Design, synthesis, and antiviral evaluation of 2-substituted 4,5- dichloro- and 4,6-dichloro-1-β-D-ribofuranosylbenzimidazoles as potential agents for human cytomegalovirus infections

Article abstract of DOI:10.1021/jm960533b

The syntheses of 2,4,6-trichlorobenzimidazole (4a) and 2-bromo-4,6- dichlorobenzimidazole (4b) were accomplished via the 2-amino intermediate (3) using a mild diazotization procedure. Ribosylation of 4a and 4b and subsequent deprotection afforded the corr

Full text of DOI:10.1021/jm960533b

8
02
J . Med. Chem. 1997, 40, 802-810
Design , Syn th esis, a n d An tivir a l Eva lu a tion of 2-Su bstitu ted 4,5-Dich lor o- a n d
4
,6-Dich lor o-1-â-D-r ibofu r a n osylben zim id a zoles a s P oten tia l Agen ts for Hu m a n
Cytom ega lovir u s In fection s1
†
Ruiming Zou, J ohn C. Drach, and Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature, Sciences and Arts,
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109-1065
X
Received J uly 24, 1996
The syntheses of 2,4,6-trichlorobenzimidazole (4a ) and 2-bromo-4,6-dichlorobenzimidazole (4b)
were accomplished via the 2-amino intermediate (3) using a mild diazotization procedure.
Ribosylation of 4a and 4b and subsequent deprotection afforded the corresponding 2,4,6-
trichloro-1-â-D-ribofuranosylbenzimidazole (7a ) and 2-bromo-4,6-dichloro-1-â-D-ribofuranosyl-
benzimidazole (7b ). The 2-azido (10), 2-amino (11), 2-thione (13), 2-methylthio (14a ), and
2
-benzylthio (14b) derivatives were prepared via displacement reactions at the 2-position of
the 2,3,5-tri-O-acetyl derivative of 7a . 2,4,5-Trichlorobenzimidazole (17a ) and 2-bromo-4,5-
dichlorobenzimidazole (17b) were synthesized from the corresponding 1,2-phenylenediamines
via successive cyclization with cyanogen bromide and diazotization in the presence of an
appropriate cupric halide. Ribosylation of compounds 17a and 17b was followed by deprotection
to afford 2,4,5-trichloro-1-â-D-ribofuranosylbenzimidazole (20a ), and 2-bromo-4,5-dichloro-1-
â-D-ribofuranosylbenzimidazole (20b). Heterocycles (3, 4a , 17a ) and nucleosides (7a ,b, 8, 10,
1
1, 13, 14a ,b, 20a ,b) were evaluated for activity against human cytomegalovirus (HCMV) and
herpes simplex virus type 1 (HSV-1) and for cytotoxicity. The 2-chloro but not the 2-amino
heterocycles were active against HCMV (IC50’s ) 5-8 µM) but not HSV-1; both also were
somewhat cytotoxic to uninfected cells (IC50’s ) 32-100 µM). Among the nucleosides, the
2
-chloro and 2-bromo analogs in both the 4,5- and 4,6-dichloro series (20a ,b, 7a ,b, respectively)
were active against HCMV (IC50’s ) 1-10 µM) and noncytotoxic in their antiviral dose ranges.
The 2-bromo compounds were more active than the 2-chloro analogs; the 2-azido and
2
-thiobenzyl analogs (10, 14b) were weakly active against HCMV, but this activity was not
well separated from cytotoxicity. None of the nucleosides were active against HSV-1. This
pattern of activity and cytotoxicity is similar to that of the 2-chloro- and 2-bromo-5,6-dichloro
analogs (TCRB, BDCRB) which we reported previously. Although these new 4,5- and 4,6-
dichloro analogs are potent and selective inhibitors of HCMV, they are not as potent at TCRB
and BDCRB.
In tr od u ction
number of benzimidazole nucleosides previously pre-
1
6,17
pared in our laboratory.
benzimidazole nucleoside analogs, e.g., 2,5,6-trichloro-
-â-D-ribofuranosylbenzimidazole (TCRB) and 2-bromo-
We found that certain
Human cytomegalovirus (HCMV) is the most common
sight- and life-threatening opportunistic viral infection
in immunocompromised individuals such as AIDS pa-
1
tients.2,3 HCMV infection is also the primary cause of
death in recipients of allogeneic bone marrow and renal
5,6-dichloro1-â-D-ribofuranosylbenzimidazole (BDCRB),
were highly active and selective against HCMV in vitro
1
7,18
4
-6
and were essentially noncytotoxic.
Both compounds
transplantation.
is difficult because few therapeutic options are available.
Many well-known antiviral drugs, such as vidarabine,
The treatment of HCMV infection
act by a new mechanism involving inhibition of DNA
1
9
processing. As part of our comprehensive structure-
activity relationship study of those benzimidazole ribo-
nucleosides, we now have synthesized isomers of TCRB,
interferons, and acyclovir, have been tested and were
not efficacious against HCMV.7-10 Only ganciclovir
2
,4,5- and 2,4,6-trichloro-1-â-D-ribofuranosylbenzimid-
(DHPG), foscarnet (PFA), and cidofovir (HPMPC) have
azole, the corresponding isomers of BDCRB, and certain
2-substituted derivatives. The present work describes
the synthesis and antiviral activity of these TCRB and
BDCRB analogs.
been approved by the FDA for the treatment of HCMV
1
1-14
diseases.
Although the treatment of HCMV infec-
tion with these drugs has produced clinical improve-
ment in a large proportion of patients, the drugs suffer
from poor oral bioavailability, low potency, and certain
adverse effects.1
2-14
In addition, drug-resistant HCMV
has emerged in some patients.15 Therefore, more active
and less toxic HCMV agents which act by new mecha-
nisms to circumvent resistance are still needed.
In our search for a more potent and nontoxic agent
for the treatment of HCMV infection, we evaluated a
*
Author to whom correspondence should be addressed.
Present address: Gilead Sciences, 344 Lakeside Dr, Foster City,
Ch em istr y
†
2
-Chlorobenzimidazole derivatives have been fre-
CA 94404.
X
Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
quently synthesized via the chlorination of benzimid-
S0022-2623(96)00533-X CCC: $14.00 © 1997 American Chemical Society

Substituted 1-â-D-ribofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 803
Sch em e 1
tection of the â nucleosides 5a and 5b gave 2,4,6-
trichloro-1-â-D-ribofuranosylbenzimidazole (an isomer of
TCRB) and 2-bromo-4,6-dichloro-1-â-D-ribofuranosyl-
benzimidazole (an isomer of BDCRB), respectively.
Similar deprotection of the R anomers 6a and 6b
resulted in a removal of the acetyl protecting groups and
a concomitant cyclization to give a 2,2′-O-cyclonucleoside
(8).
The major products (5a ,b) of the glycosylation reac-
tions were initially assigned as the â anomers since the
preferential formation of the â anomer was expected
with a protected sugar bearing a participating group at
2
5
the 2-position. This was also in agreement with the
assignment of an R configuration to the minor products
6
a ,b whose R configuration was established by a facile
formation of a 2,2′-O-cyclonucleoside (8) upon deprotec-
tion. Additional support for the anomeric assignments
were provided by comparing the 1′-H chemical shifts of
2
6
the anomeric pairs.
The 1′-H signals of the major
products 5a (δ 6.26) and 5b (δ 6.23) were found at
higher (∼0.46 ppm) field than those of the corresponding
anomers 6a (δ 6.72) and 6b (δ 6.69), and therefore it
was consistent with the assignment of 5a ,b as â ano-
mers. The unequivocal assignments were based on a
nuclear Overhauser enhancement (NOE) experiment in
which the 1′-H of 7a was irradiated and the NOE at
the 4′-H and 3′-H was measured. The observation of
an NOE of the 4′-H signal and no NOE at the 3′-H
2
7,28
proved its â configuration.
The assignment of the
sugar attachment to the heterocycle as 1-ribosyl-2,4,6-
trichloro (vs 1-ribosyl-2,5,7-trichloro) was based on an
NOE experiment in which a 5.5% of enhancement of the
azol-2-ones with phosphoryl chloride.20,21 However, the
application of this method to certain halogenated ben-
2
′-H signal of 7a was observed when 7-H was irradiated.
Successive treatment of 5a with lithium azide at an
zimidazol-2-ones has provided more capricious re-
elevated temperature and deprotection with methanolic
ammonia gave 2-azido-4,6-dichloro-1-â-D-ribofuranosyl-
benzimidazole (10) in good yield. Subsequent hydroge-
nation of 10 over Raney nickel furnished 2-amino-4,6-
dichloro-1-â-D-ribofuranosylbenzimidazole (11). Treat-
ment of 5a with thiourea followed by deprotection
afforded the 2-thione derivative (13). Alkylation of 13
with methyl iodide and benzyl bromide gave excellent
yields of the corresponding 2-methylthio (14a ) and the
sults.2
2,23
Dimerization and other side reactions have
resulted in diminished yields and difficulties in product
purification. This prompted us to initiate an investiga-
tion designed to provide a generally applicable proce-
dure for the synthesis of not only the 2-chlorobenzimid-
azoles but also the 2-bromobenzimidazoles.
The route we envisaged for the 2-substituted 4,6-
dichloro series of compounds used 2,4-dichloro-6-nitro-
aniline (1) as our starting material. Hydrogenation of
2
-benzylthio (14b) analogs.
1
over Raney nickel has been reported24 to give 3,5-
The route we envisaged for the 2-substituted 4,5-
dichloro-1,2-phenylenediamine (2) in 58% yield (Scheme
). Subsequent cyclization of 2 with cyanogen bromide
dichloro series of compounds used 3,4-dichloro-1,2-
phenylenediamine (15) as our starting material. Com-
pound 15 was cyclized with cyanogen bromide to give
1
2
2
has also been reported to afford 2-amino-4,6-dichlo-
robenzimidazole (3), but no yield was given. With
modifications of the literature procedures, we were able
to prepare 3 from 1 in 95% overall yield. Diazotization
of 3 with sodium nitrite in aqueous cupric chloride
solution afforded the desired 2,4,6-trichlorobenzimid-
azole (4a ). This mild diazotization in aqueous cupric
chloride medium alleviated the problem of forming a
hydrolysis side product under the typical conditions of
an aqueous diazotization reaction in which concentrated
HCl solution was employed as the reaction medium.
Similar diazotization of 3 in aqueous cupric bromide
solution gave the corresponding 2-bromo-4,6-dichlo-
robenzimidazole (4b) in a good yield. Glycosylation of
2
-amino-4,5-dichlorobenzimidazole (16) in an excellent
overall yield (Scheme 3). Diazotization of 16 in the
presence of cupric chloride afforded the desired 2,4,5-
trichlorobenzimidazole (17a ). Parallel diazotization of
1
6 in the presence of cupric bromide gave the corre-
sponding 2-bromo-4,5-dichlorobenzimidazole (17b). Si-
lylation of 17a and 17b with BSA and subsequent
coupling with 1,2,3,5-tetra-O-acetyl-â-D-ribofuranose
yielded the â nucleosides 19a and 19b as the major
products along with a small amount of the R anomers
(18a and 18b). Deprotection of the â nucleosides 19a
and 19b with sodium carbonate in an aqueous alcoholic
medium gave 2,4,5-trichloro-1-â-D-ribofuranosylbenzi-
midazole (20a , an isomer of TCRB) and 2-bromo-4,5-
dichloro-1-â-D-ribofuranosylbenzimidazole (20b, an iso-
mer of BDCRB), respectively.
4
a and 4b with 1,2,3,5-tetra-O-acetyl-â-D-ribofuranose
yielded what were subsequently established as the â
nucleosides 5a and 5b as the major products along with
a small amount of the R anomers (6a and 6b). Depro-

8
04 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Zou et al.
Sch em e 2
against HCMV and was not cytotoxic at concentrations
up to 100 µM (data not shown).
Among the ribonucleosides, activity against HCMV
was observed in both the 4,5- and 4,6-dichloro series but
only with the 2-chloro and 2-bromo analogs 7a ,b and
2
0a ,b (Table 2). None of the compounds were active
against HSV-1. Antiviral activity of the 2-thiomethyl
and 2-thiobenzyl analogs 14a ,b was observed only at
higher concentrations; consequently it was probably a
consequence of cytotoxicity. These results are very
similar to those obtained by us in a previous study29
wherein the 2-thiobenzyl analog in the 5,6-dichloro
series of benzimidazole nucleosides was somewhat ac-
tive against HCMV but the activity was not well
separated from cytotoxicity. In contrast, the activity of
the four 2-halogen-substituted ribonucleosides (7a ,b and
2
0a ,b) was well separated from cytotoxicity. Activity
against HCMV in both plaque and yield reduction
assays was similar to that of the 2,5,6-trichloro and
2
-bromo-5,6-dichloro analogs TCRB and BDCRB, which
we reported previously (see ref 17a and Table 2).
Similar to the relative potencies of TCRB and BDCRB,
the 2-bromo analogs 7b and 20b were the more active
compounds in both HCMV assays. Also like TCRB and
BDCRB, both compounds in both the 4,5- and 4,6-
dichloro series effected multi-log10 reductions in HCMV
titers at noncytotoxic concentrations. Specifically all
four compounds reduced HCMV titers 10000-fold at 10-
1
00 µM. These concentrations, however, are higher
than those required for TCRB and BDCRB to produce
a similar effect: due to dose-response curves with
4
5
greater slopes, TCRB gave 10 -10 -fold titer reductions
at 32 µM compared to 3-10 µM for BDCRB and 100
The major products (19a ,b) of the glycosylation reac-
tions were assigned as the â anomers since the prefer-
ential formation of the â anomer was expected with a
protected sugar bearing a participating group at the
17a
µM for ganciclovir.
Taken together, the data in Tables 1 and 2 confirm
17a
our prior observation
that the addition of ribose to a
polyhalogenated benzimidazole reduces cytotoxicity and
retains or increases activity against HCMV. Further-
more, our results show that chlorine in the 4-position
is nearly as effective as chlorine in the 5-position for
producing activity against HCMV. We conclude, there-
fore, that the activity of the new 2-halo-4,6-dichloro and
2
5
2
-position (Baker’s trans rule). Additional support for
the anomeric assignments was provided by comparing
2
6
the 1′-H chemical shifts of the anomeric pairs.
The
1
′-H signals of the major products were found at a
higher (∼0.45 ppm) field than those of the corresponding
anomers and therefore the major products are the â
anomers. In the case of compounds 19a ,b and 20a ,b,
the sugar attachment to the heterocycle was assigned
as 1-ribosyl-2,4,5-trichloro (vs 1-ribosyl-2,6,7-trichloro)
because the steric hindrance of the 2-chloro and the
2
-halo-4,5-dichloro analogs 7a ,b and 20a ,b against
HCMV is greater than that of ganciclovir but is slightly
less than that of the 2-halo-5,6-dichloro analogs TCRB
and BDCRB.
4
-chloro would be expected to prevent the attack of the
Exp er im en ta l Section
sugar on the N-3. This assignment was also in agree-
ment with our observation on the glycosylation products
of other 2,4-disubstituted benzimidazoles, whose at-
tachment was unequivocally established by the differ-
ence NOE experiments.2
Gen er a l Meth od . Melting points (MP) were taken on a
Thomas-Hoover Unimelt apparatus and were uncorrected.
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker WM-360 spectrometer operating in FT mode. The
chemical shift values were reported in parts per million (ppm)
relative to tetramethylsilane as an internal standard. Mass
(MS) spectra were determined by the Mass Spectrometry
Laboratory of the Chemistry Department, University of Michi-
gan. High-resolution MS measurements were obtained on a
VG 70-250-S MS spectrometer using a direct probe for sample
introduction. Elemental analyses were performed by M-H-W
Laboratories, Phoenix, AZ. Chemical reactions and column
chromatographic separations were followed by thin layer
chromatography (TLC) on silica gel precoated glass plates
7,28
An tivir a l Stu d ies
The benzimidazole heterocycles, cyclonucleoside, and
ribosides were evaluated for activity against HCMV and
HSV-1 and for cytotoxicity in two cell lines. The
2
-chloro heterocycles 4a and 17a were active against
HCMV at concentrations similar to that for ganciclovir
Table 1). In contrast to ganciclovir, both compound
were inactive against HSV-1 and more cytotoxic (IC50
32 µM). Thus the activity against HCMV was less
(
(layer thickness 0.2 mm) purchased from Analtech, Inc. The
TLC plates were observed under UV light (254 nm). Evapora-
tions were effected using a Buchler flash-evaporator equipped
with a Dewar “dry ice” condenser under water aspirator or
mechanical oil pump vacuum at 40 °C or cooler unless
otherwise specified.
)
specific than that of ganciclovir. The 2-amino analog 3
was inactive against the viruses and was not cytotoxic
(Table 1). Similarly, the cyclonucleoside 8 was inactive

Substituted 1-â-D-ribofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 805
Sch em e 3
Ta ble 1. Antiviral Activity and Cytotoxicity of 2-Substituted
Ta ble 2. Antiviral Activity and Cytotoxicity of 2-Substituted
Dichlorobenzimidazoles
Dichlorobenzimidazole Ribonucleosides
a
Plaque and yield reduction assays were performed in duplicate
as described in the text. Results from plaque assays are reported
as IC50’s, those for yield reduction experiments as IC90’s. b A plaque
assay was used to determine the activity of DHPG against HSV-
1
; all other compounds were assayed by ELISA in quadruplicate
c
wells. 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.
Results are presented as IC50’s. d >100 indicates IC50 or IC90 not
a-d
See Table 1 for footnotes a-d. e Average derived from a
e
reached at the noted (highest) concentration tested. Average
single experiment. All other data are averages of two to four
experiments. f TCRB and BDCRB, the 2,5,6-trichloro and 2-bromo-
5,6-dichloro analogs respectively, published previously as com-
pounds 9 and 11 in ref 17a. g Averages derived from three to six
experiments for each assay listed.
f
derived from two experiments. Average ( standard deviation
g
from 15 experiments. Average ( standard deviation from 108,
3
3, and 3 experiments, respectively.
3
,5-Dich lor o-1,2-p h en ylen ed ia m in e (2) a n d 2-Am in o-
4
,6-d ich lor oben zim id a zole (3). A mixture of 1 (purchased
from MTM Research Chemicals, Lancaster Synthesis Inc., 8.28
g, 40 mmol) and 0.80 g (wet wt) of Raney nickel in 200 mL of
analytical sample was obtained by recrystallization from
2
2
MeCN: mp 224-226 °C (lit. mp 221-224 °C); HRMS: (EI)
+
1
EtOH was hydrogenated at 50 psi of H
2
at room temperature
m/ z 200.9866 (100, M ) 200.9861); H NMR (DMSO-d
11.42, 11.08 (2 br s*, 1, 1-NH), 7.10, 6.99 (d, s, 2, 5-H and 7-H,
5-7 ) 2.0 Hz), 6.62 (br s, 2, 2-NH ). Anal. (C Cl ) C, H,
6
) δ
for 7 h. The reaction mixture was filtered, and the solid was
washed with portions of MeOH. The filtrate and washings
were combined, evaporated, and coevaporated with MeOH to
give a brown foam (2). This product was directly used in the
subsequent reaction without further purification. An analyti-
cal sample was obtained by recrystallization of the brown foam
J
2
7
H
5
2 3
N
N. [*The two broad singlets (combined integration as one
proton) for 1-NH indicated the existence of two tautomers.]
2,4,6-Tr ich lor oben zim id a zole (4a ). To a solution of
2 2 2 2
CuCl /H O (67.23 g/250 mL), a solution of NaNO /H O (10.35
2
1
from CCl
4
: mp 60-62 °C (lit. mp 60-61 °C); HRMS (EI) m/ z
g, 150 mmol/50 mL) was added dropwise and this was followed
by a solution of compound 3/MeOH (10.10 g, 50 mmol/50 mL).
After the addition was complete, stirring was continued at
room temperature for 1 h. The reaction mixture was then
heated on a steam bath for 1 h. During this period of heating,
1
1
6
1
75.9918 (100, M+ ) 175.9908); H NMR (DMSO-d
.50 (2 d, 2, 4-H and 6-H, J 4-6 ) 2.5 Hz), 5.15, 4.77 (2 br s, 4,
-NH and 2-NH ).
This brown foam (2) was dissolved in 80 mL of MeOH, and
this solution was added dropwise to a stirred solution of 5 M
BrCN/MeCN (8.8 mL) in 80 mL of H O. After the addition
6
) δ 6.54,
2
2
2 2
an additional fresh NaNO /H O solution (31.05 g, 450 mmol/
2
150 mL) was added dropwise. The reaction mixture was cooled
to room temperature, diluted with 500 mL of 1 N HCl solution,
and extracted with EtOAc (500 mL). The EtOAc solution was
filtered and the filtrate was washed with a 1 N HCl solution
had been completed, stirring was continued at room temper-
ature for 4 h. The reaction mixture was concentrated to ∼80
mL and then was washed with EtOAc (100 mL). The EtOAc
phase was extracted with H
extracts were neutralized with a saturated NaHCO
2
O (100 mL). The combined H
2
O
(300 mL), a sat. NaCl solution (300 mL), dried (Na
evaporated. The residue was chromatographed on a silica
, v/v).
2 4
SO ), and
3
solution
to ∼pH 8, and the resulting suspension was extracted with
column (4 × 20 cm, eluted with 1%, 2% MeOH/CHCl
3
EtOAc (400 mL). The EtOAc phase was washed successively
Evaporation of fractions 15-75 (20 mL per fraction) and
recrystallization from MeCN gave 5.156 g (47%) of 4a as a
crystalline compound. MP: 231-232 °C. HRMS: (EI) m/ z
with H
dried (Na
of 3 as a black foam. This product showed a single spot on
TLC (R ) 0.3, MeOH/CHCl , v/v) and was used directly in
the subsequent reaction without further purification. An
2
O (200 mL) and a saturated NaCl solution (200 mL),
2
SO ), and evaporated to give 7.698 g (95% from 1)
4
+
1
219.9357 (100%, M ) 219.9362). H NMR (DMSO-d
13.92 (br s, 1, 1-NH), 7.58, 7.44 (s, d, 2, 5-H and 7-H). Anal.
(C Cl ) C, H, N.
6
): δ
f
3
7
H
3
3 2
N

8
06 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Zou et al.
2
-Br om o-4,6-d ich lor oben zim id a zole (4b). To a solution
of CuBr /H O (5.584 g, 25 mmol/10 mL) was added dropwise
a solution of NaNO /H O (0.518 g, 7.5 mmol/5 mL). The
75 °C for 30 min. The reaction mixture was cooled to room
temperature and diluted with EtOAc (100 mL). The EtOAc
2
2
2
2
solution was washed with a saturated NaHCO
mL × 2), dried (Na SO ), and evaporated. The residue was
chromatographed on a silica column (1.9 × 40 cm, eluted with
CHCl ). Evaporation of fractions 10-24 (20 mL per fraction)
3
solution (100
reaction mixture was stirred at room temperature for 10 min.
Compound 3 (1.01 g, 5 mmol) was added portionwise over 5
min, and stirring was continued at room temperature for 2 h
2
4
3
with the dropwise addition over a 2 h period of a fresh NaNO
2
/
and recrystallization from MeCN gave 0.678 g of 5b as white
crystals. A second crop of 0.187 g was obtained by recrystal-
lization of the mother liquor from MeOH. The total yield of
5b was 0.865 g (83%): mp 194-197 °C (MeCN); HRMS (EI
H
2
O solution (1.36 g, 15 mmol/10 mL). The reaction mixture
was then heated on a steam bath for 10 min, cooled to room
temperature, and treated with 25 mL of a 1 N HBr solution
and 100 mL of EtOAc for 5 min with vigorous stirring. The
mixture was then transferred to a separatory funnel, and the
two layers were separated. The EtOAc layer was washed
successively with a 0.5 N HBr solution (100 mL) and a
+
1
with a DCI probe) m/ z 521.9589 (4, M ) 521.9596); H NMR
(DMSO-d
6
) δ 7.88 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.57 (d, 1, 5-H),
6.23 (d, 1, 1′-H, J 1′-2′ ) 7.0 Hz), 5.54 (t, 1, 2′-H, J 2′-3′ ) 7.5
Hz), 5.44 (dd, 1, 3′-H, J 3′-4′ ) 4.5 Hz), 4.50, 4.37 (2 m, 3, 4′-H
1
3
saturated NaHCO
3
solution (100 mL), dried (Na
2
SO
4
), and
6
and 5′-H), 2.15, 2.14, 2.01 (3 s, 9, 3 Ac); C NMR (DMSO-d )
evaporated. The residue was chromatographed on a silica
column (1.9 × 20 cm, eluted with 1:9 EtOAc/hexane). Evapo-
ration of fractions 9-23 (15 mL per fraction) and recrystalli-
zation from MeCN gave 0.745 g (two crops, 56%) of 4b as
brownish crystals: mp 207-209 °C; HRMS (EI) m/ z 263.8844
3
δ 170.0, 169.5, 169.2 (3 COCH ), 139.1 (C3a), 134.4 (C7a), 131.3
(C2), 128.4 (C6), 123.6 (C4), 123.2 (C5), 111.1 (C7), 87.9 (C1′),
79.7 (C4′), 70.4 (C2′), 68.6 (C3′), 62.6 (C5′), 20.7, 20.3, 20.0 (3
× COCH
3 2 2 7
). Anal. (C18H17BrCl N O ) C, H, N.
Evaporation of fractions 29-39 (20 mL per fraction) gave
+
1
(
63, M ) 263.8857); H NMR (DMSO-d
6
) δ 13.88 (br s, 1,
0.028 g (3%) of 6b as a syrup: HRMS (EI with a DCI probe)
+
1
1
-NH), 7.58, 7.41 (s, d, 2, 5-H and 7-H, J 5-7 ) 1.5 Hz). Anal.
m/ z 521.9600 (6, M ) 521.9596); H NMR (DMSO-d
6
) δ 7.72
(C
7
H
3
BrCl
2 2
N ) C, H, N.
(d, 1, 7-H, J 7-5 ) 1.5 Hz), 7.53 (d, 1, 5-H), 6.69 (d, 1, 1′-H,
2
,4,6-Tr ich lor o-1-(2,3,5-tr i-O-a cetyl-â-D-r ibofu r a n osyl)-
J 1′-2′ ) 4.5 Hz), 5.70 (t, 1, 2′-H, J 2′-3′ ) 5.0 Hz), 5.53 (dd, 1,
b en zim id a zole (5a ) a n d 2,4,6-Tr ich lor o-1-(2,3,5-t r i-O-
a cetyl-r-D-r ibofu r a n osyl)ben zim id a zole (6a ). To a sus-
pension of 4a (1.362 g, 6.15 mmol) in 31 mL of dry MeCN was
added 1.52 mL (6.15 mmol) of BSA. The reaction mixture was
stirred at 80 °C for 15 min to give a clear solution. This
solution was treated with 2.153 g (6.675 mmol) of 1,2,3,5-tetra-
O-acetyl-â-D-ribofuranose and 1.426 mL (7.380 mmol) of tri-
methylsilyl triflate (TMSOTf) at 80 °C for 1 h. The reaction
mixture was cooled and diluted with EtOAc (150 mL). The
3′-H, J 3′-4′ ) 7.0 Hz), 4.85 (m, 1, 4′-H), 4.36 (dd, 1, 5′-H, J 5′-4′
) 3.5 Hz, J 5′-5′′ ) 12.5 Hz), 4.28 (dd, 1, 5′′-H, J 5′′-4′ ) 5.5 Hz),
1
3
2.09, 2.04, 1.54 (3 s, 9, 3 Ac); C NMR (DMSO-d
169.4, 168.37 (3 COCH
6
) δ 170.2,
), 138.8 (C3a), 135.4 (C7a), 130.7 (C2),
3
127.9 (C6), 123.1 (C4), 122.7 (C5), 112.2 (C7), 87.6 (C1′), 78.4
(C4′), 71.0 (C2′), 70.6 (C3′), 63.0 (C5′), 20.6, 20.2, 19.6 (3
COCH
2,4,6-Tr ich lor o-1-â-D-r ibofu r a n osylben zim id a zole (7a ).
To a solution of Na CO (0.053 g, 0.5 mmol) in H O (1 mL)
3
).
2
3
2
EtOAc solution was washed with a saturated NaHCO
3
solution
150 mL × 2) and a saturated NaCl solution (150 mL), dried
Na SO ), and evaporated. The residue was co-evaporated
were added successively 4.5 mL of EtOH, 4.5 mL of MeOH,
and 0.24 g (0.5 mmol) of 5a . The reaction mixture was stirred
at room temperature for 2 h. AcOH (0.06 mL) was added, and
stirring was continued at room temperature for 15 min.
Volatile materials were removed by evaporation. The residue
(
(
2
4
with MeOH and then suspended in 50 mL of hot MeOH. The
suspension was cooled and filtered, and the solid product was
washed with MeOH to give 2.103 g of 5a as white crystals.
was triturated with H
MeOH/H O to give 0.135 g (76%) of 7a as white crystals: mp
176-177 °C; HRMS (EI) m/ z 351.9795 (13, M ) 351.9784);
2
O (10 mL × 2) and recrystallized from
(
This product showed a single spot on TLC, R
MeOH/CHCl , v/v.) The mother liquor was evaporated to
dryness, and the residue was chromatographed on a silica
column (2.5 × 20 cm, eluted with CHCl and 0.5% MeOH/
CHCl , v/v). Evaporation of fractions 20-24 (20 mL per
fraction) and recrystallization from MeOH gave an additional
.258 g of 5a as white crystals. The total yield of 5a was 2.361
g (80%): mp 198-200 °C; HRMS (EI) m/ z 480.0069 (4, for
f
) 0.75, 2%
2
+
3
1
H NMR (DMSO-d
1, 5-H), 5.89 (d, 1, 1′-H, J 1′-2′ ) 8.0 Hz), 5.49 (d, 1, 2′-OH,
J 2′-2′OH ) 6.5 Hz), 5.38 (t, 1, 5′-OH, J 5′-5′OH ) 4.5 Hz), 5.28 (d,
6
) δ 8.36 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.52 (d,
3
3
1, 3′-OH, J 3′-3′OH ) 4.5 Hz), 4.41 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz),
4.14 (m, 1, 3′-H, J 3′-4′ ) 2.0 Hz), 4.02 (m, 1, 4′-H, J 4′-5′ ) J 4′-5′′
) 2.5 Hz), 3.71 (m, 2, 5′-H and 5′′-H, J 5′-5′′ ) 12.0 Hz); 13C
0
3
5
37
+
1
C
7
J
3
2
1
18
H
17 Cl
2
ClN
2
O
7
: M ) 480.0072); H NMR (DMSO-d
6
) δ
NMR (DMSO-d
6
) δ 141.6 (C2), 137.5 (C3a), 134.2 (C7a), 128.0
.89 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.58 (d, 1, 5-H), 6.26 (d, 1, 1′-H,
1′-2′ ) 7.0 Hz), 5.54 (t, 1, 2′-H, J 2′-3′ ) 7.0 Hz), 5.44 (dd, 1,
′-H, J 3′-4′ ) 4.5 Hz), 4.49, 4.38 (2 m, 3, 4′-H and 5′-H), 2.139,
(C6), 123.2 (C4), 122.8 (C5), 112.6 (C7), 89.4 (C1′), 86.5 (C4′),
3 2 4
71.7 (C2′), 69.7 (C3′), 61.0 (C5′). Anal. (C12H11Cl N O ) C, H,
N.
1
3
.137, 2.02 (3 s, 9, 3 Ac); C NMR (DMSO-d
6
) δ 169.8, 169.4,
2-Br om o-4,6-d ich lor o-1-â-D-r ib ofu r a n osylb en zim id -
69.1 (3 COCH ), 140.9 (C2), 137.4 (C3a), 134.1 (C7a), 128.5
3
a zole (7b). To a solution of Na CO (0.106 g, 1 mmol) in H O
2
3
2
(
7
C6), 123.7 (C4), 123.3 (C5), 111.1 (C7), 86.9 (C1′), 79.6 (C4′),
0.5 (C2′), 68.6 (C3′), 62.5 (C5′), 20.5, 20.2, 19.9 (3 COCH ).
Anal. (C18 ) C, H, N.
(2 mL) were added successively 9 mL of EtOH, 9 mL of MeOH,
and 0.524 g (1 mmol) of 5b. The reaction mixture was stirred
at room temperature for 2 h. AcOH (0.12 mL) was added, and
stirring was continued at room temperature for 15 min.
Volatile materials were removed by evaporation. The residue
3
H
3 2 7
17Cl N O
Evaporation of fractions 29-39 (20 mL per fraction) gave
0
.09 g (3%) of 6a as a syrup: HRMS: (EI) m/ z 480.0069 (6,
3
5
37
+
1
for C18
H
17 Cl
2
ClN
2
O
7
: M ) 480.0072); H NMR (DMSO-
was triturated with H
MeCN to give 0.31 g (3 crops, 78%) of 7b as white crystals:
2
O (20 mL × 2) and recrystallized from
d
1
1
J
6
) δ 7.74 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.53 (d, 1, 5-H), 6.72 (d, 1,
+
′-H, J 1′-2′ ) 4.5 Hz), 5.72 (t, 1, 2′-H, J 2′-3′ ) 5.0 Hz), 5.51 (dd,
, 3′-H, J 3′-4′ ) 7.0 Hz), 4.84 (m, 1, 4′-H), 4.37 (dd, 1, 5′-H,
5′-4′ ) 3.5 Hz, J 5′-5′′ ) 12.0 Hz), 4.27 (dd, 1, 5′′-H, J 5′′-4′ ) 5.5
mp 175-176 °C; HRMS (EI) m/ z 395.9290 (5, M ) 395.9279);
1
H NMR (DMSO-d ) δ 8.37 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.50 (d,
6
1, 5-H), 5.90 (d, 1, 1′-H, J 1′-2′ ) 8.0 Hz), 5.49 (d, 1, 2′-OH,
J 2′-2′OH ) 6.5 Hz), 5.40 (t, 1, 5′-OH, J 5′-5′OH ) 4.5 Hz), 5.29 (d,
1
3
Hz), 2.09, 2.04, 1.58 (3 s, 9, 3 Ac); C NMR (DMSO-d
6
) δ 170.0,
), 140.6 (C2), 137.2 (C3a), 134.9 (C7a),
27.9 (C6), 123.2 (C4), 122.7 (C5), 112.1 (C7), 86.5 (C1′), 78.4
1
1
69.1, 168.3 (3 COCH
3
1, 3′-OH, J 3′-3′OH ) 4.0 Hz), 4.42 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz),
4.14 (m, 1, 3′-H, J 3′-4′ ) 2.0 Hz), 4.02 (m, 1, 4′-H, J 4′-5′ ) J 4′-5′′
) 2.5 Hz), 3.71 (m, 2, 5′-H and 5′′-H, J 5′-5′′ ) 12.0 Hz); 13C
(
C4′), 70.9 (C2′), 70.4 (C3′), 62.7 (C5′), 20.4, 20.0, 19.4 (3
COCH ).
-Br om o-4,6-d ich lor o-1-(2,3,5-t r i-O-a cet yl-â-D-r ib ofu -
r a n osyl)ben zim id a zole (5b) a n d 2-Br om o-4,6-d ich lor o-
-(2,3,5-tr i-O-acetyl-r-D-r ibofu r an osyl)ben zim idazole (6b).
3
NMR (DMSO-d
6
) δ 139.1 (C3a), 134.5 (C7a), 132.2 (C2), 127.9
2
(C6), 123.1 (C4), 122.7 (C5), 112.5 (C7), 90.4 (C1′), 86.5 (C4′),
2 2 4
71.6 (C2′), 69.7 (C3′), 61.1 (C5′). Anal. (C12H11BrCl N O ) C,
H, N.
4,6-Dich lor o-1-r-D-r ibofu r a n osylben zim id a zole 2,2′-O-
cyclon u cleosid e (8). A mixture of 0.084 g (0.175 mmol) of
6a in 10 mL of NH /MeOH (saturated at 0 °C) was stirred in
3
a pressure bottle at room temperature for 5 h. Volatile
materials were removed by evaporation and coevaporation
1
To a suspension of 4b (0.532 g, 2 mmol) in 20 mL of dry MeCN
was added 0.5 mL (2 mmol) of BSA. The reaction mixture was
stirred at 75 °C for 10 min to give a clear solution. This
solution was treated with 0.7 g (2.2 mmol) of 1,2,3,5-tetra-O-
acetyl-â-D-ribofuranose and 0.58 mL (3 mmol) of TMSOTf at

Substituted 1-â-D-ribofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 807
solution (100 mL), dried (Na SO ), evaporated, and coevapo-
rated with MeOH to give 12 as a white foam. This material
was directly used in the subsequent reaction without further
purification. An analytical sample of 12 (as white crystals)
was obtained by a combination of silica column chromatogra-
with MeOH (3×). The residue was recrystallized from MeOH/
2
4
H
2
O to give 38 mg (68%) of 8 as yellowish crystals: mp 204-
+
1
2
06 °C; HRMS (EI) m/ z 316.0005 (62, M ) 316.0018); H
NMR (DMSO-d ) δ 7.57 (d, 1, 7-H, J 7-5 ) 1.5 Hz), 7.35 (d, 1,
-H), 6.55 (d, 1, 1′-H, J 1′-2′ ) 5.0 Hz), 5.79 (d, 1, 3′-OH, J 3′-3′OH
6
5
)
J
5
5
1
7.0 Hz), 5.76 (t, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.87 (t, 1, 5′-OH,
3
phy (using CHCl as eluant) and recrystallization (from
5′-5′OH ) 5.0 Hz), 4.08 (m, 1, 3′-H, J 3′-4′ ) 9.0 Hz), 3.67 (dd, 1,
MeOH): mp 195-197 °C; HRMS (EI with a DCI probe) m/ z
+
1
′-H, J 4′-5′ ) 0 Hz, J 5′-5′′ ) 12.0 Hz), 3.55 (m, 1, 4′-H, J 4′-5′′
)
476.0200 (12, M ) 476.0212); H NMR (DMSO-d ) δ 13.79
6
1
3
.0 Hz), 3.48 (m, 1, 5′′-H); C NMR (DMSO-d
6
) δ 164.3 (C2),
(s, 1, 3-NH), 7.65 (d, 1, 7-H, J 7-5 ) 1.5 Hz), 7.49 (d, 1, 5-H),
6.67 (d, 1, 1′-H, J 1′-2′ ) 7.5 Hz), 5.67 (t, 1, 2′-H, J 2′-3′ ) 7.0
42.2 (C3a), 130.5 (C7a), 125.1 (C6), 121.6 (C5 and C4), 109.1
(
C7), 91.4 (C2′), 84.8 (C1′), 81.1 (C4′), 69.5 (C3′), 59.44 (C5′).
Anal. (C12 ) C, H, N.
-Azid o-4,6-d ich lor o-1-(2,3,5-t r i-O-a cet yl-â-D-r ib ofu -
Hz), 5.46 (dd, 1, 3′-H, J 3′-4′ ) 4.5 Hz), 4.49 (dd, 1, 5′-H, J 5′-4′
4.5 Hz, J 5′-5′′ ) 12.0 Hz), 4.43 (m, 1, 4′-H), 4.32 (dd, 1, 5′′-H,
5′′-4′ ) 2.5 Hz), 2.15, 2.14, 1.98 (3 s, 9, 3 Ac); 13C NMR (DMSO-
d ) δ 171.0 (C2), 170.1, 169.6, 169.3 (3 COCH ), 131.9, 128.3,
)
H
2 2 4
10Cl N O
2
J
r a n osyl)ben zim id a zole (9). To a mixture of 0.198 g (0.413
mmol) of 5a in 5 mL of MeCN was added 0.202 g (4.13 mmol)
6
3
127.5 (C3a, C7a, and C6), 123.1 (C5), 114.7 (C4), 109.5 (C7),
86.2 (C1′), 79.5 (C4′), 69.3 (C2′), 69.0 (C3′), 62.8 (C5′), 20.7,
20.4, 20.2 (3 COCH ). Anal. (C H Cl N O S) C, H, N.
of LiN
The mixture was then cooled to room temperature and
partitioned between EtOAc/H O (50 mL/50 mL). The EtOAc
layer was washed with a saturated NaCl solution (50 mL ×
), dried (Na SO ), and evaporated. The residue was recrys-
3
. The reaction mixture was stirred at 80°C for 2 days.
3
18 18
2
2
7
2
3
The foam of crude 12 was treated with MeOH/NH (satu-
rated at 0 °C, 50 mL) in a pressure bottle at room temperature
for 1 day. The reaction mixture was evaporated and coevapo-
rated with MeOH (3×). The residue was recrystallized from
2
2
4
tallized from MeOH to give 0.161 g (2 crops, 80%) of 9 as a
slightly yellowish crystalline compound: mp 140-142 °C;
HRMS (EI) m/ z 485.0494 (7, M ) 485.0505); 1H NMR
3
MeOH/CHCl (dissolving the residue in 10 mL of hot MeOH,
+
diluting it with 90 mL of CHCl
3
, and allowing it to stand) to
(
6
DMSO-d
6
) δ 7.81 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.49 (d, 1, 5-H),
give 1.509 g (2 crops, 86%) of 13 as white crystals: mp 213-
.04 (d, 1, 1′-H, J 1′-2′ ) 6.5 Hz), 5.59 (t, 1, 2′-H, J 2′-3′ ) 6.5
+
2
3
1
8
J
15 °C; HRMS (EI with a DCI probe) m/ z 349.9892 (17, M )
Hz), 5.43 (dd, 1, 3′-H, J 3′-4′ ) 4.5 Hz), 4.38 (m, 3, 4′-H and
5
1
1
49.9895). H NMR (DMSO-d
6
) δ 13.61 (s, 1, 3-NH), 8.13 (d,
, 7-H, J 7-5 ) 1.5 Hz), 7.41 (d, 1, 5-H), 6.48 (d, 1, 1′-H, J 1′-2′
1
3
′-H), 2.13, 2.11, 2.03 (3 s, 9, 3 Ac); C NMR (DMSO-d
69.9, 169.4, 169.2 (3 COCH ), 148.7 (C2), 137.1 (C3a), 134.1
6
) δ
)
3
.0 Hz), 5.35 (t, 1, 5′-OH, J 5′-5′OH ) 4.5 Hz), 5.22 (d, 1, 2′-OH,
2′-2′OH ) 6.5 Hz), 5.16 (d, 1, 3′-OH, J 3′-3′OH ) 4.0 Hz), 4.42
(
C7a), 126.9 (C6), 122.8 (C5), 122.4 (C4), 110.5 (C7), 85.6 (C1′),
9.3 (C4′), 70.6 (C2′). 69.0 (C3′), 62.7 (C5′), 20.5, 20.2, 20.1 (3
COCH ). Anal. (C18 ) C, H, N.
-Azid o-4,6-d ich lor o-1-â-D -r ib ofu r a n osylb e n zim id -
a zole (10). A mixture of compound 9 (0.875 g, 1.8 mmol) and
NH /MeOH (saturated at 0 °C, 18 mL) was stirred in a
7
(
m, 1, 2′-H, J 2′-3′ ) 6.0 Hz), 4.13 (m, 1, 3′-H, J 3′-4′ ) 2.0 Hz),
3
2 5 7
H17Cl N O
3
2
1
1
.93 (m, 1, 4′-H), 3.67 (m, 2, 5′-H, and 5′′-H, J 5′-4′ ) J 5′′-4′ )
2
13
.5 Hz, J 5′-5′′ ) 12.0 Hz); C NMR (DMSO-d ) δ 171.6 (C2),
6
32.2, 128.2, 127.3 (C3a, C7a, and C6), 122.5 (C5), 114.1 (C4),
3
11.0 (C7), 88.4 (C1′), 85.6 (C4′), 70.6 (C2′), 69.8 C3′), 61.1
pressure bottle at room temperature for 4.5 h. Volatile
materials were removed by evaporation and coevaporation
with MeOH (3×). The residue was recrystallized from MeOH
to give 0.605 g (2 crops, 93%) of 10 as a white crystalline
compound: mp ∼154 °C dec; HRMS (EI with a DCI probe)
(
C5′). Anal. (C12
,6-Dich lor o-2-m et h ylt h io-1-â-D-r ib ofu r a n osylb en zi-
m id a zole (14a ). To a solution of 13 (0.351 g, 1 mmol) in 10
mL of H O and 1 mL of concentrated NH OH was added 0.125
2 2 4
H12Cl N O S) C, H, N.
4
2
4
+
1
mL (2 mmol) of methyl iodide. The reaction mixture was
stirred at room temperature for 2 h. The resulting suspension
was filtered. The white solid was air dried and recrystallized
m/ z 359.0197 (50, M ) 359.0188); H NMR (DMSO-d
6
) δ 8.15
(
d, 1, 7-H, J 7-5 ) 1.5 Hz), 7.43 (d, 1, 5-H), 5.63 (d, 1, 1′-H,
J
5
4
1′-2′ ) 7.5 Hz), 5.41 (d, 1, 2′-OH, J 2′-2′OH ) 6.0 Hz), 5.27 (t, 1,
′-OH, J 5′-5′OH ) 5.0 Hz), 5.19 (d, 1, 3′-OH, J 3′-3′OH ) 4.5 Hz),
.39 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.11 (m, 1, 3′-H, J 3′-4′ ) 2.0
from MeOH/H
2
O to give 0.337 g (2 crops, 92%) of 14a as white
crystals: mp 202-205 °C; HRMS (EI with a DCI probe) m/ z
+
1
3
64.0030 (39, M ) 364.0051); H NMR (DMSO-d ) δ 8.15 (d,
6
Hz), 3.95 (m, 1, 4′-H, J 4′-5′ ) J 4′-5′′ ) 3.0 Hz), 3.67 (m, 2, 5′-H
1
3
1, 7-H, J 7-5 ) 2.0 Hz), 7.38 (d, 1, 5-H), 5.69 (d, 1, 1′-H, J 1′-2′
)
and 5′′-H, J 5′-5′′ ) 12.0 Hz); C NMR (DMSO-d
6
) δ 149.1 (C2),
37.3 (C3a), 133.9 (C7a), 126.5 (C6), 122.3 (C5), 122.0 (C4),
12.1 (C7), 87.9 (C1′), 86.1 (C4′), 71.4 (C2′), 69.8 (C3′), 61.2
) C, H, N.
7
J
(
3
5
d
(
6
.5 Hz), 5.44 (d, 1, 2′-OH, J 2′-2′OH ) 6.5 Hz), 5.32 (t, 1, 5′-OH,
5′-5′OH ) 5.0 Hz), 5.25 (d, 1, 3′-OH, J 3′-3′OH ) 4.5 Hz), 4.40
1
1
m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.12 (m, 1, 3′-H, J 3′-4′ ) 2.0 Hz),
(C5′). Anal. (C12
H
11Cl
2
N
5
O
4
.98 (m, 1, 4′-H, J 4′-5′ ) J 4′-5′′ ) 3.0 Hz), 3.69 (m, 2, 5′-H and
2
-Am in o-4,6-d ich lor o-1-â-D-r ib ofu r a n osylb e n zim id -
13
′′-H, J 5′-5′′ ) 12.0 Hz), 2.75 (s, 3, 2-SMe); C NMR (DMSO-
) δ 155.5 (C2), 139.5 (C3a), 135.7 (C7a), 126.3 (C6), 121.9
a zole (11). A mixture of compound 10 (0.227 g, 0.63 mmol)
6
and Raney nickel (0.05 g, wet wt) in EtOH (10 mL) was
C4), 121.7 (C5), 111.8 (C7), 88.9 (C1′), 86.3 (C4′), 71.5 (C2′),
9.9 (C3′), 61.2 (C5′), 14.7 (2-SMe). Anal. (C13 S)
C, H, N.
hydrogenated at 50 psi of H
reaction mixture was filtered, and the filtrate was evaporated.
The residue was recrystallized from H O to give 0.143 g (2
crops, 68%) of 11 as a white powder: mp 135-140 °C dec;
2
at room temperature for 6 h. The
2 2 4
H14Cl N O
2
2-(Ben zylt h io)-4,6-d ich lor o-1-â-D-r ib ofu r a n osylb en -
zim id a zole (14b). To a solution of 13 (0.351 g, 1 mmol) in
10 mL of H O and 1 mL of concentrated NH OH was added
2 4
0.238 mL (2 mmol) of benzyl bromide. The reaction mixture
was stirred at room temperature for 2 h. The resulting
suspension was filtered. The white solid was washed with
+
HRMS (EI with a DCI probe) m/ z 333.0301 (24, M
)
1
3
33.0283); H NMR (DMSO-d
Hz), 7.10 (d, 1, 5-H), 7.06 (s, 2, 2-NH
7.5 Hz), 5.57 (t, 1, 5′-OH, J 5′-5′OH ) 4.5 Hz), 5.29 (d, 1, 2′-
OH, J 2′-2′OH ) 7.5 Hz), 5.25 (d, 1, 3′-OH, J 3′-3′OH ) 4.0 Hz),
.32 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.09 (m, 1, 3′-H, J 3′-4′ ) 2.0
Hz), 3.97 (m, 1, 4′-H, J 4′-5′ ) 2.0 Hz, J 4′-5′′ ) 2.5 Hz), 3.67 (m,
6
) δ 7.50 (d, 1, 7-H, J 7-5 ) 2.0
2
), 5.73 (d, 1, 1′-H, J 1′-2′
)
4
portions of H
solution was washed with H
2
O and dissolved in 50 mL of EtOAc. The EtOAc
O, dried (Na SO ), and evapo-
2
2
4
1
3
2
(
, 5′-H and 5′′-H, J 5′-5′′ ) 12.0 Hz); C NMR (DMSO-d
C2), 139.0 (C3a), 134.8 (C7a), 122.2 (C6), 120.1 (C5), 118.5
6
) δ 155.6
rated. The residue was triturated with hexane (10 mL × 2)
and then recrystallized from EtOAc/hexane to give 0.418 g
(95%) of 14b as white crystals: mp 104-107 °C; HRMS (EI
(C4), 108.5 (C7), 87.7 (C1′), 85.8 (C4′), 71.3 (C2′), 70.1 (C3′),
+
1
6
1.0 (C5′). Anal. (C12
H
13Cl
2
N
3
O
4
) C, H, N.
with a DCI probe) m/ z 440.0364 (4, M ) 440.0364); H NMR
(DMSO-d ) δ 8.16 (d, 1, 7-H, J 7-5 ) 2.0 Hz), 7.52 (m, 2, Ph),
4
,6-Dich lor o-1-(2,3,5-t r i-O-a cet yl-â-D-r ib ofu r a n osyl)-
6
ben zim id a zole-2-th ion e (12) a n d 4,6-Dich lor o-1-â-D-r ibo-
fu r a n osylben zim id a zole-2-th ion e (13). A mixture of 5a
7.40 (d, 1, 5-H), 7.31 (m, 3, Ph), 5.68 (d, 1, 1′-H, J 1′-2′ ) 7.5
Hz), 5.43 (d, 1, 2′-OH, J 2′-2′OH ) 6.5 Hz), 5.32 (t, 1, 5′-OH,
(2.399 g, 5 mmol) and thiourea (0.76 g, 10 mmol) in EtOH (50
J
5′-5′OH ) 5.0 Hz), 5.24 (d, 1, 3′-OH, J 3′-3′OH ) 4.5 Hz), 4.64
(m, 2, CH Ph), 4.37 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.10 (m, 1,
3′-H, J 3′-4′ ) 2.0 Hz), 3.96 (m, 1, 4′-H, J 4′-5′ ) J 4′-5′′ ) 3.0 Hz),
mL) was heated at 80 °C for 1 h (it became a clear solution).
Volatile materials were then remove by evaporation. The
2
1
3
residue was partitioned between H
The H O layer was again extracted with CHCl
combined CHCl extracts were washed with a saturated NaCl
2
O/CHCl
3
(100 mL/100 mL).
3.67 (m, 2, 5′-H and 5′′-H, J 5′-5′′ ) 12.0 Hz); C NMR (DMSO-
) δ 154.1 (C2), 139.5 (C3a), 136.8 (Ph), 135.5 (C7a), 129.2,
128.5, 127.5 (Ph), 126.5 (C6), 122.1 (C4), 121.8 (C5), 111.9 (C7),
2
3
(50 mL). The
d
6
3

8
08 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
8.9 (C1′), 86.3 (C4′), 71.6 (C2′), 69.8 (C3′), 61.2 (C5′), 35.9 (2-
Zou et al.
SO ), and
8
saturated NaCl solution (100 mL × 2), dried (Na
2
4
SCH
-Am in o-4,5-d ich lor oben zim id a zole (16). To a stirred
solution of 13.2 mL of 5 M BrCN/MeCN in 120 mL of H
2
Ph). Anal. (C19
H
18Cl
2
N
2
O
4
S) C, H, N.
evaporated. The residue was heated in 50 mL of MeOH and
cooled, and the resultant suspension was filtered. The solid
products were combined and recrystallized from MeCN to give
1.898 g of 19a as white crystals. The mother liquor and the
filtrate were combined and evaporated. The residue was
chromatographed on a silica column (2 × 27 cm, eluted with
2
2
O
was added dropwise a solution of 3,4-dichloro-1,2-phenylene-
diamine (10.60 g, 59.88 mmol) in 120 mL of MeOH at room
temperature over 1 h. After the addition was complete,
stirring was continued at room temperature for 2 h. The
reaction mixture was concentrated to ∼120 mL and was
washed with EtOAc (100 mL × 2). The combined EtOAc phase
CHCl ). Evaporation of fractions 4-11 (15 mL per fraction)
3
and recrystallization from MeCN gave an additional 0.905 g
of 19a as white crystals. The total yield of 19a was 2.803 g
(73%): mp 194-197 °C; HRMS (EI with DCI probe) m/ z
was extracted with H
was neutralized with sat. NaHCO
2 2
O (100 mL). The combined H O phase
+
35
37
1
3
solution to pH ∼8 and was
480.0073 (6, M ) 480.0072 for C18
H
17 Cl
2
ClN
2 7
O ); H NMR
extracted with EtOAc (800 mL). The EtOAc phase was
washed with half-saturated NaCl solution (400 mL), dried
(DMSO-d
6
) δ 7.80 (d, 1, 7-H, J 7-6 ) 9.0 Hz), 7.61 (d, 1, 6-H),
6.27 (d, 1, 1′-H, J 1′-2′ ) 6.5 Hz), 5.53 (t, 1, 2′-H, J 2′-3′ ) 7.0
Hz), 5.42 (m, 1, 3′-H, J 3′-4′ ) 5.0 Hz), 4.42 (m, 3, 4′-H and 5′-
2 4
(Na SO ), and evaporated. The residue was recrystallized from
1
3
MeCN to give 10.06 g of 16 as beige crystals. The mother
liquor was evaporated, and the residue was suspended in a
H), 2.14, 2.11, 2.02 (3 s, 9, 3 Ac); C NMR (DMSO-d
6
) δ 170.1,
3
), 141.3 (C2), 139.7 (C3a), 132.5 (C7a),
169.6, 169.3 (3 COCH
small amount of CHCl
3
. The CHCl
3
suspension was filtered
126.3 (C5), 125.2 (C6), 121.3 (C4), 111.9 (C7), 87.1 (C1′), 79.5
(C4′), 70.7 (C2′), 68.7 (C3′), 62.7 (C5′), 20.6, 20.3, 20.1 (3 ×
to give an additional 1.18 g of 16 as a solid (pure by TLC).
The total yield of 16 was 11.24 g (93%): mp 239-241 °C;
HRMS (EI) m/ z 200.9872 (100, M ) 200.9861); 1H NMR
3 3 2 7
COCH ). Anal. (C18H17Cl N O ) C, H, N.
+
Evaporation of fractions 13-17 (15 mL per fraction) gave
(
6
DMSO-d
-H and 7-H), 6.62, 6.28 (2 br s, 2, 2-NH
C, H, N.
,4,5-Tr ich lor oben zim id a zole (17a ). To a mixture of
CuCl (1.345 g, 10 mmol) in 25 mL of acetone was added 0.99
6
) δ 11.44, 11.06 (2 br s, 1, 1-NH), 7.05, 699 (2 d, 2,
0.089 g (2%) of 18a as a yellowish syrup: HRMS (EI with DCI
+
1
2
). Anal. (C Cl
7
H
5
2
N
3
)
6
probe) m/ z 478.0089 (8, M ) 478.0101); H NMR (DMSO-d )
δ 7.70 (d, 1, 7-H, J 7-6 ) 9.0 Hz), 7.54 (d, 1, 6-H), 6.74 (d, 1,
1′-H, J 1′-2′ ) 4.0 Hz), 5.68 (t, 1, 2′-H, J 2′-3′ ) 5.0 Hz), 5.50 (dd,
1, 3′-H, J 3′-4′ ) 7.5 Hz), 4.82 (m, 1, 4′-H, J 4′-5′ ) 3.0 Hz, J 4′-5′′
2
2
mL (7.5 mmol) of 90% t-BuONO. The reaction mixture was
stirred at room temperature for 20 min. Compound 16 (1.01
g, 5 mmol) was added portionwise over 15 min, and the
reaction mixture was heated under gentle reflux for 2 h (with
addition of 0.99 mL of fresh 90% t-BuONO every 30 min). The
reaction mixture was then cooled to room temperature, treated
with 50 mL of 2 N HCl solution, and extracted with 100 mL
of EtOAc. The EtOAc layer was washed with 2 N HCl solution
) 5.5 Hz), 4.37 (dd, 1, 5′-H, J 5′-5′′ ) 12.0 Hz), 4.26 (dd, 1, 5′′-
H), 2.08, 2.02, 1.54 (3 s, 9, 3 Ac); 1 C NMR (DMSO-d
169.4, 168.5 (3 × COCH
3
) δ 170.2,
6
3
), 140.8 (C2), 139.43 (C3a), 133.3
(C7a), 125.6 (C5), 124.7 (C6), 120.7 (C4), 113.3 (C7), 86.7 (C1′),
78.2 (C4′), 71.0 (C2′), 70.4 (C3′), 62.8 (C5′), 20.6, 20.2, 19.7 (3
3
COCH ).
2-Br om o-4,5-d ich lor o-1-(2,3,5-t r i-O-a cet yl-â-D-r ib ofu -
r a n osyl)ben zim id a zole (19b) a n d 2-Br om o-4,5-d ich lor o-
1-(2,3,5-t r i-O-a ce t yl-r-D-r ib ofu r a n osyl)b e n zim id a zole
(18b). A mixture of 17b (0.532 g, 2 mmol) and BSA (0.5 mL)
in dry MeCN (20 mL) was stirred at 75 °C for 10 min to give
a clear solution. To this solution were added 0.70 g (2.2 mmol)
of 1,2,3,5-tetra-O-acetyl-â-D-ribofuranose and 0.58 mL (3
mmol) of TMSOTf. Stirring was continued at 75 °C for 30 min.
The reaction mixture was cooled to room temperature and
diluted with EtOAc (100 mL). The EtOAc solution was washed
(
50 mL × 2) and a saturated NaCl solution (50 mL), dried (Na
2
-
SO ), and evaporated. The residue was recrystallized from
4
MeCN to give 0.498 (45%) of 17a as a brownish solid.
Analytical sample was obtained via silica column chromatog-
raphy followed by recrystallization from MeCN: mp 244-247
+
°
C; HRMS (EI with DCI probe) m/ z 219.9372 (100, M
)
1
2
19.9362); H NMR (DMSO-d
m, 2, 6-H and 7-H). Anal. (C
-Br om o-4,5-d ich lor oben zim id a zole (17b). To a solu-
tion of CuBr /H O (5.584 g, 25 mmol/10 mL) was added a
solution of NaNO /H O (0.518 g, 7.5 mmol/5 mL). The reaction
6
) δ 13.89 (br s, 1, 1-NH), 7.46
(
7
H
3
Cl ) C, H, N.
3 2
N
2
with a saturated NaHCO
SO ), and evaporated. The residue was chromatographed on
). Evaporation
3
solution (100 mL × 2), dried (Na
2
-
2
2
4
2
2
a silica column (2 × 40 cm, eluted with CHCl
3
mixture was stirred at room temperature for 10 min. Com-
pound 16 (1.01 g, 5 mmol) was added portionwise over 5 min.
Stirring was continued at room temperature for 2 h [with
of fractions 9-23 (20 mL per fraction) and recrystallization
from MeCN gave 0.918 g (2 crops, 88%) of 19b as white
crystals: mp 194-197 °C; HRMS (EI with DCI probe) m/ z
+
1
dropwise addition of a fresh solution of NaNO
2
/H
2
O (1.36 g,
521.9590 (7, M ) 521.9596); H NMR (DMSO-d ) δ 7.81 (d,
6
1
5 mmol/10 mL)] and then at 100 °C for 10 min. The reaction
1, 7-H, J 7-6 ) 9.0 Hz), 7.59 (d, 1, 6-H), 6.24 (d, 1, 1′-H, J 1′-2′ )
mixture was cooled to room temperature and treated with 100
mL of EtOAc and 25 mL of 1 N HBr solution for 5 min with
vigorous stirring. The content was transferred to a separatory
funnel, and the two layers were separated. The EtOAc layer
was washed with a 0.5 N HBr solution (100 mL) and a
7.0 Hz), 5.54 (t, 1, 2′-H, J 2′-3′ ) 7.0 Hz), 5.42 (dd, 1, 3′-H, J 3′-4′
) 4.5 Hz), 4.45 (m, 3, 4′-H and 5′-H), 2.14, 2.12, 2.01 (3 s, 9, 3
Ac); 13C NMR (DMSO-d
6
3
) δ 170.0, 169.5, 169.1 (3 COCH ),
141.3 (C3a), 132.8 (C7a), 131.6 (C2), 126.2 (C5), 125.0 (C6),
121.2 (C4), 111.7 (C7), 88.1 (C1′), 79.5 (C4′), 70.5 (C2′), 68.7
saturated Na
2
CO
3
solution (100 mL), dried (Na
2
SO
4
), and
(C3′), 62.7 (C5′), 20.6, 20.3, 20.0 (3 COCH
BrCl ) C, H, N.
3 17
). Anal. (C18H -
evaporated. The residue was chromatographed on a silica
column (1.9 × 20 cm, eluted successively with 10%, 20%, 40%
EtOAc/hexane). Evaporation of fractions 11-17 (15 mL/
fraction) and recrystallization from MeCN gave 0.75 g (2 crops,
2 2 7
N O
Evaporation of fractions 27-33 (20 mL per fraction) gave
0.032 g (3%) of 18b as a yellowish syrup: HRMS (EI with DCI
+
1
6
probe) m/ z 521.9617 (4, M ) 521.9596); H NMR (DMSO-d )
5
6%) of 17b as yellowish crystals: mp 221-223 °C; HRMS
δ 7.70 (d, 1, 7-H, J 7-6 ) 9.0 Hz), 7.52 (d, 1, 6-H), 6.71 (d, 1,
1′-H, J 1′-2′ ) 4.0 Hz), 5.67 (t, 1, 2′-H, J 2′-3′ ) 5.0 Hz), 5.53 (dd,
1, 3′-H, J 3′-4′ ) 7.5 Hz), 4.82 (m, 1, 4′-H, J 4′-5′ ) 3.5 Hz, J 4′-5′′
+
1
(
EI with DCI probe) m/ z 263.8849 (65, M ) 263.8857); H
NMR (DMSO-d
-H). Anal. (C
6
) δ 13.84 (br s, 1, 1-NH), 7.45 (m, 2, 6-H and
BrCl ) C, H, N.
7
7
H
3
2
N
2
) 5.5 Hz), 4.36 (dd, 1, 5′-H, J 5′-5′′ ) 12.0 Hz), 4.28 (dd, 1, 5′′-
H), 2.08, 2.02, 1.51 (3 s, 9, 3 Ac); 1 C NMR (DMSO-d
3
) δ 170.2,
2
,4,5-Tr ich lor o-1-(2,3,5-tr i-O-a cetyl-â-D-r ibofu r a n osyl)-
6
ben zim id a zole (19a ) a n d 2,4,5-Tr ich lor o-1-(2,3,5-tr i-O-
a cetyl-r-D-r ibofu r a n osyl)ben zim id a zole (18a ). To a sus-
pension of 17a (1.772 g, 8 mmol) in dry MeCN (40 mL) was
added 3 mL (12 mmol) of BSA. The reaction mixture was
stirred at ambient temperature for 15 min. This was treated
with 1,2,3,5-tetra-O-acetyl-â-D-ribofuranose (3.055 g, 9.6 mmol)
and TMSOTf (3.092 mL, 16 mmol) at ambient temperature
for 30 min. (It became a clear solution and then turned into
a suspension again.) The reaction mixture was diluted with
EtOAc (120 mL) and filtered. The solid was pure product 19a .
3
169.4, 168.5 (3 COCH ), 140.9 (C3a), 133.8 (C7a), 130.8 (C2),
125.5 (C5), 124.5 (C6), 120.6 (C4), 113.3 (C7), 87.8 (C1′), 78.2
(C4′), 70.9 (C2′), 70.5 (C3′), 63.0 (C5′), 20.6, 20.2, 19.7 (3
COCH
2,4,5-Tr ich lor o-1-â-D-r ibofu r an osylben zim idazole (20a).
To a solution of Na CO (0.106 g, 1 mmol) in H O (2 mL) were
3
).
2
3
2
added successively 9 mL of EtOH, 9 mL of MeOH, and 0.480
g (1 mmol) of 19a . The reaction mixture was stirred at room
temperature for 2 h. AcOH (0.12 mL) was added, and stirring
was continued at room temperature for 15 min. Volatile
materials were removed by evaporation. The residue was
3
The filtrate was washed with 1:1 saturated NaHCO solution/

Substituted 1-â-D-ribofuranosylbenzimidazoles
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 809
triturated with H
to give 0.308 g (2 crops, 87%) of 20a as white crystals: mp
2
O (20 mL) and recrystallized from MeCN
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.
1
78-180 °C; HRMS (EI with DCI probe) m/ z 351.9774 (10,
+
1
M
)
5
)
J
) 351.9784); H NMR (DMSO-d
6
): δ 8.09 (d, 1, 7-H, J 7-6
HCMV Yield Red u ction Assa y. The effect of compounds
on the replication of HCMV also was measured using a yield
reduction assay. HFF cells were planted as described above
in 96-well cluster dishes and incubated overnight, medium was
removed, and the cultures were inoculated with HCMV in 0.1
9.0 Hz), 7.48 (d, 1, 6-H), 5.90 (d, 1, 1′-H, J 1′-2′ ) 7.5 Hz),
.53 (d, 1, 2′-OH, J 2′-2′OH ) 6.5 Hz), 5.30 (d, 1, 3′-OH, J 3′-3′OH
4.5 Hz), 5.29 (t, 1, 5′-OH, J 5′-5′OH ) 5.0 Hz), 4.40 (m, 1, 2′-H,
2′-3′ ) 6.0 Hz), 4.13 (m, 1, 3′-H, J 3′-4′ ) 2.0 Hz), 4.00 (m, 1,
′-H, J 4′-5′ ) 3.5 Hz, J 4′-5′′ ) 3.0 Hz), 3.70 (m, 2, 5′-H and 5′′-
3
1
4
mL at a moi of 0.5-1 pfu/cell as reported elsewhere. After
virus adsorption, 0.1 mL of fresh medium containing test
compounds in twice the desired final concentration (2×) was
added. This was accomplished by preparing a 2× solution of
the highest desired concentrations and then serially diluting
1:3 along the eight wells. In this manner, six compounds could
be tested in duplicate on a single plate with concentrations
from 100 to 0.05 µM. Plates were incubated at 37 °C for 7
days and subjected to one cycle of freezing and thawing;
aliquots from each of the eight wells of a given column were
transferred to the first column of a fresh 96-well monolayer
culture of HFF cells. Contents were mixed and serially diluted
1:3 across the remaining eleven columns of the secondary
plate. Each column of the original primary plate was diluted
across a separate plate in this manner. Cultures were
incubated, plaques were enumerated, and titers were calcu-
lated as described above.
1
3
H, J 5′-5′′ ) 12.0 Hz); C NMR (DMSO-d
6
) δ 142.0 (C2), 139.8
(
(
C3a), 132.6 (C7a), 125.8 (C5), 124.6 (C6), 120.9 (C4), 113.2
C7), 89.4 (C1′), 86.5 (C4′), 71.8 (C2′), 69.7 (C3′), 61.2 (C5′).
) C, H, N.
-Br om o-4,5-d ich lor o-1-â-D-r ib ofu r a n osylb en zim id -
a zole (20b). To a solution of Na CO (0.106 g, 1 mmol) in
O (2 mL) were added successively 9 mL of EtOH, 9 mL of
3 2 4
Anal. (C12H11Cl N O
2
2
3
H
2
MeOH, and 0.524 g (1 mmol) of 19b. The reaction mixture
was stirred at room temperature for 2 h. AcOH (0.12 mL) was
added, and stirring was continued at room temperature for
1
5 min. Volatile materials were removed by evaporation. The
residue was triturated with H
from MeCN to give 0.338 g (2 crops, 85%) of 20b as white
2
O (20 mL × 2) and recrystallized
+
crystals: mp 174-175 °C; HRMS (EI) m/ z 395.9276 (11, M
1
)
395.9279); H NMR (DMSO-d
6
) δ 8.09 (d, 1, 7-H, J 7-6 ) 9.0
Hz), 7.46 (d, 1, 6-H), 5.90 (d, 1, 1′-H, J 1′-2′ ) 8.0 Hz), 5.50 (d,
, 2′-OH, J 2′-2′OH ) 6.5 Hz), 5.30 (d, 1, 3′-OH, J 3′-3′OH ) 4.5
Hz), 5.29 (t, 1, 5′-OH, J 5′-5′OH ) 5.0 Hz), 4.43 (m, 1, 2′-H, J 2′-3′
HSV-1 ELISA. An ELISA was employed32 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 concentration
of 100 pfu/well were added. Following a 3-day incubation at
37 °C, medium was removed, plates were blocked and rinsed,
and horse radish peroxidase conjugated rabbit anti-HSV-1
antibody was added. Following removal of the antibody
containing solution, plates were rinsed and then developed by
adding 150 µL per well of a solution of tetramethylbenzidine
1
)
5.5 Hz), 4.13 (m, 1, 3′-H, J 3′-4′ ) 2.0 Hz), 4.00 (m, 1, 4′-H,
J
4′-5′ ) J 4′-5′′ ) 3.0 Hz), 3.70 (m, 2, 5′-H and 5′′-H, J 5′-5′′
)
1
3
1
2.0 Hz); C NMR (DMSO-d
6
) δ 141.4 (C3a), 132.9 (C7a), 132.5
(C2), 125.7 (C5), 124.4 (C6), 120.8 (C4), 112.9 (C7), 90.4 (C1′),
8
6.4 (C4′), 71.7 (C2′), 69.7 (C3′), 61.1 (C5′). Anal. (C12
11
H -
BrCl ) C, H, N.
2
2 4
N O
Cell Cu ltu r e P r oced u r es. The routine growth and pas-
sage of KB cells (human oral epidermoid carcinoma), BSC-1
cells (African green monkey kidney cells), and human foreskin
fibroblasts (HFF cells) was performed in monolayer cultures
using minimal essential medium (MEM) with either Hanks
salts [MEM(H)] or Earle salts [MEM(E)] supplemented with
as substrate. The reaction was stopped with H
2 4
SO 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 absor-
bance obtained with virus in the absence of drug.
1
0% calf serum or 10% fetal bovine serum (HFF cells). The
sodium bicarbonate concentration was varied to meet the
buffering capacity required. Cells were passaged at 1:2 to 1:10
dilutions according to conventional procedures by using 0.05%
trypsin plus 0.02% EDTA in a HEPES buffered salt solution.
Vir ologica l P r oced u r es. The Towne strain, plaque-puri-
Cytotoxicity Assa ys. Two different assays were used to
explore cytotoxicity of selected compounds using methods we
have detailed previously. (i) Cytotoxicity produced in station-
ary HFF cells was determined by microscopic inspection of cells
not infected by HCMV used in plaque assays.30 (ii) The effect
of compounds during two population doublings of KB cells was
determined by crystal violet staining and spectrophotometric
quantitation of dye eluted from stained cells as described
earlier.33 Briefly, 96-well cluster dishes were planted with KB
cells at 3000-5000 cells per well. After overnight incubation
at 37 °C, test compound was added in quadruplicate at six to
eight concentrations. Plates were incubated at 37 °C for 48 h
fied isolate P
o
, 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. High titer HSV-1 stocks were prepared by infecting
3
0
KB cells at an m.o.i. of <0.1 also as detailed previously.30
Virus titers were determined using monolayer cultures of HFF
cells for HCMV and monolayer cultures of BSC-1 cells for
2
in a CO 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.
3
l
HSV-1 as described earlier. Briefly, HFF or BSC-1 cells were
planted as described above in 96-well cluster dishes and
incubated overnight at 37 °C. The next day cultures were
inoculated with HCMV or HSV-1 and serially diluted 1:3
across the remaining eleven columns of the 96-well plate.
After virus adsorption the inoculum was replaced with fresh
medium and cultures were incubated for 7 days for HCMV, 2
or 3 days for HSV-1. Plaques were enumerated under 20-fold
magnification in wells having the diludon which gave 5-20
plaques per well. Virus titers were calculated according to the
following formula: Titer (pfu/mL) ) number of plaques × 5 ×
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 concentrations (IC50’s)
or IC90’s were calculated from the regression lines. Samples
containing positive controls (acyclovir for HSV-1, ganciclovir
for HCMV, and 2-acetylpyridine thiosemicarbazone for cyto-
toxicity) were used in all assays.
n
Ack n ow led gm en t. The authors thank members of
Dr. Drach’s research group, especially J ulie M. Breiten-
bach, Roger G. Ptak, and Dr. Allison C. Westerman, for
the antiviral evaluations. We also appreciate the
contributions of J . Hinkley for the preparation of chemi-
cal intermediates and Ms. Marina Savic for preparation
of the manuscript. This research work was supported
by federal funds from the Department of Health and
Human Services Research Contract NOl-AI-72641, Re-
3
; where n represents the nth dilution of the virus used to
infect the well in which plaques were enumerated.
HCMV P la qu e Red u ction Assa y. HFF cells in 24-well
cluster dishes were infected with approximately 100 pfu of
2
HCMV/cm cell sheet using the procedures detailed above.
Following virus adsorption, compounds dissolved in growth
medium were added to duplicate wells in four to eight selected
concentradons. After incubation at 37 °C for 7-10 days, cell
sheets were fixed and stained with crystal violet and micro-
scopic plaques were enumerated as described above. Drug

8
10 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Zou et al.
search Grant UOl-AI31718 from the National Institute
of Allergy and Infectious Diseases, and Research Agree-
ment DRDA-942921 with Glaxo-Wellcome.
R.; Coleman, L. A.; Devivar, R. V.; Genzlinger, G; Kreske, E.
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