethanol (500 mL) and hydrogenated under an average
hydrogen pressure of 40 psi. Hydrogen consumption ceased
once the reduction was complete. The reaction mixture was
filtered and evaporated to dryness to obtain 4,5-dichloro-1,2-
phenylenediamine (2): yield 44.55 g (98%); mp 163-165 °C
(lit.25 mp 162-163 °C).
2-Am in o-5,6-d ich lor ob en zim id a zole (3). 4,5-Dichloro-
1,2-phenylenediamine (2) (10.0 g, 57 mmol) was added to a
mixture of water (120 mL), methanol (120 mL), and cyanogen
bromide (11.3 mL of a 5.0 M solution, 57 mmol) and left to
stir overnight. This mixture was decolorized with charcoal and
adjusted to pH >9, with concentrated NH4OH, to precipitate
orangish-brown crystals of 2-amino-5,6-dichlorobenzimidazole
(3): yield 10.38 g (91%); mp 259-261 °C (lit.18 mp 250-251
°C).
2-Am in o-5,6-d ich lor o-1-(su bstitu ted ben zyl)ben zim id -
a zole (4). 2-Amino-5,6-dichlorobenzimidazole (3) (0.152 g,
0.75 mmol) and sodium hydroxide (0.03 g, 0.75 mmol) were
dissolved in acetonitrile (40 mL). After approximately 1 h of
stirring, the appropriately substituted benzyl halide (0.75
mmol) was added and the mixture was heated at reflux. When
only one spot was present on TLC, the reaction mixture was
cooled to room temperature, evaporated to dryness, dissolved
in methanol, decolorized with charcoal, filtered, and evapo-
rated to dryness. The product was dissolved in hot methanol
and recrystallized from a mixture of methanol and water to
obtain pure 4a -o.
s,), 7.06-7.08 (1H, d), 7.23 (1H, s), 7.30-7.36 (3H, m), 7.42
(1H, s); 13C NMR (DMSO-d6) δ 44.44, 109.46, 115.83, 120.00,
123.26, 125.71, 126.92, 127.75, 130.86, 133.00, 134.18, 139.18,
143.14, 157.00. Anal. (C14H10N3Cl3) C, H, N.
2-Am in o -1-(p -t er t -b u t y lb e n zy l)-5,6-d ic h lo r o b e n z-
im id a zole (4g). 3 was reacted with p-tert-butylbenzyl bro-
mide (0.17 g, 0.75 mmol): yield 0.19 g (72%); Rf 0.61; white
1
crystals; mp 263-265 °C; H NMR (DMSO-d6) δ 1.21 (9H, s),
5.23 (2H, s), 6.91 (2H, s), 7.07-7.10 (2H, 2 s), 7.29-7.37 (4H,
2d); 13C NMR (DMSO-d6) δ 31.03, 34.00, 44.45, 109.22, 115.41,
119.67, 122.50, 125.36, 126.63, 133.54, 134.22, 143.18, 149.92,
156.96. Anal. (C18H20N3Cl2) C, H, N.
2-Am in o-1-(p -(t r iflu or om et h yl)b en zyl)-5,6-d ich lor o-
ben zim id a zole (4h ). 3 was reacted with p-trifluoromethyl-
benzyl bromide (0.18 g, 0.75 mmol): yield 0.13 g (48%); Rf 0.45;
light brown needles; mp 248-250 °C; 1H NMR (DMSO-d6) 5.45
(2H, s), 6.95 (2H, s), 7.29-7.35 (4H, m), 7.71 (1H, s), 7.84 (1H,
s). Anal. (C15H10N3Cl2F3) C, H, N.
2-Am in o-1-(m -m et h ylb en zyl)-5,6-d ich lor ob en zim id -
a zole (4i). 3 was reacted with m-methylbenzyl chloride (0.11
g, 0.75 mmol): yield 0.13 g (58%); Rf 0.52; light brownish
1
crystals; mp 189-191 °C; H NMR (DMSO-d6) δ 2.24 (3H, s),
5.22 (2H, s), 6.89-6.93 (3H, m), 6.99 (1H, s), 7.05-7.07 (1H,
d), 7.17-7.21 (1H, t), 7.29-7.31 (2H, d); 13C NMR (DMSO-d6)
δ 21.23, 45.03, 109.43, 115.66, 120.00, 122.92, 124.17, 127.64,
128.35, 128.77, 134.44, 136.71, 138.01, 143.43, 157.22. Anal.
(C15H13N3Cl2) C, H, N.
2-Am in o-1-ben zyl-5,6-d ich lor oben zim id a zole (4a ).
3
2-Am in o-1-(m -(t r iflu or om et h yl)b en zyl)-5,6-d ich lor o-
ben zim id a zole (4j). 3 was reacted with m-trifluoromethyl-
benzyl chloride (0.18 g, 0.75 mmol): yield 0.19 g (70%); Rf:
0.39; light brownish crystals; mp 220-223 °C; 1H NMR
(DMSO-d6) δ 5.34 (2H, s), 6.97 (2H, s), 7.31 (1H, s), 7.39-7.40
(1H, d), 7.45 (1H, s), 7.50-7.65 (3H, m); 13C NMR (DMSO-d6)
δ 44.47, 109.31, 115.74, 120.20, 123.66, 124.37, 125.65, 127.56,
129.94, 130.90, 134.06, 138.03, 143.04, 156.80, 129.26. Anal.
(C15H10N3Cl2F3) C, H, N.
was reacted with benzyl chloride (0.127 g, 1.0 mmol): yield
1
0.14 g (64%); Rf 0.52; yellow solid; mp 255-257 °C; H NMR
(DMSO-d6) δ 5.29 (2H, s, CH2), 6.94 (2H, s, NH2) 7.17 (2H, d,
C2′-H and C6′-H), 7.27 (1H, t, C4′-H), 7.31 (1H, s, C4-H), 7.34
(2H, dd, C3′-H and C5′-H), 7.36 (1H, s, C7-H); 13C NMR
(DMSO-d6) δ 45.62 (CH2), 110.07 (C4), 116.27 (C7), 120.50 (C3a
or C5), 123.55 (C6), 127.76 (C2′, C6′), 128.32 (C4′), 129.50 (C3′,
C5′), 135.03 (C7a), 137.42 (C1′), 144.06 (C3a), 157.83 (C2);
Anal. (C14H11N3Cl2) C, H, N.
2-Am in o-1-(o-ch lor ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (4k ). 3 was reacted with o-chlorobenzyl chloride (0.12
g, 0.75 mmol): yield 0.17 g (69%); Rf 0.61; light brownish
2-Am in o-1-(p -ch lor ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (4b). 3 was reacted with p-chlorobenzyl chloride (0.12
g, 0.75 mmol): yield 0.11 g (45%); Rf 0.46; light brown crystals;
mp 247-249 °C; 1H NMR (DMSO-d6) δ 5.26 (2H, s), 6.92 (2H,
s), 7.15-7.18 (2H, d), 7.30 (1H, s), 7.37-7.39 (2H, s), 7.40 (1H,
s); 13C NMR (DMSO-d6) δ 44.95, 110.04, 116.26, 121.20, 123.97,
129.33, 129.61, 133.02, 134.36, 135.69, 142.89, 157.20. Anal.
(C14H10N3Cl3) C, H, N.
1
crystals; mp 231-233 °C; H NMR (DMSO-d6) δ 5.34 (2H, s),
6.42-6.45 (1H, d), 6.91 (2H, s), 7.22-7.23 (2H, m), 7.28-7.34
(3H, m), 7.50-7.53 (1H, d); 13C NMR (DMSO-d6) δ 43.52,
109.43, 115.80, 120.06, 123.22, 126.92, 127.70, 129.25, 129.83,
132.12, 133.81, 134.32, 143.49, 157.37. Anal. (C14H10N3Cl3)
C, H, N.
2-Am in o-1-(p -m et h oxyb en zyl)-5,6-d ich lor ob en zim id -
a zole (4c). 3 was reacted with p-methoxybenzyl chloride (0.12
g, 0.75 mmol): yield 0.11 g (45%); Rf 0.52; light yellow powder;
mp 199-200 °C; 1H NMR (DMSO-d6) δ 3.70 (3H, s), 5.17 (2H,
s), 6.86-6.90 (4H, m), 7.13-7.15 (2H, d), 7.27 (1H, s), 7.35 (1H,
s); 13C NMR (DMSO-d6) δ 45.00, 55.66, 110.04, 114.67, 116.03,
120.85, 123.61, 128.65, 128.98, 129.16, 142.93, 157.15, 159.29;
Anal. (C15H13N3Cl2O) C, H, N.
2-Am in o-1-(p -m e t h ylb e n zyl)-5,6-d ich lor ob e n zim id -
a zole (4d ). 3 was reacted with p-methylbenzyl chloride (0.11
g, 0.75 mmol): yield 0.16 g (70%); Rf 0.52; light brown needles;
mp 231-233 °C; 1H NMR (DMSO-d6) δ 2.23 (3H, s), 5.22 (2H,
s), 6.93 (2H, s), 7.05 (2H, d), 7.19 (2H, d), 7.29 (1H, s), 7.32
(1H, s); 13C NMR (DMSO-d6) δ 20.58, 44.63, 109.25, 115.39,
119.68, 122.69, 126.94, 129.15, 133.47, 136.69, 143.12, 156.93.
Anal. (C15H13N4Cl2) C, H, N.
2-Am in o-1-(m ,m -b is(t r iflu or om e t h yl)b e n zyl)-5,6-d i-
ch lor oben zim id a zole (4l). 3 was reacted with m,m-bis-
(trifluoromethyl)benzyl bromide (0.23 g, 0.75 mmol): yield 0.11
g (35%); Rf 0.52; white crystals; mp 233-235 °C; 1H NMR
(DMSO-d6) δ 5.47 (2H, s), 7.00 (2H, s), 7.32 (1H, s), 7.54 (1H,
s), 7.83 (2H, s), 8.02 (1H, s); 13C NMR (DMSO-d6) δ 44.02,
109.07, 115.65, 120.09, 121.29, 123.15, 127.64, 130.27, 130.64,
133.83, 140.08, 143.03, 156.65. Anal. (C16H9N3Cl2F6) C, H,
N.
2-Am in o -1-(p -n it r o b e n zy l)-5,6-d ic h lo r o b e n zim id -
a zole (4m ). 3 was reacted with p-nitrobenzyl chloride (0.13
g, 0.75 mmol): yield 0.11 g (44%); Rf 0.39; yellow powder; mp
1
276-279 °C; H NMR (DMSO-d6) δ 5.44 (2H, s), 6.96 (2H, s),
7.32-7.40 (4H, m), 8.19-8.20 (2H, d); 13C NMR (DMSO-d6) δ
44.58, 109.40, 115.87, 120.18, 123.27, 124.06, 128.16, 134.30,
143.44, 144.55, 147.16, 157.09. Anal. (C14H10N4Cl2O2) C, H,
N.
2-Am in o-1-(m ,p -d ich lor ob e n zyl)-5,6-d ich lor ob e n z-
im id a zole (4e). 3 was reacted with m,p-dichlorobenzyl
chloride (0.15 g, 0.75 mmol): yield 0.17 g (62%); Rf 0.52; light
2-Am in o -1-(m -n it r o b e n zy l)-5,6-d ic h lo r o b e n zim id -
a zole (4n ). 3 was reacted with m-nitrobenzyl chloride (0.13
g, 0.75 mmol): yield 0.19 g (74%); Rf 0.36; yellow powder; mp
1
yellow powder; mp 247-249 °C; H NMR (DMSO-d6) δ 5.81
1
(2H, s), 7.49 (2H, s,), 7.59-7.61 (1H, d), 7.85 (1H, s), 7.98-
7.99 (2H, s), 8.11-8.14 (1H, d); 13C NMR (DMSO-d6) δ 43.96,
109.45, 115.87, 120.38, 123.36, 127.34, 129.16, 130.46, 131.14,
131.45, 134.09, 143.11, 156.83, 137.8. Anal. (C14H9N3Cl4) C,
H, N.
296-298 °C; H NMR (DMSO-d6) δ 5.43 (2H, s), 7.02 (2H, s),
7.32 (1H, s), 7.48 (1H, s), 7.55-7.65 (2H, m), 8.07-8.13 (2H,
m); 13C NMR (DMSO-d6) δ 44.06, 109.11, 115.57, 119.96,
121.59, 122.39, 123.00, 130.18, 133.40, 133.94, 138.81, 143.08,
153.00, 156.74. Anal. (C14H10N4Cl2O2) C, H, N.
2-Am in o-1-(m -ch lor ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (4f). 3 was reacted with m-chlorobenzyl chloride (0.12
g, 0.75 mmol): yield 0.15 g (61%); Rf 0.52; light yellow crystals;
mp 229-230 °C; 1H NMR (DMSO-d6) δ 5.28 (2H, s), 6.94 (2H,
2-Am in o-1-(p -flu or ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (4o). 3 was reacted with p-fluorobenzyl chloride (0.11
g, 0.75 mmol): yield 0.16 g (70%); Rf 0.52; white fluffy needles;
mp 225-227 °C; 1H NMR (DMSO-d6) δ 5.25 (2H, s), 6.92 (2H,

Synthesis of Substituted 5,6-Dichlorobenzimidazoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1259
s), 7.15-7.29 (5H, m), 7.39 (1H, s); 13C NMR (DMSO-d6) δ
44.37, 109.53, 115.59, 115.77, 115.83, 120.24, 123.17, 129.23,
129.32, 132.82, 139.19, 143.13. Anal. (C14H10N3Cl2F) C, H,
N.
1-(p-Nitr oben zyl)-2,5,6-tr ich lor oben zim id a zole (6m ). 5
was reacted with p-nitrobenzyl chloride (0.13 g, 0.75 mmol):
yield 0.18 g (67%); Rf 0.80; yellow crystals; mp 200-204 °C.
Anal. (C14H8N3Cl3O2) C, H, N.
1-(m -Nitr oben zyl)-2,5,6-tr ich lor oben zim id a zole (6n ). 5
was reacted with m-nitrobenzyl chloride (0.13 g, 0.75 mmol):
yield 0.19 g (71%); Rf 0.80; white crystals; mp 198-200 °C.
Anal. (C14H8N3Cl3O2) C, H, N.
1-(p-F lu or oben zyl)-2,5,6-tr ich lor oben zim id a zole (6o).
5 was reacted with p-fluorobenzyl chloride (0.11 g, 0.75
mmol): yield 0.19 g (75%); Rf 0.80; white crystals; mp 215-
217 °C. Anal. (C14H8N2Cl3F) C, H, N.
1-(Su bstitu ted ben zyl)-2,5,6-tr ich lor oben zim idazole (6).
2,5,6-Trichlorobenzimidazole18 (5) (0.1661 g, 0.75 mmol) and
sodium hydroxide (0.03 g, 0.75 mmol) were dissolved in
acetonitrile (40 mL). After approximately 1 h of stirring, the
appropriately substituted benzyl halide (0.75 mmol) was added
and the mixture was heated at reflux. Once thin-layer
chromatography showed only one spot, the reaction mixture
was cooled to room temperature, evaporated to dryness,
dissolved in methanol, decolorized with charcoal, filtered, and
evaporated to dryness. The product was dissolved in hot
methanol and recrystallized from a mixture of methanol and
water to obtain pure 6a -o.
2-Br om o-5,6-d ich lor oben zim id a zole (7). To a mixture
of CuBr2 (17.87 g, 80 mmol) in dry acetone (160 mL) was added
90% tert-butyl nitrite (8 mL). The reaction mixture was stirred
at room temperature for 10 min before 2-amino-5,6-dichloro-
benzimidazole (3) (8.08 g, 40 mmol) was added portionwise
over a period of 10 min at 60 °C. After 30 min of stirring,
tert-butyl nitrite (8 mL, 67 mmol) was added and the mixture
stirred for 1.5 h. This reaction mixture was concentrated and
partitioned between 2 N HBr and ethyl acetate. The ethyl
acetate layer was washed with saturated NaCl solution, dried
over Na2SO4, decolorized with charcoal, and evaporated. The
residue was recrystallized in methanol to yield 2-bromo-5,6-
dichlorobenzimidazole (7) crystals: Rf 0.17; orange crystals;
1-Ben zyl-2,5,6-tr ich lor oben zim id a zole (6a ). 5 was re-
acted with benzyl bromide (0.17 g, 1.0 mmol): yield 0.21 g
(90%); Rf 0.90; yellow solid; mp 180-182 °C. Anal. (C14H9N2-
Cl3) C, H, N.
1-(p-Ch lor oben zyl)-2,5,6-tr ich lor oben zim id a zole (6b).
5 was reacted with p-chlorobenzyl chloride (0.12 g, 0.75
mmol): yield 0.17 g (66%); Rf 0.82; white crystals; mp 194-
1
196 °C; H NMR (DMSO-d6) δ 5.56 (2H, s, CH2), 7.24 (2H, d,
1
mp 233-234 °C (lit.18 mp 233-234 °C); H NMR (DMSO-d6)
C2′-H and C6-H), 7.42 (2H, d, C3′-H and C5′-H), 7.97 (1H, s,
C4-H), 8.12 (1H, d, C7-H); 13C NMR (DMSO-d6) δ 47.51 (CH2),
113.47 (C4), 121.07 (C7), 126.41 (C3a or C5), 126.99 (C6),
129.76 (C2′, C3′, C5′, C6′), 133.50 (C4′), 135.39 (C7a), 135.47
(C1′), 141.38 (C3a or C5), 143.40 (C2). Anal. (C14H8N2Cl4) C,
H, N.
13.62 (1H, s), 7.81 (s, 2H).
2-Br om o-5,6-d ich lor o-1-(su bstitu ted ben zyl)ben zim id -
a zole (8). 2-Bromo-5,6-dichlorobenzimidazole (7) (0.200 g,
0.75 mmol) and sodium hydroxide (0.03 g, 0.75 mmol) were
dissolved in acetonitrile (40 mL). After approximately 1 h of
stirring, the appropriately substituted benzyl halide (0.75
mmol) was added and the mixture was set to reflux. Once
thin-layer chromatography showed only one spot, the reaction
mixture was cooled to room temperature, evaporated to
dryness, dissolved in a mixture of methanol and tetrahydro-
furan, decolorized with charcoal, filtered, and evaporated to
dryness. The product was dissolved in a hot mixture of
1-(p-Meth oxyben zyl)-2,5,6-tr ich lor oben zim idazole (6c).
5 was reacted with p-methoxybenzyl chloride (0.12 g, 0.75
mmol): yield 0.17 g (65%); Rf 0.81; yellow crystals; mp 159-
160 °C. Anal. (C15H11N2Cl3O) C, H, N.
1-(p-Meth ylben zyl)-2,5,6-tr ich lor oben zim id a zole (6d ).
5 was reacted with p-methylbenzyl chloride (0.11 g, 0.75
mmol): yield 0.13 g (54%); Rf 0.81; white crystals; mp 174-
176 °C. Anal. (C15H11N2Cl3) C, H, N.
methanol and tetrahydrofuran and recrystallized from
a
mixture of methanol, tetrahydrofuran, and water to obtain
1-(m ,p -Dich lor ob en zyl)-2,5,6-t r ich lor ob en zim id a zole
(6e). 5 was reacted with m,p-dichlorobenzyl chloride (0.15 g,
0.75 mmol): yield 0.19 g (67%); Rf 0.80; white crystals; mp
224-226 °C. Anal. (C14H7N2Cl5) C, H, N.
1-(m -Ch lor oben zyl)-2,5,6-tr ich lor oben zim id a zole (6f).
5 was reacted with m-chlorobenzyl chloride (0.12 g, 0.75
mmol): yield 0.16 g (63%); Rf 0.81; white crystals; 170-172
°C. Anal. (C14H8N2Cl4) C, H, N.
pure 8a -o.
1-Ben zyl-2-br om o-5,6-d ich lor oben zim id a zole (8a ).
7
was reacted with benzyl bromide (0.128 g, 0.75 mmol): yield
0.13 g (64%); Rf 0.73; white crystals; mp 194-196 °C. Anal.
(C14H9N2Cl2Br) C, H, N.
2-Br om o-1-(p -ch lor ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (8b). 7 was reacted with p-chlorobenzyl chloride (0.12
g, 0.75 mmol): yield 0.20 g (67%); Rf 0.58; white crystals; mp
206-208 °C. Anal. (C14H8N2Cl3Br) C, H, N.
2-Br om o-1-(p -m et h oxyb en zyl)-5,6-d ich lor ob en zim id -
a zole (8c). 7 was reacted with p-methoxybenzyl chloride (0.12
g, 0.75 mmol): yield 0.14 g (48%); Rf 0.65; light brown crystals;
mp 156-158 °C. Anal. (C15H11N2Cl2BrO) C, H, N.
2-Br om o-1-(p -m e t h ylb e n zyl)-5,6-d ich lor ob e n zim id -
a zole (8d ). 7 was reacted with p-methylbenzyl chloride (0.11
g, 0.75 mmol): yield 0.20 g (72%); Rf 0.65; white powder; mp
194-195 °C. Anal. (C15H11N2Cl2Br) C, H, N.
2-Br om o-1-(m ,p -d ich lor ob e n zyl)-5,6-d ich lor ob e n z-
im id a zole (8e). 7 was reacted with m,p-dichlorobenzyl
chloride (0.15 g, 0.75 mmol): yield 0.20 g (63%); Rf 0.58; white
crystals; mp 222-224 °C. Anal. (C14H7N2Cl4Br) C, H, N.
2-Br om o-1-(m -ch lor ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (8f). 7 was reacted with m-chlorobenzyl chloride (0.12
g, 0.75 mmol): yield 0.22 g (76%); Rf 0.52; white powder; mp
166-168 °C. Anal. (C14H8N2Cl3Br) C, H, N.
1-(p-ter t-Bu tylben zyl)-2,5,6-tr ich lor oben zim idazole (6g).
5 was reacted with p-tert-butylbenzyl bromide (0.17 g, 0.75
mmol): yield 0.21 g (77%); Rf 0.81; white crystals; mp 222-
226 °C. Anal. (C18H17N2Cl3) C, H, N.
1-(p -(Tr iflu or om et h yl)b en zyl)-2,5,6-t r ich lor ob en zim -
id a zole (6h ). 5 was reacted with p-(trifluoromethyl)benzyl
bromide (0.18 g, 0.75 mmol): yield 0.19 g (67%); Rf 0.81; white
crystals; mp 198-200 °C. Anal. (C15H8N2Cl3F3) C, H, N.
1-(m -Meth ylben zyl)-2,5,6-tr ich lor oben zim id a zole (6i).
5 was reacted with m-methylbenzyl chloride (0.11 g, 0.75
mmol): yield 0.16 g (67%); Rf 0.81; yellow crystals; mp 138-
141 °C. Anal. (C15H11N2Cl3) C, H, N.
1-(m -(Tr iflu or om e t h yl)b e n zyl)-2,5,6-t r ich lor ob e n z-
im id a zole (6j). 5 was reacted with m-(trifluoromethyl)benzyl
chloride (0.18 g, 0.75 mmol): yield 0.17 g (60%); Rf 0.81; white
crystals; mp 175-177 °C. Anal. (C15H8N2Cl3F3) C, H, N.
1-(o-Ch lor oben zyl)-2,5,6-tr ich lor oben zim id a zole (6k ).
5 was reacted with o-chlorobenzyl chloride (0.12 g, 0.75
mmol): yield 0.15 g (57%); Rf 0.80; white crystals; mp 120-
122 °C. Anal. (C14H8N2Cl4) C, H, N.
2-B r o m o -1-(p -t er t -b u t y lb e n zy l)-5,6-d ic h lo r o b e n z-
im id a zole (8g). 7 was reacted with p-tert-butylbenzyl bro-
mide (0.17 g, 0.75 mmol): yield 0.21 g (67%); Rf 0.52; white
crystals; mp 230-231 °C. Anal. (C18H17N2Cl2Br) C, H, N.
2-Br om o-1-(p -(t r iflu or om et h yl)b en zyl)-5,6-d ich lor o-
ben zim id a zole (8h ). 7 was reacted with p-(trifluoromethyl)-
benzyl bromide (0.18 g, 0.75 mmol): yield 0.25 g (79%); Rf 0.58;
white crystals; mp 195-197 °C. Anal. (C15H8N2Cl2BrF3) C,
H, N.
1-(m ,m -Bis(t r iflu or om e t h yl)b e n zyl)-2,5,6-t r ich lor o-
ben zim id a zole (6l). 5 was reacted with m,m-bis(trifluoro-
methyl)benzyl bromide (0.23 g, 0.75 mmol): yield 0.25 g (75%);
Rf 0.81; white crystals; mp 173-175 °C. Anal. (C16H7N2Cl3F6)
C, H, N.

1260 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Porcari et al.
2-Br om o-1-(m -m et h ylb en zyl)-5,6-d ich lor ob en zim id -
a zole (8i). 7 was reacted with m-methylbenzyl chloride (0.11
g, 0.75 mmol): yield 0.20 g (72%); Rf 0.58; light brown powder;
mp 160-162 °C; 1H NMR (DMSO-d6) δ 2.25 (3H, s, CH3) 5.51
(2H, s, CH2), 6.91 (1H, d, C6′-H), 7.01 (1H, s, C2′-H), 7.10 (1H,
d, C4′-H), 7.23 (1H, t, C5′-H), 7.96 (1H, s, C4-H), 8.06 (1H, s,
C7-H); 13C NMR (DMSO-d6) δ 21.84 (CH3), 49.07 (CH2), 113.4
(C4), 120.84 (C7), 124.63 (C6′), 126.15 (C3a or C5), 126.76 (C6),
128.1 (C2′), 129.42 (C4′), 129.64 (C5′), 134.15 (C2), 135.94
(C7a), 136.4 (C1′), 138.98 (C3′), 142.84 (C3a or C5). Anal.
(C15H11N2Cl2Br) C, H, N.
sealed in a pressure vessel, and heated to 60 °C with constant
stirring. After 5 days, the solvent was removed and the
residue was dissolved in chloroform and eluted off a silica gel
column (3.5 × 8 cm) using chloroform as the solvent. The
fractions showing the desired compound were combined,
evaporated to dryness, and recrystallized from methanol and
water to obtain pure 11: yield 0.08 g (37%); Rf 0.30 (ethyl
acetate:hexane, 1:9); white solid; mp 175-177 °C; 1H NMR
(DMSO-d6) δ 1.20 (6H, d), 4.07 (1H, m), 5.30 (2H, s), 6.90-
9.63 (1H, d), 7.10 (1H, s), 7.14 (1H, s), 7.23-7.36 (5H, m). Anal.
(C17H17N3Cl2) C, H, N.
2-Br om o-1-(m -(t r iflu or om et h yl)b en zyl)-5,6-d ich lor o-
ben zim id a zole (8j). 7 was reacted with m-(trifluoromethyl)-
benzyl chloride (0.18 g, 0.75 mmol): yield 0.21 g (65%); Rf 0.58;
yellow crystals; mp 172-174 °C. Anal. (C15H8N2Cl2BrF3) C,
H, N.
2-Br om o-1-(o-ch lor ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (8k ). 7 was reacted with o-chlorobenzyl chloride (0.12
g, 0.75 mmol): yield 0.23 g (40%); Rf 0.80; white crystals; mp
120-122 °C. Anal. (C14H8N2Cl3Br) C, H, N.
2-Br om o-1-(m ,m -b is(t r iflu or om et h yl)b en zyl)-5,6-d i-
ch lor oben zim id a zole (8l). 7 was reacted with m,m-bis-
(trifluoromethyl)benzyl bromide (0.23 g, 0.75 mmol): yield 0.25
g (69%); Rf 0.58; white needles; mp 168-170 °C. Anal.
(C16H7N2Cl2BrF6) C, H, N.
2-B r o m o -1-(p -n it r o b e n zy l)-5,6-d ic h lo r o b e n zim id -
a zole (8m ). 7 was reacted with p-nitrobenzyl chloride (0.13
g, 0.75 mmol): yield 0.23 g (77%); Rf 0.84; yellow powder; mp
203-205 °C. Anal. (C14H8N2Cl2BrO2) C, H, N.
2-B r om o-1-(m -n it r o b e n zy l)-5,6-d ic h lo r o b e n zim id -
a zole (8n ). 7 was reacted with m-nitrobenzyl chloride (0.13
g, 0.75 mmol): yield 0.25 g (82%); Rf 0.58; light brown crystals;
mp 202-204 °C. Anal. (C14H8N2Cl2BrO2) C, H, N.
2-Br om o-1-(p -flu or ob e n zyl)-5,6-d ich lor ob e n zim id -
a zole (8o). 7 was reacted with p-fluorobenzyl chloride (0.11
g, 0.75 mmol): yield 0.19 g (67%); Rf 0.58; white crystals; mp
215-217 °C. Anal. (C14H8N2Cl2BrF) C, H, N.
1-(P h en yleth yl)-2,5,6-tr ich lor oben zim idazole (9). 2,5,6-
Trichlorobenzimidazole (5) (0.1661 g, 0.75 mmol) and sodium
hydroxide (0.03 g, 0.75 mmol) were dissolved in acetonitrile
(40 mL). After approximately 1 h of stirring, (2-bromoethyl)-
benzene (0.14 g, 0.75 mmol) was added and the mixture was
heated at reflux. Once thin-layer chromatography showed only
one spot, the reaction mixture was cooled to room temperature,
evaporated to dryness, dissolved in methanol, decolorized with
charcoal, filtered, and evaporated to dryness. The product was
dissolved in hot methanol and recrystallized from a mixture
of methanol and water to obtain pure 9: yield 0.15 g (62%);
Rf 0.82; light brown crystals; mp 179-181 °C; 1H NMR
(DMSO-d6) δ 3.00 (2H, t), 4.45 (2H, t), 7.07-7.22 (5H, m), 7.85
(1H, s), 7.90 (1H, s); 13C NMR (DMSO-d6) δ 34.57, 45.82,
112.65, 119.80, 125.05, 125.62, 126.70, 128.35, 128.96, 134.28,
137.37, 140.27, 142.40. Anal. (C15H11N2Cl3) C, H, N.
2-Br om o-1-(p h en ylet h yl)-5,6-d ich lor ob en zim id a zole
(10). 2-Bromo-5,6-dichlorobenzimidazole (7) (0.20 g, 0.75
mmol) and sodium hydroxide (0.03 g, 0.75 mmol) were dis-
solved in acetonitrile (40 mL). After approximately 1 h of
stirring, (2-bromoethyl)benzene (0.14 g, 0.75 mmol) was added
and the mixture was heated at reflux. Once thin-layer
chromatography showed only one spot, the reaction mixture
was cooled to room temperature, evaporated to dryness,
dissolved in a mixture of methanol and tetrahydrofuran,
decolorized with charcoal, filtered, and evaporated to dryness.
The product was dissolved in a mixture of hot methanol and
tetrahydrofuran and recrystallized from a mixture of metha-
nol, tetrahydrofuran, and water to obtain pure 10: yield 0.18
g (63%); Rf 0.78; white crystals; mp 179-181 °C; 1H NMR
(DMSO-d6) δ 3.00 (2H, t), 4.44 (2H, t), 7.08-7.24 (5H, m), 7.86
(1H, s), 7.90 (1H, s); 13C NMR (DMSO-d6) δ 34.72, 46.69,
112.58, 119.63, 124.86, 125.52, 126.71, 128.36, 128.98, 132.86,
134.67, 137.34, 141.72. Anal. (C15H11N2Cl2Br) C, H, N.
1-Ben zyl-2-(isop r op yla m in o)-5,6-d ich lor ob en zim id -
a zole (11). 1-Benzyl-2,5,6-trichlorobenzimidazole (6a ) (200
mg, 0.64 mmol) was dissolved in isopropylamine (20 mL),
1-B e n zy l-2-(c y c lo p r o p y la m in o )-5,6-d ic h lo r o b e n z-
im id a zole (12). 1-Benzyl-2,5,6-trichlorobenzimidazole (6a )
(700 mg, 2.25 mmol) was dissolved in cyclopropylamine (30
mL), sealed in a pressure vessel, and heated to 60 °C with
constant stirring. After 5 days, the solvent was removed and
the residue was washed with chloroform to obtain a white
precipitate. The precipitate was recrystallized from methanol
and water to obtain pure 12: yield 0.20 g (27%); Rf 0.10
(chloroform); white crystals; mp 194-196 °C; 1H NMR (DMSO-
d6) δ 0.53 (2H, m), 0.72 (2H, m), 2.80 (1H, m), 3.35 (1H, s),
5.27 (2H, s), 7.09 (1H, s), 7.11 (1H, s), 7.25-7.45 (5H, m). Anal.
(C17H15N3Cl2) C, H, N.
Cell Cu ltu r e P r oced u r es. The routine growth and pas-
sage of KB and human foreskin fibroblasts (HFF cells) were
performed in monolayer cultures using minimal essential
medium (MEM) with either Hanks salts [MEM(H)] or Earle
salts [MEM(E)] supplemented with 10% calf serum (KB cells)
or 10% fetal bovine serum (HFF cells). The sodium bicarbon-
ate concentration was varied to meet the buffering capacity
required. Cells were passaged at 1:2 to 1:10 dilutions accord-
ing to conventional procedures by using 0.05% trypsin plus
0.02% EDTA in a HEPES-buffered salt solution. Similar
suspension culture conditions were employed for CEM-SS cells.
Vir ologica l P r oced u r es. The Towne strain, plaque-puri-
fied isolate Po, of HCMV was kindly provided by Dr. Mark
Stinski, University of Iowa. The KOS strain of HSV-1 was
used in most experiments and was provided by Dr. Sandra K.
Weller, University of Connecticut. Stock HCMV was prepared
by infecting HFF cells at a multiplicity of infection (moi) of
<0.01 plaque-forming units (pfu) per cell as detailed previ-
ously.27 High-titer HSV-1 stocks were prepared by infecting
KB cells at an moi of <0.1 also as detailed previously.27 Virus
titers were obtained using monolayer cultures of HFF cells
for HCMV and monolayer cultures of BSC-1 cells for HSV-1
as described earlier.28 The HIV-1 strain IIIB producer cell line
H9IIIB was obtained through the courtesy of Dr. R. C. Gallo.
Matched pairs of pre-AZT and post-AZT human treatment
isolates of HIV-1 which were sensitive and resistant to AZT
were obtained from Dr. D. D. Richman29 through the AIDS
Research and Reference Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health, Bethesda, MD. The A17 variant of HIV-1,
resistant to nonnucleoside RT inhibitors including TIBO,30 was
obtained from Dr. A. M. Emini through the AIDS Research
and Reference Program; HIV-2 also was obtained from the
latter program. HIV-1 strain IIIB was assayed in CEM-SS
cells as described previously by Kucera et al.31,32
An tivir a l Assa ys, HCMV. A plaque assay was used for
HCMV. HFF cells in 24-well cluster dishes were infected with
approximately 100 pfu of HCMV per cm2 cell sheet using the
procedures detailed above. Following virus adsorption, com-
pounds dissolved in growth medium were added to duplicate
wells in four to eight selected concentrations. After incubation
at 37 °C for 7 days, cell sheets were fixed and stained with
crystal violet and microscopic plaques enumerated as described
above. Drug effects were calculated as a percentage of
reduction in number of plaques in the presence of each drug
concentration compared to the number observed in the absence
of drug. For yield reduction assay, the procedure devised by
us28 was used. HFF cells were planted as described above in
96-well cluster dishes, incubated overnight, and infected with
HCMV at a moi of 0.5-1 pfu per cell. After virus adsorption,
innoculum was replaced with 0.2 mL of fresh medium contain-

Synthesis of Substituted 5,6-Dichlorobenzimidazoles
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1261
ing test compounds in a manner so that six compounds could
be tested in duplicate on a single plate with concentrations
from 100 to 0.14 µM. Plates were incubated at 37 °C for 7
days and subjected to one cycle of freezing and thawing, and
aliquots from each of the wells were transferred to a fresh 96-
well monolayer culture of HFF cells. Each column of the
original drug-containing plate was diluted across a new
separate plate in this manner. Cultures were incubated,
plaques were enumerated, and titers were calculated as
described.28
HSV-1. An ELISA developed by us33 was employed to detect
HSV-1. Ninety-six-well cluster dishes were planted with
10 000 BSC-1 cells per well in 200 µL per well of MEM(E) plus
10% calf serum. After overnight incubation at 37 °C, selected
drug concentrations in quadruplicate and HSV-1 at a concen-
tration of 100 pfu/well were added. Following a 3-day incuba-
tion at 37 °C, medium was removed, plates were blocked and
rinsed, and horseradish peroxidase conjugated rabbit anti-
HSV-1 antibody was added. Following removal of the antibody
containing solution, plates were rinsed and then developed by
adding a solution of tetramethylbenzidine as substrate. The
reaction was stopped with H2SO4, and absorbance was read
at 450 and 570 nm. Drug effects were calculated as a
percentage of the reduction in absorbance in the presence of
each drug concentration compared to absorbance obtained with
virus in the absence of drug.
Da ta An a lysis. Dose-response relationships were con-
structed by linearly regressing the percent inhibition of
parameters derived in the preceding sections against log drug
concentrations. Fifty-percent inhibitory (IC50) concentrations
were calculated from the regression lines. Samples containing
positive controls (acyclovir for HSV-1, ganciclovir for HCMV,
zidovudine for HIV-1, and 2-acetylpyridine thiosemicarbazone
for cytotoxicity) were used in all assays.
Ack n ow led gm en t. The authors thank Chirag Patel,
J ulie M. Breitenbach, Nathan P. Iyer, Dr. Mary S.
Ludwig, Carolyn Oh, and Roger G. Ptak for expert
technical performance of biological assays, J ack Hinkley
for his large-scale preparation of starting materials, and
Marina Savic for assistance with manuscript prepara-
tion. This study was supported by research grants U01-
25739, U01-AI31718, R01-AI33332, and R01-AI-36872
from the National Institute of Allergy and Infectious
Diseases, Research Agreement DRDA-942921, and by
training grant T32-GM07767 from the National Insti-
tutes of Health.
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activities of compounds in cells acutely infected with HIV-1.
The syncytial plaque assay performed as previously described
by us32 was used to measure the effect of compounds on HIV-
1. Briefly, CEM-SS cells rendered adherent to substrate by
means of poly-L-lysine were infected with HIV-1 strain IIIB in
the presence and absence of selected concentrations of com-
pounds. Syncytial plaques were enumerated at 30-fold mag-
nification 7 days postinfection. In the second assay, reverse
transcriptase (RT) was employed as a marker for HIV-1.
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pfu per cell with strain IIIB of HIV-1 in a minimal volume of
stock virus in growth medium. Cultures were incubated at
37 °C for 2 h to permit virus adsorption, washed and then
diluted to 5 × 105 cells/mL with RPMI 1640 containing 10%
fetal bovine serum. One-tenth milliliter was then added to
each well of a 96-well cluster dish. Fresh medium (0.1 mL
with 10% fetal bovine serum) containing test compounds in
twice the desired final concentration was added to triplicate
wells at seven concentrations ranging from 100 to 0.14 µM.
After 6 days of incubation, supernatant samples were taken
and the amount of RT activity was measured by the incorpora-
tion of [3H]dTTP into acid insoluble material using the assay
described by White et al.34 Drug effects were calculated as a
percentage of the reduction in counts per minute (cpm) in the
presence of each drug concentration compared to the cpm
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incubated at 37 °C for 48 h in a CO2 incubator, rinsed, fixed
with 95% ethanol, and stained with 0.1% crystal violet.
Acidified ethanol was added and plates read at 570 nm in a
spectrophotometer designed to read 96-well ELISA assay
plates. (iii) Inhibition of DNA synthesis in CEM-SS cells was
measured by determining the effect of compounds on the
uptake of [3H]dThd into total acid precipitable material as
detailed earlier.32
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Discrete Sites For Premature RNA.-Chain Termination Late in
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76, 2571-2575.

1262 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Porcari et al.
(14) (a) Harlow, P.; Molloy, G. Effect of 5,6-Dichloro-1-â-D-ribofuran-
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