Synthesis and antiviral evaluation of halogenated β-D- and -L- erythrofuranosylbenzimidazoles

Article abstract of DOI:10.1021/jm990195p

A series of 2-substituted benzimidazole D- and L-erythrofuranosyl nucleosides were synthesized and tested for activity against herpesviruses and for cytotoxicity. The D-nucleosides 2,5,6-trichloro-1-(β-D- erythrofuranosyl)benzimidazole (8a) and 2-bromo-5,6-dichloro-1-(β-D- erythrofuranosyl)benzimidazole (8b) were prepared by coupling 1,2,3-tri-O- acetyl-β-D-erythrofuranose (D-6) with the appropriate benzimidazole, followed by removal of the acetyl protecting groups. The 2-isopropylamino (9), 2-cyclopropylamino (10), and 2-mercaptobenzyl (11) derivatives were synthesized by nucleophilic displacements of the C-2 chlorine in the benzimidazole moiety of 8a. The v-nucleoside 4-bromo-5,6-dichloro-2- isopropylamino-1-(β-v-erythrofuranosyl)benzimidazole (17) was prepared by coupling D-6 with the appropriate benzimidazole. The L-erythrofuranosyl derivatives, 5,6-dichloro-2-isopropylamino-1-(β-L- erythrofuranosyl)benzimidazole (21a), its 2-cyclopropylamino analogue (21b), and the 2-isopropylamino analogue (25), were prepared by coupling L-6 with the appropriate benzimidazole. Several of these new derivatives had very good activity against HCMV in plaque and yield reduction assays (IC₅₀ = 0.05-19 μM against the Towne strain of HCMV) and DNA hybridization assays. Very little activity was observed against other herpesviruses. This pattern is similar to the antiviral activity profile observed for the corresponding ribofuranosides 2,5,6-trichloro-1-(β-D-ribofuranosyl)benzimidazole (4a), its 2-bromo analogue (4b), and the 2-cyclopropylamino analogue (4c). In comparison, 8a was 15-fold more active against HCMV than 4a, and 8b was 4- fold more active against HCMV than 4b. The 5,6-dichloro-2-isopropylamino-1- (βL-erythrofuranosyl)benzimidazole (21a) was less active than 4c, which is now in clinical trials for HCMV infection. Both 8a,b had comparable HCMV activity to 4c. Mode of action studies with the v-erythrose analogues established that 8b acted by inhibition of viral DNA processing whereas 9 and 10 may act via a different mechanism. The lack of a 5'-hydroxymethyl group in all members of this series established that antiviral activity occurred without 5'-phosphorylation, a feature required for the activity of most nucleoside analogues.

Full text of DOI:10.1021/jm990195p

2464
J . Med. Chem. 2000, 43, 2464-2472
Syn th esis a n d An tivir a l Eva lu a tion of Ha logen a ted â-D- a n d
-L-Er yth r ofu r a n osylben zim id a zoles
Kristjan S. Gudmundsson,† J effrey Tidwell,§ Nicole Lippa, George W. Koszalka,§ Nanine van Draanen,§
Roger G. Ptak,‡ 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 Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan 48109-1065, and Division of Chemistry, Glaxo Wellcome Inc., 5 Moore Drive,
Research Triangle Park, North Carolina 27709
Received April 21, 1999
A series of 2-substituted benzimidazole D- and L-erythrofuranosyl nucleosides were synthesized
and tested for activity against herpesviruses and for cytotoxicity. The D-nucleosides 2,5,6-
trichloro-1-(â-D-erythrofuranosyl)benzimidazole (8a ) and 2-bromo-5,6-dichloro-1-(â-D-erythro-
furanosyl)benzimidazole (8b) were prepared by coupling 1,2,3-tri-O-acetyl-â-D-erythrofuranose
(D-6) with the appropriate benzimidazole, followed by removal of the acetyl protecting groups.
The 2-isopropylamino (9), 2-cyclopropylamino (10), and 2-mercaptobenzyl (11) derivatives were
synthesized by nucleophilic displacements of the C-2 chlorine in the benzimidazole moiety of
8a . The D-nucleoside 4-bromo-5,6-dichloro-2-isopropylamino-1-(â-D-erythrofuranosyl)benz-
imidazole (17) was prepared by coupling D-6 with the appropriate benzimidazole. The
L-erythrofuranosyl derivatives, 5,6-dichloro-2-isopropylamino-1-(â-L-erythrofuranosyl)benz-
imidazole (21a ), its 2-cyclopropylamino analogue (21b), and the 2-isopropylamino analogue
(25), were prepared by coupling L-6 with the appropriate benzimidazole. Several of these new
derivatives had very good activity against HCMV in plaque and yield reduction assays (IC50
)
0.05-19 µM against the Towne strain of HCMV) and DNA hybridization assays. Very little
activity was observed against other herpesviruses. This pattern is similar to the antiviral
activity profile observed for the corresponding ribofuranosides 2,5,6-trichloro-1-(â-D-ribofur-
anosyl)benzimidazole (4a ), its 2-bromo analogue (4b), and the 2-cyclopropylamino analogue
(4c). In comparison, 8a was 15-fold more active against HCMV than 4a , and 8b was 4-fold
more active against HCMV than 4b. The 5,6-dichloro-2-isopropylamino-1-(â-L-erythrofuranosyl)-
benzimidazole (21a ) was less active than 4c, which is now in clinical trials for HCMV infection.
Both 8a ,b had comparable HCMV activity to 4c. Mode of action studies with the D-erythrose
analogues established that 8b acted by inhibition of viral DNA processing whereas 9 and 10
may act via a different mechanism. The lack of a 5′-hydroxymethyl group in all members of
this series established that antiviral activity occurred without 5′-phosphorylation, a feature
required for the activity of most nucleoside analogues.
In tr od u ction
effects, they have limited oral bioavailability, or they
must be injected intra-vitrally. Moreover, virus strains
resistant to most of these drugs are emerging.9 Conse-
quently, there is a need for a more potent and selective
antiviral drug to treat HCMV infections. As part of our
Human cytomegalovirus (HCMV) is one of eight
human herpesviruses. It is estimated that by adulthood,
more than half of all Americans will have been infected
with HCMV.1 HCMV infections in immunocompetent
individuals are usually asymptomatic. However, in
immunocompromised patients, HCMV infections are
often life-threatening. Transplant recipients2 and indi-
viduals with acquired immune deficiency syndrome
(AIDS)3 are vulnerable to these infections. HCMV is also
a leading cause of birth defects as a consequence of in
utero fetal infection. Currently, there are four FDA-
approved drugs available for the treatment of HCMV
infections: ganciclovir (1),5 foscarnet (2),6 cidofovir (3),7
and the 21-mer antisense oligonucleotide fomivirsen.8
Unfortunately, these drugs can produce significant side
search for new anticancer10 and antiviral drugs,11,12
a
number of benzimidazole nucleosides have been syn-
thesized. Certain compounds, including 2,5,6-trichloro-
1-(â-D-ribofuranosyl)benzimidazole (TCRB, 4a ) and 2-
bromo-5,6-dichloro-1-(â-D-ribofuranosyl)benzimidazole
(BDCRB, 4b), have potent activity against HCMV with
low cellular toxicity at concentrations inhibiting viral
growth.12 Biological evaluation of 4a ,b has established
that their antiviral activity involves neither phospho-
rylation of the compound nor inhibition of DNA, RNA,
or protein synthesis.13 Rather, they act by a unique
mechanism, which involves inhibition of viral DNA
processing and virus assembly.14 Related L-ribofuranosyl
analogues, such as 2-isopropylamino-5,6-dichloro-1-(â-
L-ribofuranosyl)benzimidazole (4c, 1263W94), also have
been shown to have very good activity against HCMV.
These L-ribofuranosyl derivatives inhibit HCMV DNA
* To whom correspondence should be addressed. Tel: 734-764-7547.
Fax: 734-763-5633. E-mail: ltownsen@umich.edu.
§ Glaxo Wellcome Inc.
† Present address: Glaxo Wellcome Inc., 5 Moore Dr., Research
Triangle Park, NC 27709.
‡ Present address: Southern Research Institute, 431 Aviation Way,
Frederick, MD 21701.
10.1021/jm990195p CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/17/2000

â-D- and -L-Erythrofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2465
meric proton of 7a with the chemical shift and coupling
constants of related 2,3-di-O-acetyl-â-D-benzimidazole
nucleosides12,18,19 supported the â-assignment. Com-
pound 7a was deprotected with sodium carbonate in
aqueous ethanol to give 2,5,6-trichloro-1-(â-D-erythro-
furanosyl)benzimidazole (8a ) in 81% yield. Similarly,
2-bromo-5,6-dichloro-1-(â-D-erythrofuranosyl)benzimid-
azole (8b) was synthesized by a condensation of 2-bromo-
5,6-dichlorobenzimidazole (5b)12 with D-6, followed by
deprotection with sodium carbonate in aqueous ethanol.
The chlorine at C-2 of compound 7a can be readily
displaced with a variety of nucleophiles. A series of C-2
substituted D-erythrofuranosides 9-11 were formed by
the treatment of 7a with the appropriate nucleophiles,
under mild conditions, resulting in substitution of the
2-chloro group and concomitant deacetylation of the
carbohydrate moiety. The choice of nucleophiles was
based on the previously established activity of C-2
substituted benzimidazole ribonucleosides. Thus, com-
pound 7a was treated with cyclopropylamine, isopropyl-
amine, and sodium mercaptobenzylate to afford 2-cy-
clopropylamino-5,6-dichloro-1-(â-D-erythrofuranosyl)-
benzimidazole (9), 2-isopropylamino-5,6-dichloro-1-(â-
D-erythrofuranosyl)benzimidazole (10), and 5,6-di-
chloro-2-mercaptobenzyl-1-(â-D-erythrofuranosyl)benz-
imidazole (11), respectively.
F igu r e 1. Structures of ganciclovir (1), foscarnet (2), cidofovir
(3), and the benzimidazole nucleosides TCRB (4a ), BDCRB
(4b), and 1263W94 (4c).
In the synthesis of 4-bromo-5,6-dichloro-2-isopropyl-
amino-1-(â-D-erythrofuranosyl)benzimidazole (17), 4-bro-
mo-5,6-dichloro-2-isopropylaminobenzimidazole (15) was
synthesized first and then coupled with D-6. Thus,
2-nitro-4,5-dichloroaniline (12) was brominated using
NBS to give 13 and then reduced (Fe, HCl) to 3-bromo-
4,5-dichloro-1,2-phenylenediamine (14). Phenylenedi-
amine 14 was condensed with isopropyl isothiocyanate
in pyridine in the presence of the desulfurizing agent,
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-
p-toluenesulfonate,24 to give 15. Condensation of 15 with
D-6 under Vorbruggen conditions, followed by deprotec-
tion, gave 17. The L-isomers 5,6-dichloro-2-isopropyl-
amino-1-(â-L-erythrofuranosyl)benzimidazole (21a ), 5,6-
dichloro-2-cyclopropylamino-1-(â-L-erythrofuranosyl)-
benzimidazole (21b), and 4,5,6-trichloro-2-isopropylamino-
1-(â-L-erythrofuranosyl)benzimidazole (25) were syn-
thesized in a similar fashion by condensing 1,2,3-tri-O-
acetyl-â-L-erythrofuranose (L-6)25 with the appropriate
benzimidazole. The cyclopropylamino and isopropyl-
amino groups were chosen as C-2 substituents based
on structure-activity relationships from the L-ribofur-
anosyl benzimidazole series.
synthesis by a mechanism that does not require phos-
phorylation and does not involve viral DNA poly-
merase.15 Thus they have a different mode of action
from the D-ribofuranose derivatives.15 Compound 4c is
in clinical development for the treatment of HCMV
infections.15,16
Numerous structure-activity relationship studies of
derivatives of 4a -c, modified in either the heterocycle
or carbohydrate moiety, have been undertaken in our
laboratories10-12,17,18 In this article we describe studies
on the effect of replacing a ribofuranosyl moiety with a
D- or L-erythrofuranosyl moiety.
Resu lts a n d Discu ssion
Ch em istr y. As part of our studies on the structure-
activity relationships for the benzimidazole nucleosides,
2,5,6-trichloro-1-(5-deoxy-â-D-ribofuranosyl)benzimid-
azole18 and various 5′-modified analogues have been
prepared.19 Several of these compounds had better
antiviral activity than TCRB. This prompted the syn-
thesis and antiviral evaluation of the erythrofuranosyl
analogues described herein.
Biology. This new series of compounds was evaluated
for activity against HCMV and HSV-1 as well as for
cytotoxicity in a number of cell lines (Table 1). Like the
lead compounds (4a -c), none of the compounds in this
study had significant activity against HSV-1. Several
of the tested compounds were more cytotoxic than 4a -
c, but for the most active compounds (e.g. 8a ,b) cyto-
toxicity was well-separated from antiviral activity.
D-Erythrose20 was acetylated with acetic anhydride
in pyridine to give 1,2,3-tri-O-acetyl-D-erythrofuranose
(D-6).21 Silylation of 2,5,6-trichlorobenzimidazole (5a )22
with bis(trimethylsilyl)acetamide (BSA) was followed by
the addition of D-6 and trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) to the reaction mixture to give
exclusively the desired 2,5,6-trichloro-1-(2,3-di-O-acetyl-
â-D-erythrofuranosyl)benzimidazole (7a ) in 75% yield.
The desired â-anomer (7a ) was the only isolated product
since a condensation of the acetylated carbohydrate D-6
and 2,5,6-trichlorobenzimidazole (5a), under Vorbruggen
conditions, follows Bakers rule,23 due to the 2′-O-acetyl
participation during the condensation. Comparison of
the chemical shifts and coupling constants of the ano-
Evaluation of the compounds against HCMV revealed
that the erythro compounds 8a ,b were approximately
10 times more active than the ribo-4a and 4 times more
active than the ribo-4b. Compounds 8a ,b were about
40 times more active than ganciclovir (Table 1).
Interestingly, the antiviral activity observed for the
di-O-acetates (7a ,b) was nearly the same as the activity

2466 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Gudmundsson et al.
Sch em e 1. Synthesis of D-Erythrofuranosylbenzimidazole Derivatives
observed for the deprotected erythrosides (8a ,b). We
speculate that this may be due to deacetylation of 7a ,b
by serum esterases in the cell culture medium. Results
from yield reduction assays established more firmly the
greater activity of 7a ,b and 8a ,b against HCMV than
the activity of 4a ,b and ganciclovir. The 2-isopropyl-
amino derivative 9 and the 2-cyclopropylamino deriva-
tive 10 were considerably less active than the 2-halo
analogues and also less active than ganciclovir against
HCMV with less separation between antiviral activity
and cytotoxicity (Table 1). The same was true for the
mercaptobenzyl derivative 11.
The nucleosides 7b, 8b, and 9 were also tested against
the AD169 strain of HCMV in a plaque assay. All three
compounds were 4-5-fold more active against AD169
than against the Towne strain. The 2-isopropylamino
analogue 9 was less active than the 2-halo analogues
against both strains of HCMV in the plaque assays but
had equivalent activity in viral cytopathic effect (CPE)
and DNA hybridization assays.
respectively). Furthermore, whereas the D-erythrofur-
anosyl derivatives were more active than the corre-
sponding D-ribose derivatives (compare 8a ,b to 4a ,b),
this was not the case for the L-erythrofuranosyl deriva-
tives, which were less active than the corresponding
L-ribose derivatives. These differences in structure-
activity trends are not surprising in view of the fact that
L-benzimidazole nucleosides have a different mode of
action (inhibit viral DNA synthesis)15 than the D-
benzimidazole nucleosides (inhibit viral DNA process-
ing).14
Because two separate modes of action have been
discovered with the ribosylbenzimidazoles, initial mode
of action studies were performed with the erythro-
furanosylbenzimidazoles. Compounds 8b, 9, and 10
were selected for study based upon the different sub-
stituents in the 2-position. Their activity was compared
in strains of HCMV resistant to one or the other
mechanism of benzimidazole action. Strain C4 was
derived by selection with 4a and acquired resistance to
4a ,b by mutation of genes UL56 and UL89.26 Strain
2916r was derived by selection with an analogue of 4c
The L-erythrofuranosyl derivatives 21a ,b and 25 were
less active than their D-analogues (9, 10, and 17,

â-D- and -L-Erythrofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2467
Sch em e 2. Synthesis of
L-Erythrofuranosylbenzimidazoles
F igu r e 2. Time of drug action in the HCMV replication cycle.
Each compound was added at each of the indicated times to
duplicate cultures of infected cells to give a final concentration
of 100 µM. An identical aliquot of control medium also was
added to separate cultures in duplicate. Incubation was
continued for one replication cycle (96 h), and all the cultures
were frozen. HCMV titers were determined subsequently in
separate assays and are presented as percent of control titer
at each time point. Ganciclovir is a known inhibitor of HCMV
DNA synthesis;5 TCRB is an inhibitor of DNA processing.14
viral DNA synthesis inhibitor, could be added to HCMV-
infected cultures as late as 56-64 h without a signifi-
cant loss of activity. Activity of compound 4a was lost
even later, consistent with ganciclovir inhibition of viral
DNA synthesis and 4a inhibition of viral DNA process-
ing once DNA was synthesized. Activity of compound 9
was lost at approximately the same time as observed
with ganciclovir and 4a thereby establishing that the
compound acted late in the viral replication cycle. Even
though the results for 9 do not unambiguously dif-
ferentiate between the two mechanisms, the results,
together with data in Table 2, show that compound 9
affects either DNA synthesis, DNA processing, or both.
Whatever the reason, the change from a Cl or Br to an
iso- or cyclopropylamine in the 2-position not only
affected the potency of the compounds but also may
affected the mechanism by which the compounds act.
Regardless of mechanism, it is clear that the compounds
cannot be phosphorylated at the 5′-position as a conse-
quence of their design, thereby establishing that they
act differently than many nucleoside analogues.
and acquired resistance by mutation of gene UL97.27
In plaque assays, compound 8b was very active against
wild-type Towne and AD169 viruses as well as against
strain 2916r (Table 2). In contrast, strain C4 was as or
more resistant to 8b as it was to 4b (Table 2). As
expected, all four strains were equally sensitive to
ganciclovir. Together these data strongly suggest that
8b acts by the same mechanism as 4b: namely, inhibi-
tion of viral DNA processing via the proteins specified
by genes UL56 and UL89.
A more limited examination of the activity of com-
pounds 9 and 10 by the more sensitive yield reduction
assay revealed that in contrast to 8b, both wild-type and
C4 HCMV strains had about the same sensitivity to 9
and 10 (Table 2). The observation that C4 was resistant
to 4b in this assay but not to 4c suggests either 9 and
10 interact with a UL56/UL89 protein complex very
differently than does 4b or 9 and 10 may act in a
manner similar to 4c and not inhibit viral DNA proc-
essing. To explore this possibility further, a drug time
of addition study was accomplished with compound 9
along with control compounds that act by related
mechanisms. Figure 2 shows that ganciclovir, a known
Exp er im en ta l Section
Gen er a l Ch em ica l P r oced u r es. Melting points were
taken on a Thomas-Hoover Unimelt apparatus and are uncor-
rected. Nuclear magnetic resonance (NMR) spectra were
obtained at 300, 360, or 500 MHz. Flash column chromatog-
raphy was performed using silica gel 60 230-400 mesh (ICN)
and the technique described by Still et al.28 Thin-layer chro-
matography (TLC) was performed on prescored silica gel
GHLF plates (Analtech, Newark, DE). Compounds were
visualized by illumination under UV light (254 nm) or by
spraying with 20% methanolic sulfuric acid followed by char-
ring on a hot plate.
2,5,6-Tr ich lor o-1-(2,3-d i-O-a cet yl-â-D-er yt h r ofu r a n o-
syl)ben zim id a zole (7a ). 2,5,6-Trichlorobenzimidazole22 (5a ;
350 mg, 1.6 mmol) was suspended in dry CH3CN (5 mL). To
this suspension was added BSA (0.6 mL, 2.4 mmol) and the
resulting solution stirred at room temperature for 20 min.
1,2,3-Tri-O-acetyl-D-erythrofuranose (D-6; 390 mg, 1.6 mmol)

2468 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Gudmundsson et al.
Ta ble 1. Antiviral Activity and Cytotoxicity of Erythrofuranosylbenzimidazole Nucleosides
IC50 or IC90 (µΜ)
antiviral activity
HCMVa
HSV-1b
ELISA
cytotoxicityc
compd
plaque (Towne) plaque (AD169) yield (Towne)
CPE
DNA hybrid
visual growth MOLT 4
7a
8a
7b
8b
9
10
11
17
21a
21b
25
0.25
0.24
0.22
0.24
7.9
0.10
0.12
0.05
0.06
5.0d
6.8d
0.1
0.1
0.05
0.05
0.8
0.6
0.33
0.1
>100e
>100
>100
50
>100
>100
65
41
32
59
45
60
60
0.05d
0.04d
1.8
0.21
0.23
0.42
0.26
>10
0.16
0.8
32
50
47
84
132
>100
>100
32
>100
>100
60
19
32
>10
1.0
2.3
2.3
0.8
13
15
43
2.0
0.15
4a (TCRB)f
4b (BDCRB)f
4c (1263W94)
1 (GCV)g
2.8
0.70
1.3 ( 0.8
0.20
102
130
>100
238
118
>100
210
0.3
2
0.52
0.1
0.88
>100
0.45
0.60d
65d
7.4 ( 6.5
1.6 ( 1.2
3.5 ( 2.1 >100
>100
a
Plaque and yield reduction assays were performed in duplicate wells as described in the Experimental Section. Results from plaque
assays are reported as IC50’s, those for yield reduction experiments as IC90’s. CPE is cytophathic effect and measures microscopically
visable effects of virus on cells. The DNA hybridizaton assay was performed as described.33 b HSV-1 was assayed by ELISA in quadruplicate
wells. c Visual cytotoxicity was scored on HFF cells unaffected by HCMV at the time of plaque enumeration. Inhibition of KB cell growth
was determined in quadruplicate assays. Inhibition of MOLT 4 cells was determined as described in the literature.36 Results are presented
d
as IC50’s. Average of duplicate or triplicate experiments. e >100 µM indicates that IC50 not reached at the noted (highest) concentration
tested. f Results for compounds 4a ,b, also reported in ref 12 as compounds 9 and 11; averages ( SD from 5 and 15 experiments. g Averages
( SD derived from 108, 33, and 3 experiments, respectively, in which ganciclovir was used as a positive control.
Ta ble 2. Activity of D-Erythrofuranosylbenzimidazole
Nucleosides in Drug-Resistant Strains of HCMV
DMSO-d6) δ 169.64, 169.27, 141.54, 140.85, 132.18, 126.55,
126.20, 120.41, 118.49, 85.81, 71.98, 71.94, 70.75, 20.56, 20.11.
Anal. Calcd for C15H13Cl3N2O5: C, H, N.
2,5,6-Tr ich lor o-1-(â-D-e r yt h r ofu r a n osyl)b e n zim id -
a zole (8a ). Compound 7a (0.35 g, 0.85 mmol) was dissolved
in EtOH (40 mL). To this solution was added Na2CO3 (0.62 g,
5.84 mmol) in water (40 mL) and the resulting mixture stirred
for 4 h. Acetic acid was then added until a pH of 7 was obtained
and the resulting suspension was concentrated in vacuo. The
suspension was triturated with water and the solid collected
by filtration and recrystallized from MeOH to give 225 mg
(81%) of 8a as a white crystalline solid: mp 115-117 °C; Rf
0.19 (EtOAc/hexane 1:1); 1H NMR (360 MHz, DMSO-d6) δ 8.00
(s, 1H), 7.90 (s, 1H), 5.93 (d, 1H, 1′-H, J ) 7.8 Hz), 5.59 (d,
1H, D2O exchangeable), 5.37 (d, 1H, D2O exchangeable), 4.55
(m, 2H), 4.24 (d, 1H), 3.90 (d, 1H); 13C NMR (90 MHz, DMSO-
d6) δ 142.14, 141.01, 132.41, 126.16, 125.85, 120.36, 113.45,
89.31, 74.42, 73.31, 70.13. Anal. Calcd for C11H9Cl3N2O3: C,
H, N.
IC50 or IC90 (µM)
HCMV straina
Towne AD169
wild- wild-
type C-4b type 2916r c
compound
substituents
R1 R2
no.
name
Plaque Reduction Assay
4b BDCRB
4c 1263W94 â-L-ribose
â-D-ribose
Br
NHC3H7
1.7d 31d
0.34d 0.41d
0.33d >100d
0.03 0.03
8b
1
â-D-erythrose Br
0.23d 12d
4.5
ganciclovir
4.1 3.3
3.6
Yield Reduction Assay
â-D-ribose Br 0.3
4c 1263W94 â-L-ribose NHC3H7 0.59
2-Br om o-5,6-d ich lor o-1-(2,3-d i-O-a cet yl-â-D-er yt h r o-
fu r a n osyl)ben zim id a zole (7b). 2-Bromo-5,6-dichlorobenz-
imidazole12 (5b; 1.0 g, 3.8 mmol) was suspended in dry CH3CN
(20 mL). BSA (1.3 mL, 5.2 mmol) was added to this suspension
and the resulting solution stirred at room temperature for 20
min. 1,2,3-Tri-O-acetyl-D-erythrofuranose (D-6; 1.0 g, 4.1 mmol)
in CH3CN (15 mL) was added and this was followed by
TMSOTf (1.3 mL, 7.0 mmol). The reaction mixture was stirred
at ambient temperature for 2 h. Saturated NaHCO3(aq)
solution (100 mL) was added and the resulting mixture
extracted with EtOAc (3 × 50 mL). The combined organic
extracts were dried over magnesium sulfate, filtered and
evaporated in vacuo to give the crude product as a foam, which
was purified using flash column chromatography (SiO2, EtOAc/
hexane 1:2). The appropriate fractions were combined and the
solvent removed in vacuo to yield, after recrystallization from
MeOH, 1.3 g (75%) of 7b as white crystals: mp 168-170 °C;
4b BDCRB
8.0
0.70
8.5
9
10
â-D-erythrose NHC3H7 6.9
â-D-erythrose NHC3H5 10
5.0
a
Plaque and yield reduction assays were performed in duplicate
as described in the text. Results from plaque assays are reported
b
as IC50’s, those for yield reduction experiments as IC90’s. HCMV
resistant to 4a ,b - see ref 26. c HCMV resistant to 4c - see ref
d
27. Average derived from two or three experiments.
in CH3CN (4 mL) was added to the solution and this was then
followed by TMSOTf (0.3 mL, 1.6 mmol). The reaction mixture
was stirred at ambient temperature for 2 h. Saturated
NaHCO3(aq) solution was then added and the resulting
mixture extracted with EtOAc (3 × 50 mL). The combined
organic extracts were dried over magnesium sulfate, filtered
and evaporated under reduced pressure to give the crude
product as a foam. This foam was purified using flash column
chromatography (SiO2, CHCl3). The appropriate fractions were
combined and the solvent was removed in vacuo to yield, after
recrystallization from MeOH, 400 mg (61%) of 7a as white
1
Rf 0.2 (EtOAc/hexane 1:2); H NMR (360 MHz, DMSO-d6) δ
8.11 (s, 1H), 7.99 (s, 1H), 6.17 (d, 1H, J ) 7.9 Hz, 1′-H), 5.81
(m, 1H), 5.61 (t, 1H), 4.79 (dd, 1H), 4.12 (d, 1H), 2.17 (s, 3H),
1.98 (s, 3H). Anal. Calcd for C15H13BrCl2N2O5: C, H, N.
1
crystals: mp 166-168 °C; Rf 0.52 (2% MeOH in CHCl3); H
2-Br om o-5,6-d ich lor o-1-(â-D-er yt h r ofu r a n osyl)b en z-
im id a zole (8b). Compound 7b (0.4 g, 0.88 mmol) was dis-
solved in EtOH (40 mL). To this solution was added Na2CO3
(0.62 g, 5.84 mmol) in water (40 mL) and the resulting mixture
NMR (360 MHz, DMSO-d6) δ 8.13 (s, 1H), 8.00 (s, 1H), 6.19
(d, 1H, 1′-H, J ) 7.8 Hz), 5.81 (m, 1H), 5.60 (t, 1H), 4.79 (dd,
1H), 4.12 (d, 1H), 2.16 (s, 3H), 1.98 (s, 3H); 13C NMR (90 MHz,

â-D- and -L-Erythrofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2469
stirred for 4 h, then neutralized with acetic acid and concen-
trated under reduced pressure. The suspension was triturated
with water and the solid collected by filtration. This solid was
recrystallized from MeOH to give 275 mg (85%) of 8b as a
white crystalline solid: mp 138-139 °C; Rf 0.5 (EtOAc/hexane
2:1); 1H NMR (360 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.88 (s, 1H),
5.93 (d, 1H, 1′-H, J ) 7.9 Hz), 5.56 (d, 1H, D2O exchangeable),
5.35 (d, 1H, D2O exchangeable), 4.55 (m, 2H), 4.24 (m, 1H),
3.88 (d, 1H); 13C NMR (90 MHz, DMSO-d6) δ 142.60, 132.58,
132.43, 125.93, 125.68, 120.17, 113.23, 90.19, 74.31, 73.10,
70.10. Anal. Calcd for C11H9BrCl2N2O3: C, H, N.
DMSO-d6) δ 8.3 (s, 1H), 7.5 (br s, 2H). Anal. Calcd for C6H3-
BrCl2N2O2: C, H, N.
3-Br om o-4,5-d ich lor o-1,2-p h en ylen ed ia m in e (14). Com-
pound 13 (48.3 g, 169 mmol) was suspended in ethanol (1 L)
and the resulting suspension cooled to 0 °C. Concentrated HCl
(37%, 193 mL, 2.36 mol) was added dropwise to this suspension
over a period of 15 min. The resulting reaction mixture was
heated at reflux temperature for 1 h and subsequently cooled
to room temperature. The mixture was diluted with water (1.5
L) and the pH of this mixture was adjusted to 8 by the addition
of solid NaHCO3. The mixture was extracted with EtOAc (3
× 300 mL), the extracts were dried over magnesium sulfate
and filtered, and the filtrate was concentrated in vacuo to give
a solid. Recrystallization of the solid from aqueous methanol
gave 34.2 g (79%) of 14 as a solid: mp 128-129 °C dec; 1H
NMR (300 MHz, DMSO-d6) δ 6.69 (s, 1H), 5.24 (broad s, 2H),
5.06 (broad s, 2H). Anal. Calcd for C6H5BrCl2N2: C, H, N.
2-Isopr opylam in o-5,6-dich lor o-1-(â-D-er yth r ofu r an osyl)-
ben zim id a zole (9). Compound 7a (0.66 g, 1.6 mmol) was
treated with isopropylamine (5 mL) and the resulting solution
stirred at room temperature for 7 days. The solution was
concentrated under reduced pressure and purified by flash
column chromatography (SiO2, EtOAc/hexane 2:1). Fractions
containing the product were pooled, solvent was removed in
vacuo and the resulting solid was recrystallized from aqueous
ethanol to give 450 mg (80%) of 9 as a white solid: mp 185-
4-Br om o-5,6-d ic h lor o-2-isop r op yla m in ob e n zim id -
a zole (15). Compound 14 (25.0 g, 97.7 mmol) and isopropyl
isothiocyanate (11.2 g, 110.5 mmol) were added to anhydrous
pyridine (0.5 L). The resulting mixture was heated to 80 °C
for 30 min, then 1-cyclohexyl-3-(2-morpholinoethyl)carbodi-
imide metho-p-toluenesulfonate (56.8 g, 134.1 mmol) was
added as a solid in one portion. The resulting mixture was
allowed to stir at 80-90 °C for 1 h. The reaction was
subsequently allowed to cool to ambient temperature. Solids
were removed from the mixture by filtration and the filtrate
was concentrated under reduced pressure to afford a solid. This
solid was dissolved in EtOAc (200 mL) and the organic phase
washed with water (100 mL) and saturated NaCl(aq) (100 mL),
dried over magnesium sulfate, filtered and concentrated to a
solid. This solid was purified using flash column chromatog-
raphy (SiO2, EtOAc/hexane 7:3). The appropriate fractions
were combined and evaporated to give 19.2 g (62%) of 15 as a
1
187 °C dec; Rf 0.33 (EtOAc/hexane 1:1); H NMR (360 MHz,
DMSO-d6) δ 7.40 (s, 1H), 7.30 (s, 1H), 6.60 (d, 1H, D2O
exchangeable), 5.76 (d, 1H, 1′-H, J ) 7.8 Hz), 5.33 (d, 1H, D2O
exchangeable), 5.23 (d, 1H, D2O exchangeable), 4.42 (m, 2H),
4.20 (m, 1H), 4.01 (m, 1H), 3.82 (d, 1H), 1.21 (s, 3H), 1.19 (s,
3H); 13C NMR (90 MHz, DMSO-d6) δ 155.78, 143.66, 131.88,
123.10, 119.95, 115.92, 110.96, 86.49, 73.64, 72.36, 69.72,
44.58, 22.50, 22.40. Anal. Calcd for C14H17Cl2N3O3: C, H, N.
2-Cyclop r op yla m in o-5,6-d ich lor o-1-(â-D-er yt h r ofu r -
a n osyl)ben zim id a zole (10). Compound 7a (0.7 g, 1.7 mmol)
was added to cyclopropylamine (5 mL) and treated as described
for 9 above to give 480 mg (87%) of 10 as a white crystalline
1
solid: mp 223-225 °C; Rf 0.23 (EtOAc/hexane 2:1); H NMR
(360 MHz, DMSO-d6) δ 7.47 (s, 1H), 7.33 (s, 1H), 7.08 (d, 1H,
D2O exchangeable), 5.69 (d, 1H, 1′-H, J ) 7.8 Hz), 5.29 (d, 1H,
D2O exchangeable), 5.20 (d, 1H, D2O exchangeable), 4.42 (m,
2H), 4.19 (m, 1H), 3.81 (d, 1H), 2.75 (m, 1H), 0.70 (m, 2H),
0.51 (m, 2H); 13C NMR (90 MHz, DMSO-d6) δ 156.84, 143.51,
132.00, 123.19, 120.30, 116.33, 111.07, 86.60, 73.62, 72.33,
69.68, 25.22, 6.55, 6.20. Anal. Calcd for C14H15Cl2N3O3: C, H,
N.
1
tan solid: mp 247-249 °C; H NMR (300 MHz, DMSO-d6) δ
11.1 (broad s, 1H), 7.28 (s, 1H), 7.17 (s, 1H), 3.87 (m, 1H), 1.17
(d, 6H). Anal. Calcd for C10H10BrCl2N3‚0.08C6H14: C, H, N.
4-Br om o-5,6-d ich lor o-2-isop r op yla m in o-1-(â-D-er yth r o-
fu r a n osyl)ben zim id a zole (17). A mixture of 4-bromo-5,6-
dichloro-2-isopropylaminobenzimidazole (15; 1.19 g, 3.69 mmol),
ClCH2CH2Cl (30 mL) and BSA (0.96 mL, 3.87 mmol) was
heated to 80 °C for 30 min. TMSOTf (0.46 mL, 2.39 mmol)
was added and the mixture stirred for 15 min, then 1,2,3-tri-
O-acetyl-D-erythrofuranose (D-6; 1.0 g, 4.1 mmol) was added
and the reaction stirred for 12 h at 80 °C. The reaction mixture
was allowed to cool to room temperature and was then diluted
with 5% NaHCO3 (35 mL) and CH2Cl2 (20 mL). This mixture
was stirred for 30 min, the phases were separated and the
aqueous phase was extracted with additional CH2Cl2 (2 × 20
mL). The combined organic phases were dried over magnesium
sulfate, filtered and concentrated in vacuo. The resulting
residue was purified using flash column chromatography (SiO2,
CH2Cl2/MeOH 60:1). The appropriate fractions were combined
and evaporated to give 0.76 g of 4-bromo-5,6-dichloro-2-
isopropylamino-1-(2,3-di-O-acetyl-â-D-erythrofuranosyl)benz-
imidazole, 16). Compound 16 was deprotected using Na2CO3
(0.24 g, 2.29 mmol) in aqueous ethanol in a similar fashion as
described above. This gave, after purification using flash
column chromatography (SiO2, EtOAc/hexane 7:3), 0.13 g
(25%) of 17 as a white solid: mp 200-205 °C dec; 1H NMR
(300 MHz, DMSO-d6) δ 7.41 (s, 1H), 6.89 (d, 1H), 5.83 (d, 1H),
5.39 (d, 1H), 5.32 (d, 1H), 4.51 (m, 2H), 4.23 (m, 2H), 3.88 (d,
5,6-Dich lor o-2-m er ca p toben zyl-1-(â-D-er yth r ofu r a n o-
syl)ben zim id a zole (11). Compound 7a (0.57 g, 1.4 mmol) was
dissolved in THF (5 mL) and to this solution were added
mercaptobenzyl alcohol (2 mL) and sodium hydride (80%
sodium hydride, 220 mg, 5.6 mmol). The resulting solution was
stirred at ambient temperature for 8 h. Water was added to
the reaction mixture and extracted with EtOAc (3 × 50 mL).
The organic phase was extracted several times with 10%
NaOH(aq), then dried over magnesium sulfate and concen-
trated in vacuo to give a solid. This solid was purified by flash
chromatography (SiO2, EtOAc/hexane 1:2), fractions containing
product were combined and evaporated to a solid and the solid
was recrystallized from aqueous ethanol to give 500 mg (87%)
of 11 as a white solid: mp 136-138 °C dec; Rf 0.2 (EtOAc/
hexane 1:2); 1H NMR (360 MHz, DMSO-d6) δ 7.89 (s, 1H), 7.77
(s, 1H), 7.47 (m, 2H), 7.31 (m, 3H), 5.78 (d, 1H, 1′-H, J ) 7.7
Hz), 5.50 (d, 1H, D2O exchangeable), 5.33 (m, 1H, D2O
exchangeable), 4.61 (dd, 2H), 4.45 (m, 2H), 4.20 (m, 1H), 3.85
(d, 1H); 13C NMR (90 MHz, DMSO-d6) δ 154.35, 142.87, 136.82,
134.00, 129.10, 128.55, 127.54, 124.73, 124.34, 118.94, 112.43,
88.76, 74.09, 73.47, 70.08, 35.76. Anal. Calcd for C18H16
-
Cl2N2O3S‚C2H5OH: C, H, N.
1H), 1.28 (d, 6H). Anal. Calcd for
0.35C6H14: C, H, N.
C14H16BrCl2N3O3‚
2-Br om o-3,4-d ich lor o-6-n itr oa n ilin e (13). 4,5-Dichloro-
2-nitroaniline (12; 41.0 g, 198 mmol) was dissolved in DMF
(200 mL) and to this solution was added N-bromosuccinimide
(42.9 g, 241 mmol). The resulting solution was stirred at 100
°C for 1 h. The mixture was allowed to cool to room temper-
ature and then poured into a flask containing ice-water (1
L). A yellow solid formed and was collected by filtration. This
solid was dissolved in CH2Cl2 (2 L) and the organic phase
washed with water (3 × 500 mL). The organic phase was dried
over magnesium sulfate, filtered and concentrated in vacuo
to give 48.6 g (86%) of 13 as a solid: 1H NMR (300 MHz,
1,2,3-Tr i-O-a cetyl-L-er yth r ofu r a n ose (L-6). L-Erythro-
furanose29 (5.0 g, 41.6 mmol), DMAP (0.2 g, 1.67 mmol) and
acetic anhydride (23.61 mL, 249.5 mmol) were added to
pyridine (150 mL) and the reaction stirred for 3 h at room
temperature. The reaction mixture was then poured into a
flask containing saturated NaHCO3 (100 mL). The product was
extracted with CH2Cl2 (500 mL) and concentrated. The re-
sidual syrup was dissolved in EtOAc (2 × 250 mL) and washed
with 0.1 N HCl (200 mL), and the organic phase was dried
over MgSO4, filtered and concentrated. The product was

2470 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Gudmundsson et al.
purified using flash column chromatography (SiO2, hexane/
EtOAc 3:1). Appropriate fractions were obtained to give 4.35
g (42%) of L-6 as a yellow syrup: Spectral data was identical
with that for the referenced D-enantiomer. Anal. Calcd for
C10H14O7: C, H.
5,6-Dich lor o-2-isop r op yla m in oben zim id a zole30 (19a ).
4,5-Dichloro-1,2-phenylenediamine (18; 80.02 g, 452 mmol),
isopropyl isothiocyanate (48.86 g, 483 mmol), 1-cyclohexyl-3-
(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (249
g, 587 mmol) and pyridine (1.15 L) were used as previously
described for the synthesis of 15. The product was recrystal-
lized from dioxane to afford 79 g (72%) of 19a as a tan solid:
mp 218-220 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.7 (broad
s, 1H), 7.25 (s, 2H), 6.80 (d, 1H), 3.87 (m, 1H), 1.16 (d, 6H).
Anal. Calcd for C10H11Cl2N3: C, H, N.
solid Na2CO3. The aqueous phase was extracted with EtOAc
(3 × 700 mL) and the organic phase dried (MgSO4), filtered
and concentrated under reduced pressure. Purification using
flash column chromatography (SiO2, EtOAc/hexane 35:65) gave
10.9 g (72%) of 22 as a tan solid: mp 113-115 °C dec; 1H NMR
(300 MHz, DMSO-d6) δ 6.66 (s, 1H), 5.24 (broad s, 2H), 5.10
(broad s, 2H). Anal. Calcd for C6H5Cl3N2: C, H, N.
2-Isop r op yla m in o-4,5,6-tr ich lor oben zim id a zole (23).
3,4,5-Trichloro-1,2-phenylenediamine (22; 3.12 g, 14.8 mmol),
isopropyl isothiocyanate (1.62 g, 16.0 mmol) and 1-cyclohexyl-
3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate
(8.4 g, 19.9 mmol) in anhydrous pyridine (50 mL) were used
as described for 19a ,b to give, after recrystallization from
dioxane, 2.9 g (72%) of 23 as a tan solid: 1H NMR (300 MHz,
DMSO-d6) δ 11.2 (s, 1H), 7.34 (s, 1H), 7.20 (d, 1H), 3.93 (m,
1H), 1.25 (d, 6H). Anal. Calcd for C10H10Cl3N3‚0.10C4H8O2: C,
H, N.
2-Isop r op yla m in o-4,5,6-tr ich lor o-1-(â-L-er yth r ofu r a n o-
syl)b en zim id a zole (25). 2-Isopropylamino-4,5,6-trichloro-
benzimidazole (23; 0.86 g, 3.10 mmol), BSA (0.80 mL, 3.26
mmol), TMSOTf (0.39 mL, 2.01 mmol), 1,2,3-tri-O-acetyl-L-
erythrofuranose (L-6; 0.84 g, 3.41 mmol) and ClCH2CH2Cl (25
mL) were used as described above for 21a ,b to give, after flash
column chromatography (SiO2, CH2Cl2/MeOH 60:1), 0.28 g
(20%) of 24 as a white foam. This foam (0.05 g, 0.12 mmol)
was deprotected using Na2CO3 (0.02 g, 0.17 mmol) in an
aqueous mixture of methanol and ethanol in a similar fashion
as described above for 21a ,b. This gave, after flash column
chromatography (SiO2, CH2Cl2/MeOH 98:2), 0.04 g (91%) of
25 as a white solid: mp 199-200 °C dec; 1H NMR (300 MHz,
DMSO-d6) δ 7.30 (s, 1H), 6.80 (d, 1H), 5.76 (d, 1H), 5.31 (d,
1H), 5.23 (d, 1H), 4.44 (m, 2H), 4.19 (m, 1H), 4.09 (m, 1H),
3.80 (d, 1H), 1.2 (d, 6H). Anal. Calcd for C14H16Cl3N3O3: C, H,
N.
In Vitr o An tivir a l Eva lu a tion . Cell lines were subcul-
tured according to conventional procedures previously de-
scribed.31 Stock preparations of HCMV and HSV-1 were
prepared as described elsewhere.26,31 The effect of compounds
on the replication of HCMV was measured using plaque
reduction assays,31 a yield reduction assay using a multiplicity
of infection ) 0.5 PFU/mL,32 and a DNA hybridization assay.33
Effects on HSV-1 replication were measured in an ELISA
assay.34 Three different assays were used to explore cytotox-
icity of selected compounds using methods we have detailed
previously: (i) cytotoxicity produced in stationary HFF cells
was determined by microscopic inspection of cells not affected
by the virus used in the plaque assays;17 (ii) effect of com-
pounds on proliferation of KB cells during two population
doublings of the controls was determined by crystal violet
staining and spectrophotometric quantification of dye eluted
from stained cells as described earlier;35 (iii) cytotoxicity in
MOLT 4 cells (human T-cell lymphocytic leukemia cell line)
was performed as described elsewhere.36
5,6-Dich lor o-2-isopr opylam in o-1-(â-L-er yth r ofu r an osyl)-
b en zim id a zole (21a ). 5,6-Dichloro-2-isopropylaminobenz-
imidazole (19a ; 1.0 g, 4.09 mmol), BSA (1.06 mL, 4.29 mmol),
TMSOTf (0.51 mL, 2.66 mmol), 1,2,3-tri-O-acetyl-L-erythro-
furanose (L-6; 1.10 g, 4.49 mmol) and ClCH2CH2Cl (25 mL)
were used as described above for 17 to give, after flash column
chromatography (SiO2, CH2Cl2/MeOH 30:1), 0.45 g (26%) of
20a as a brown solid. This solid (0.34 g, 0.79 mmol) was
deprotected using Na2CO3 (0.13 g, 1.23 mmol) in an aqueous
mixture of ethanol and methanol in a similar fashion as
described above for 17. This gave 0.17 g (63%) of 21a as a white
1
solid: mp 180-181 °C; H NMR (300 MHz, DMSO-d6) δ 7.38
(s, 1H), 7.28 (s, 1H), 6.58 (d, 1H), 5.75 (d, 1H), 5.30 (d, 1H),
5.21 (d, 1H), 4.41 (m, 2H), 4.19 (m, 1H), 4.02 (m, 1H), 3.80 (d,
1H), 1.19 (d, 6H). Anal. Calcd for C14H17Cl2N3O3: C, H, N.
2-Cyclop r op yla m in o-5,6-d ich lor oben zim id a zole (19b).
4,5-Dichloro-1,2-phenylenediamine (18; 6.04 g, 34.1 mmol),
cyclopropyl isothiocyanate (3.69 g, 37.2 mmol), 1-cyclohexyl-
3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate
(20.1 g, 47.4 mmol) and pyridine (135 mL) were used as
previously described for 19a . The product was recrystallized
from acetonitrile to afford 5.82 g (70%) of 19b as a yellow
1
solid: mp 223-225 °C; H NMR (300 MHz, DMSO-d6) δ 11.2
(s, 1H), 7.43 (s, 1H), 7.27 (s, 2H), 2.49 (m, 1H), 0.70 (m, 2H),
0.49 (m, 2H). Anal. Calcd for C10H9Cl2N3: C, H, N.
2-Cyclop r op yla m in o-5,6-d ich lor o-1-(â-L-er yt h r ofu r -
a n osyl)b en zim id a zole (21b ). 2-Cyclopropylamino-5,6-di-
chlorobenzimidazole (19b; 1.0 g, 4.13 mmol), BSA (1.00 mL,
4.30 mmol), TMSOTf (0.52 mL, 2.68 mmol), 1,2,3-tri-O-acetyl-
L-erythrofuranose (L-6; 1.12 g, 4.54 mmol) and ClCH2CH2Cl
(25 mL) were used as described above for 21a to give, after
flash column chromatography (SiO2, CH2Cl2/MeOH 98:2), 1.81
g of 20b as a solid. Compound 20b (1.81 g) was deprotected
using Na2CO3 (0.67 g, 6.34 mmol) in an aqueous mixture of
methanol and ethanol in a similar fashion as described above
for 21a . This gave, after flash column chromatography (SiO2,
CH2Cl2/MeOH 99:1), 0.18 g (13%) of 21b as a white solid: mp
201-202 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.54 (s, 1H), 7.39
(s, 1H), 7.15 (s, 1H), 5.74 (d, 1H), 5.36 (d, 1H), 5.26 (d, 1H),
4.47 (m, 2H), 4.24 (br s, 1H), 3.86 (d, 1H), 2.81 (br s, 1H), 0.74
(m, 2H), 0.55 (m, 2H). Anal. Calcd for C14H15Cl2N3O3: C, H,
N.
3,4,5-Tr ich lor o-1,2-p h en ylen ed ia m in e (22). 4,5-Dichloro-
2-nitroaniline (12; 15.2 g, 73.2 mmol) and N-chlorosuccinimide
(12.2 g, 91.6 mmol) were dissolved in DMF (150 mL). The
resulting orange solution was heated to 100 °C for 1 h. After
cooling to room temperature this solution was poured into ice
water (1.2 L). A yellow solid formed which was collected by
filtration and dissolved in CH2Cl2, and the organic phase was
washed with water, dried (MgSO4), filtered and concentrated
to give 17.6 g (98%) of 2,3,4-trichloro-6-nitroaniline as a yellow
solid. The solid (17.6 g, 72.8 mmol) was dissolved in EtOH (400
mL), iron powder (14.0 g, 250 mmol) was added and the
resulting suspension was cooled to 0 °C. Concentrated HCl (93
mL, 1.14 mol) was added dropwise over a period of 15 min.
When the addition was complete, the suspension was heated
at reflux for 3.5 h, then allowed to cool to room temperature.
The resulting suspension was diluted with water (2 L) and
the pH of the mixture adjusted to 8 by the slow addition of
Dose-response relationships for most assays were con-
structed by linearly regressing the percent inhibition of
parameters derived in the preceding sections against log drug
concentrations. For yield reduction experiments, the log of
virus titer was regressed against log drug concentration. Fifty-
percent inhibitory concentrations (IC50’s) or IC90’s (yield ex-
periments) were calculated from the regression lines. Samples
containing positive controls (acyclovir for HSV-1, ganciclovir
for HCMV) were used in all assays.
Tim e of Dr u g Ad d ition P r otocol. Ninety-six-well culture
plates were seeded at 12 500 HFF cells/well in MEM(E) with
10% fetal bovine serum (FBS) and penicillin plus streptomycin
(pen/strep) the evening before infection. On the day of infection,
medium was removed from the plates and replaced with 180
µL of virus inoculum. The inoculum was prepared by diluting
previously titered stock virus (Towne strain) in MEM(E) with
5% FBS + pen/strep to obtain a multiplicity of infection of 0.5
PFU/cell. At each time point, 20 µL of 10× drug prepared in
MEM(E), 5% FBS + pen/strep was added to a subset of wells
on the plates. Each drug was tested in duplicate at each time
point. The drugs were prepared prior to infection and were

â-D- and -L-Erythrofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2471
Edwards, C. A.; Nassiri, M. R.; Townsend, L. B.; Drach, J . C.
Benzimidazole Ribonucleosides: Mode of Action of TCRB in
HCMV-Infected Cells. Fifth International Conference on An-
tiviral Research, Vancouver, British Columbia, Canada, March
1992.
stored at -80 °C between time points. In addition, duplicate
virus control cultures received medium without drug at each
time point. At 96 h post-infection, all plates were frozen at
-80 °C. Subsequently the plates were thawed and virus titer
in each well was determined by end-point dilution and plaque
enumeration as detailed previously.32
(14) Underwood, M. R.; Harvey, R. J .; Stanat, S. C.; Hemphilll, M.
L.; Miller, T.; Drach, J . C.; Townsend, L. B.; Biron, K. K.
Inhibition of Human Cytomegalovirus DNA maturation by a
benzimidazole ribonucleoside is mediated through the UL89 gene
product. J . Virol. 1998, 72, 717-725.
(15) (a) Koszalka, G. W.; Chamberlain, S. D.; Harvey, R. J .; Frick,
L. W.; Good, S. S.; Davis, M. L.; Smith, A.; Drach, J . C.;
Townsend, L. B.; Biron, K. K. Benzimidazoles for the Treatment
of Human Cytomegalovirus. Antiviral Res. 1996, 30, A43. (b)
Chamberlain, S. D.; Chan, J . H.; Tidwell, J . H.; Peckham, G.
E.; Harvery, R. J .; Dornsife R. E.; Frick, L. W.; Townsend, L.
B.; Drach, J . C.; Koszalka, G. W. Synthesis and Evaluation of a
Series of Carbohydrate Modified Derivatives of the Anti-HCMV
Agent, 5,6-Dichloro-2-isopropylamino-1-(â-L-ribofuranosyl)benz-
imidazole (1263W94). 214th ACS National Meeting, San Fran-
sisco, CA, April 1997.
(16) Drew, W. L.; Lalezari, J . P.; Wang, L. H.; Miner, R. C.; Aberg,
J . A.; Wire, M. B.; J acobson, M. A. In Vivo Anti-CMV Activity
and Safety of Oral 1263W94 in HIV-Infected Subjects with
Asymptomatic CMV Shedding. Interscience Conference on An-
timicrobial Agents and Chemotherapy, San Diego, CA, 1998.
(17) (a) Saluja, S.; Zou, R.; Drach, J . C.; Townsend, L. B. Structure-
Activity Relationships Among 2-Substituted 5,6-Dichloro-, 4,6-
Dichloro-, and 4,5-Dichloro-1-[(2-hydroxyethoxy)methyl)- and
1-[(1,3-dihydroxy-2-propoxy)methyl]-benzimidazoles. J . Med.
Chem. 1996, 39, 881-891. (b) Migawa, M. T.; Girardet, J .-L.;
Walker, J . A. II; Koszalka, G. W.; Chamberlain, S. D.; Drach, J .
C.; Townsend, L. B. Design, Synthesis and Antiviral Activity of
R-Nucleosides: D- and L-Isomers of Lyxofuranosyl- and (5-
Deoxylyxofuranosyl)benzimidazoles. J . Med. Chem. 1998, 41,
1242-1251. (c) Girardet, J .-L.; Drach, J . C.; Chamberlain, S. D.;
Koszalka, G. W.; Townsend, L. B. Design, Synthesis and
Antiviral Activity of R-L-Arabinofuranosyl Derivatives of 2-Sub-
stituted-5,6-dichlorobenzimidazoles. Nucleosides Nucleotides 1998,
17, 2389-2401.
Ack n ow led gm en t. We thank J ulie M. Breitenbach,
Mark Underwood, R. E. Dornsife, Dean Selleseth, and
Robert J . Harvey for expert performance of antiviral and
cytotoxicity assays. We also thank Ms. Kimberly J .
Barrett for her assistance with the preparation of this
manuscript. These studies were supported by Research
Grant UOI-AI31718 from the National Institute of
Allergy and Infectious Diseases and Research Agree-
ment DRDA-942921 with Glaxo Wellcome. K.S.G. was
supported in part by a NATO Fellowship from Visin-
darad, Iceland.
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J M990195P