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

Article abstract of DOI:10.1021/jm960462g

2-Chloro-5,6-difluorobenzimidazole (8) was prepared from 4,5-difluoro- 2-nitroaniline (5) via successive reduction, cyclization, and diazotization reactions. 2-Chloro-5,6-dibromobenzimidazole (10) was obtained by a direct bromination of 2-chlorobenzimidazole (9) with bromine-water. 2-Chloro-5,6- diiodobenzimidazele (15) was synthesized by a stepwise transformation of the nitre functions of 2-chloro-5,6-dinitrobenzimidazole (11) into iodo groups via diazotization reactions. Ribosylation of 8, 10, and 15 gave the respective β nucleosides 16a-c as the major products along with a small amount of the α anomers 17a-c. Deprotection of 16a-c afforded the corresponding free β nucleosides 2-chloro-5,6-difluoro-1-β-D- ribofuranosylbenzimidazole (2), 2-chloro-5,6-dibromo-1-β-D- ribofuranosylbenzimidazole (3), and 2-chloro-5,6-diiodo-1-β-D- ribofuranosylbenzimidazole (4). Similar deprotection of the α anomers (17a- c) resulted in a removal of the acetyl protecting groups and a concomitant cyclization to give the 2,2'-O-cyclonucleosides (18a-c). Mast of the benzimidazole heterocycles, but not the difluoro analog, were active against human cytomegalovirus (HCMV) (IC₅₀'s = 3-40 μM) and herpes simplex virus type 1 (HSV-1) (IC₅₀'s = 50-90 μM). This activity, however, was not well separated from cytotoxicity, IC₅₀'s = 10-100 μM. The corresponding unsubstituted, the 5,6-dimethyl, and the 5,6-difluoro ribonucleosides (19, 20, and 2, respectively), were inactive against both viruses. Similar to the previously reported 2,5,6-trichloro analog (TCRB), the 5,6-dibromo ribonucleoside 3 was active against HCMV (IC₅₀ ? 4 μM) but more cytotoxic than TCRB. The 5,6-diiodo analog 4 also was active (IC₅₀ ? 2 μM) but more cytotoxic (IC₅₀ = 10-20 μM) than either 3 or TCRB. The cyclonucleosides were inactive against both viruses and not cytotoxic, or slightly active with corresponding cytotoxicity. The order of activity against HCMV of the dihalobenzimidazole ribonucleosides was I ? Br ? CI >> F > H = CH₃. The order of cytotoxicity among the most active compounds, however, was I > Br > Cl, thereby establishing that TCRB had the best antiviral properties.

Full text of DOI:10.1021/jm960462g

J . Med. Chem. 1997, 40, 811-818
811
Notes
Design , Syn th esis, a n d An tivir a l Eva lu a tion of
2-Ch lor o-5,6-d ih a lo-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
Received J une 27, 1996X
2-Chloro-5,6-difluorobenzimidazole (8) was prepared from 4,5-difluoro-2-nitroaniline (5) via
successive reduction, cyclization, and diazotization reactions. 2-Chloro-5,6-dibromobenzimi-
dazole (10) was obtained by a direct bromination of 2-chlorobenzimidazole (9) with bromine-
water. 2-Chloro-5,6-diiodobenzimidazole (15) was synthesized by a stepwise transformation
of the nitro functions of 2-chloro-5,6-dinitrobenzimidazole (11) into iodo groups via diazotization
reactions. Ribosylation of 8, 10, and 15 gave the respective â nucleosides 16a -c as the major
products along with a small amount of the R anomers 17a -c. Deprotection of 16a -c afforded
the corresponding free â nucleosides 2-chloro-5,6-difluoro-1-â-D-ribofuranosylbenzimidazole (2),
2-chloro-5,6-dibromo-1-â-D-ribofuranosylbenzimidazole (3), and 2-chloro-5,6-diiodo-1-â-D-ribo-
furanosylbenzimidazole (4). Similar deprotection of the R anomers (17a -c) resulted in a
removal of the acetyl protecting groups and a concomitant cyclization to give the 2,2′-O-
cyclonucleosides (18a -c). Most of the benzimidazole heterocycles, but not the difluoro analog,
were active against human cytomegalovirus (HCMV) (IC50’s ) 3-40 µM) and herpes simplex
virus type 1 (HSV-1) (IC50’s ) 50-90 µM). This activity, however, was not well separated
from cytotoxicity, IC50’s ) 10-100 µM. The corresponding unsubstituted, the 5,6-dimethyl,
and the 5,6-difluoro ribonucleosides (19, 20, and 2, respectively), were inactive against both
viruses. Similar to the previously reported 2,5,6-trichloro analog (TCRB), the 5,6-dibromo
ribonucleoside 3 was active against HCMV (IC50 ≈ 4 µM) but more cytotoxic than TCRB. The
5,6-diiodo analog 4 also was active (IC50 ≈ 2 µM) but more cytotoxic (IC50 ) 10-20 µM) than
either 3 or TCRB. The cyclonucleosides were inactive against both viruses and not cytotoxic,
or slightly active with corresponding cytotoxicity. The order of activity against HCMV of the
dihalobenzimidazole ribonucleosides was I = Br = Cl . F > H ) CH3. The order of cytotoxicity
among the most active compounds, however, was I > Br > Cl, thereby establishing that TCRB
had the best antiviral properties.
In tr od u ction
and nontoxic HCMV drugs with good oral availability.
Moreover, as virus strains resistant to current drugs
emerge,14 drugs which act by new mechanisms will be
needed to treat HCMV infections.
Human cytomegalovirus (HCMV) is the most common
sight- and life-threatening opportunistic viral infection
in immunocompromised individuals such as AIDS pa-
tients.2,3 HCMV infection is also the primary cause of
death in recipients of allogeneic bone marrow trans-
plantation and renal transplantation.4,5 The treatment
of HCMV infection is difficult because only a few
therapeutic options are available. While many well-
known antiviral drugs, such as vidarabine, interferons,
and acyclovir, have been tested and proven not to be
efficacious against HCMV,6-9 only ganciclovir (DHPG),10
foscarnet (PFA),11 and cidofovir12 have been approved
by the FDA for the treatment of HCMV diseases.
Although the treatment of HCMV infection with these
drugs has produced clinical improvement in a large
proportion of patients, the compounds suffer from poor
oral bioavailability and produce adverse effects.10-13
Therefore, there still is an urgent need for more active
In our search for more potent and nontoxic agents for
the treatment of HCMV infection, we evaluated a
number of benzimidazole nucleosides previously pre-
pared in our laboratory.15 Unlike 5,6-dichloro-1-â-D-
ribofuranosylbenzimidazole (DRB) which has antiviral
and cytotoxic activities,16-18 we discovered that other
substituted benzimidazole ribosides, e.g., the 2-chloro
analog of DRB (TCRB) and the 2-bromo analog (BD-
CRB), were highly active and selective against HCMV
in vitro and were essentially noncytotoxic.18,19 Both
compounds act by a new mechanism involving inhibition
of viral DNA processing.20 As part of a comprehensive
structure-activity relationship study related to the
antiviral activity of TCRB, we have synthesized the
ribosides of 2-chloro-5,6-difluoro-, 2-chloro-5,6-dibromo-,
and 2-chloro-5,6-diiodobenzimidazole (2-4). The present
work describes the synthesis and evaluation of these
compounds for their activity against HCMV.
* Author to whom correspondence should be addressed.
† Present address: Gilead Sciences, 344 Lakeside Dr., Foster City,
CA 94404.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00462-1 CCC: $14.00 © 1997 American Chemical Society

812 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
17a -c whose R configuration was established by the
facile formation of 2,2′-O-cyclonucleosides 18a -c upon
deprotection. Additional support for the anomeric as-
signments was provided by comparing the H-1′ chemical
shifts of the anomeric pairs.19 The H-1′ signals of the
major products 16a -c were observed at a higher (∼0.45
ppm) field than those of the corresponding R anomers
(17a -c), and therefore it was consistent with the
assignment of 16a -c as the â anomers.25 An alterna-
tive synthesis of 2-chloro-5,6-dibromo-1-â-D-ribofurano-
sylbenzimidazole (3) was also achieved via a direct
bromination of 2-chloro-1-â-D-ribofuranosylbenzimida-
zole26 (19) with bromine-water.27 This procedure not
only provided a better synthesis of compound 3 but also
gave further support for our assignments of anomeric
configuration. Compounds 2, 3, 4, 8, 10, and 15 were
evaluated for their activities against human cytomega-
lovirus.
Resu lts a n d Discu ssion
Ch em ist r y. The reduction of 4,5-difluoro-2-nitro-
aniline (5) with iron powder in 1 N HCl gave 4,5-
difluoro-1,2-phenylenediamine21 (6), which was treated
with cyanogen bromide to afford 2-amino-5,6-difluo-
robenzimidazole (7) in a good overall yield (Scheme 1).
Diazotization of 7 with sodium nitrite in aqueous cupric
chloride solution furnished 2-chloro-5,6-difluorobenz-
imidazole (8). The 5,6-dibromo derivative 10 was
prepared via a direct bromination of 2-chlorobenzimi-
dazole22 (9) with bromine-water. The bromination
reaction was controlled to give the desired 5,6-dibromo
derivative as the major product by using mild reaction
conditions and monitoring carefully the reaction process
by TLC. The direct iodination of 9 with iodine monochlo-
ride was unsuccessful using a variety of reaction condi-
tions. The introduction of iodine atoms into specific
positions of the benzimidazole moiety was accomplished
via stepwise diazotization and replacement reactions.
Thus, the treatment of 2-chloro-5,6-dinitrobenzimida-
zole23 (11) with iron powder in acetic acid effected a
selective reduction of only one of the nitro groups and
gave the 5(6)-amino-6(5)-nitro derivative 12. Diazoti-
zation of the amino group and subsequent replacement
with potassium iodide afforded the corresponding 5(6)-
iodo-6(5)-nitro derivative 13. Although the intermediate
product 12 could be isolated in a pure form (41%) by
successive column chromatography and recrystalliza-
tion, it is experimentally preferable to carry out these
reaction steps without purification of 12. A selective
reduction of the remaining nitro function of 13 to an
amino group was achieved by hydrogenation over Raney
nickel to yield the 5(6)-iodo-6(5)-amino derivative 14.
This reaction was closely monitored by TLC since
prolonged hydrogenation also removed the iodine atom.
Compound 14 was again subjected to the diazotization
and replacement reactions and gave the desired 2-chloro-
5,6-diiodobenzimidazole (15) in 87% yield. Glycosyla-
tion of 8, 10, and 15 with 1,2,3,5-tetra-O-acetyl-â-D-
ribofuranose yielded the respective â nucleosides 16a -c
as the major products along with a small amount of the
R anomers (17a -c) (Scheme 2). A removal of the
protecting groups from 16a -c gave the corresponding
free â nucleosides 2-chloro-5,6-difluoro-1-â-D-ribofura-
nosylbenzimidazole (2), 2-chloro-5,6-dibromo-1-â-D-ri-
bofuranosylbenzimidazole (3), and 2-chloro-5,6-diiodo-
1-â-D-ribofuranosylbenzimidazole (4). Similar depro-
tection of the R anomers 17a -c resulted in a removal
of the acetyl protecting groups and a concomitant
cyclization to give the 2,2′-O-cyclonucleosides (18a -c).
The major products (16a -c) of the glycosylation
reactions were assigned as the â anomers since the
preferential formation of the â anomer was expected
with a protected sugar bearing a participating group at
the 2-position.24,25 This was also in agreement with the
assignment of an R configuration to the minor products
An tivir a l Stu d ies. The benzimidazole heterocycles,
ribosides, and cyclonucleosides were evaluated for activ-
ity against HCMV and HSV-1 and for cytotoxicity in
two cell lines. Three of the benzimidazole heterocycles
(TCB,18a 10, and 13) were active against HCMV at
concentrations similar to that found for ganciclovir
(IC50’s ) 3-9 µM, Table 1). In contrast to ganciclovir,
these compounds were less active against HSV-1 (IC50’s
) 25-65 µM) and much more cytotoxic (IC50’s ) 10-35
µM). Thus the activity against HCMV was much less
specific than that of ganciclovir and the activity against
HSV-1 was most likely a manifestation of cytotoxicity.
The dinitro and diiodo compounds 11 and 15 were less
active against HCMV with the antiviral activity not well
separated from cytotoxicity (Table 1). The difluoro
analog 8 and compounds 12 and 14 were essentially
inactive against the viruses and not cytotoxic.
The activity of ribonucleosides 3 and 4 against HCMV
was somewhat similar to that of the trichloro analog
TCRB, which we have reported previously.18a Specifi-
cally, the dibromo and diiodo analogs were active
against HCMV at low micromolar concentrations, but
both analogs were more cytotoxic than TCRB (Table 2).
The diiodo analog was most unlike TCRB; it was the
most cytotoxic and exhibited some activity against HSV-
1. In contrast, the 5,6-unsubstituted26 analog 19, the
5,6-dimethyl28 analog 20, and the 5,6-difluoro analog 2
were not cytotoxic and were inactive against HCMV and
HSV-1 in the plaque assay and ELISA, respectively. The
5,6-difluoro analog was weakly active against HCMV
in the yield assay (Table 2). The three cyclonucleosides
18a -c were inactive against HSV-1 (IC50’s >100 µM).
They were inactive or weakly active against HCMV
(IC50’s ) >100, 82, and 32 µM respectively for the 5,6-
difluoro, 5,6-dibromo, and 5,6-diiodo analogs) and this
activity closely paralleled their cytotoxicity in uninfected
HFF cells. This would indicate that the activity of the
cyclonucleosides was a manifestation of cytotoxicity.
Taken together, these data confirm our prior
observation18a that the addition of ribose to a polyha-
logenated benzimidazole reduces cytotoxicity and re-
tains or increases activity against HCMV. The order
for activity against HCMV among the 5,6-dihalo ribo-
nucleosides was I = Br = Cl . F > H compared to I >
Br > Cl > F, H for cytotoxicity. Thus, our results show
that the dichloro analog (TCRB) is the most potent and
selective of these compounds and establish that the size

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 813
Sch em e 1
Sch em e 2
Evaporations 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.
and electronegativity of substituents in the 5- and
6-positions are critical for activity against HCMV and
for toxicity to uninfected cells.
4,5-Diflu or o-1,2-p h en ylen ed ia m in e (6). To a solution of
4,5-difluoro-2-nitroaniline (5, 1.106 g, 6.352 mmol, purchased
from Aldrich Chemical Co.) in MeOH (20 mL) were added 50
mL of 1 N HCl and 1.774 g (31.764 mmol) of iron powder. The
reaction mixture was stirred at room temperature for 3 h and
then filtered. The filtrate was neutralized with concentrated
NH4OH to about pH 8. The resulting suspension was filtered
again, and the filter cake was washed thoroughly with cold
MeOH. The filtrate and washings were combined, concen-
trated to about 50 mL, and extracted with CHCl3 (50 mL ×
Exp er im en ta l Section
Gen er a l Meth od s. 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 (HRMS) measurements were ob-
tained 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 (layer thickness 0.2 mm) purchased from Analtech, Inc.
The TLC plates were observed under UV light (254 nm).
3). The CHCl3 extracts were combined, washed with
a
saturated NaCl solution (100 mL × 2), dried (Na2SO4), and
evaporated. The residue was coevaporated with CHCl3 to give
0.78 g (85%) of 6 as a red solid. This material was used in
the subsequent reaction without further purification. A brown
crystalline sample of 6 was obtained by recrystallization from
CCl4: mp 126-129 °C [lit.21 mp 130-131 °C (EtOAc/hexane)];

814 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
(br s, 1, 1-NH), 7.06 (dd, 2, 4-H and 7-H, 3J F-H ) 11.0 Hz, 4J F-H
) 7.5 Hz), 6.30 (br s, 2, 2-NH2). Anal. (C7H5F2N3) C, H, N.
2-Ch lor o-5,6-d iflu or oben zim id a zole (8). To a stirred
mixture of an aqueous CuCl2 solution (40 mL, 60% by weight)
and an aqueous NaNO2 solution (2.08 g, 30 mmol/10 mL) in
H2O (20 mL) was added portionwise compound 7 (1.69 g, 10
mmol) over a period of 5 min. The reaction mixture was
stirred at room temperature for 1 h. A fresh CuCl2 solution
(30 mL, 60% by weight) was added, and stirring was continued
at 100 °C for 10 min (a small amount of MeOH was added to
suppress the formation of foam). The mixture was cooled to
room temperature and extracted with EtOAc (150 mL × 2).
The EtOAc solution was washed with a saturated NaCl
solution (100 mL × 2), dried (Na2SO4), and evaporated. The
residue was chromatographed on a silica column (3 × 15 cm)
using 2% and 3% MeOH/CHCl3 as eluants. Evaporation of
the appropriate fractions gave a brownish solid. This solid
was washed with Et2O and dried to give 0.962 g of 8. The
Et2O washings were evaporated, and the residue was repuri-
fied on a silica column (2 × 10 cm, eluted successively with
1%, 2% MeOH/CHCl3, v/v). Evaporation of the appropriate
fractions and washing of the residue with Et2O gave an
additional 0.152 g of 8. The total yield of 8 was 1.114 g
Ta ble 1. Antiviral Activity and Cytotoxicity of
2-Chloro-5,6-disubstituted Benzimidazoles
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. The
b
plaque assay was used to determine the activity of DHPG against
HSV-1; all other compounds were assayed by ELISA in quadru-
plicate wells. c Visual cytotoxicity was scored on HFF cells at time
of HCMV plaque enumeration. Inhibition of KB cell growth was
determined as described in the text in quadruplicate assays.
(59%): mp ∼274 °C dec; HRMS: m/ z 187.9954 (100, M+
)
d
187.9953); 1H NMR (DMSO-d6) δ 13.5 (br s, 1, 1-NH), 7.62 (t,
2, 4-H and 7-H, J F-H ) 9.0 Hz). Anal. (C7H3ClF2N2) C, H, N.
2-Ch lor o-5,6-d ibr om oben zim id a zole (10). To a mixture
of 2-chlorobenzimidazole22 (9, 0.763 g, 5 mmol) in MeOH (25
mL) was added dropwise a solution of Br2/MeOH (1 mL/10
mL). The reaction mixture was stirred at room temperature
for 5 h. H2O (25 mL) was added, and stirring was continued
at room temperature for 18 h. The resulting suspension was
filtered, and the filter cake was washed thoroughly with
portions of cold H2O until the washings were neutral. The
solid was air-dried and recrystallized from MeOH to give 1.115
g (3 crops, 72%) of 10 as a crystalline compound: mp 228-
229 °C; HRMS (EI) m/ z 307.8348 (46, M+ ) 307.8351); 1H
NMR (DMSO-d6) δ 13.70 (br s, 1, 1-NH), 7.93 (s, 2, 4-H and
7-H). Anal. (C7H3Br2ClN2) C, H, N.
Results are presented as IC50’s. >100 indicates IC50 or IC90 not
reached at the noted (highest) concentration tested. e2,5,6-Trichlo-
robenzimidazole, published previously as compound 4 in ref 18a.
f Average derived from two to four experiments for each parameter
g
studied. Average ( standard deviation from 15 experiments.
h
Average ( standard deviation from 108, 33, and three experi-
ments, respectively.
Ta ble 2. Antiviral Activity and Cytotoxicity of
2-Chloro-5,6-disubstituted-benzimidazole Ribonucleosides
5(6)-Am in o-2-ch lor o-6(5)-n it r ob en zim id a zole (12).
A
mixture of 2-chloro-5,6-dinitrobenzimidazole23 (11, 1.213 g, 5.0
mmol) and iron powder (1.398 g, 25 mmol) in AcOH (50 mL)
was stirred at room temperature for 4 h. The reaction mixture
was diluted with 100 mL of EtOAc and filtered, and the solid
was washed with portions of EtOAc (∼100 mL). The filtrate
and washings were combined and washed with H2O (100 mL
× 3). The H2O layer was extracted with 100 mL of EtOAc.
The EtOAc solutions were combined, evaporated, and coevapo-
rated with toluene (10 mL × 3) and MeOH (10 mL × 2) to
give a brown solid. The brown solid was purified on a silica
column (3 × 25 cm, eluted successively with 2%, 4%, 8%
MeOH/CHCl3, v/v). Evaporation of fractions 30-58 (20 mL
per fraction) and recrystallization from MeOH gave 0.431 g
(two crops, 41%) of 12 as red crystals: mp >250 °C dec;
HRMS: (EI) m/ z 212.0096 (100, M+ ) 212.0101); 1H NMR
(DMSO-d6) δ 13.07 (br s, 1, 1-NH), 8.15 (s, 1, 7-H), 7.06 (s, 2,
5-NH2), 6.91 (s, 1, 4-H). Anal. (C7H5ClN4O2) C, H, N.
a-d
See Table 1 for footnotes a-d. e Average derived from two
or three experiments. f The trichloro analog, published previously
as compound 9 in ref 18a. Averages derived from three to six
experiments for each assay listed. The dimethyl analog 20,
synthesized as reported in ref 28. Compound 19 was synthesized
g
h
i
and reported in ref 26.
HRMS (EI) m/ z 144.0490 (100, M+ ) 144.0499); 1H NMR
(DMSO-d6) δ 6.44 (t, 2, 3-H and 6-H, J F-H ) 10.5 Hz), 4.59 (br
s, 4, 2 NH2). Anal. (C6H6F2N2) C, H, N.
2-Am in o-5,6-d iflu or oben zim id a zole (7). To a stirred
solution of 5 M BrCN/MeCN (4.9 mL, 24.5 mmol) and H2O
(50 mL) was added dropwise a solution of 6 (3.515 g, 24.389
mmol) in MeOH (50 mL) over 20 min. After the addition was
complete, stirring was continued at room temperature for 2
h. The reaction mixture was concentrated to about 50 mL and
then washed with EtOAc (50 mL × 3). The combined EtOAc
solution was extracted with 100 mL of H2O and then discarded.
The combined H2O phase was made basic with a saturated
NaHCO3 solution (precipitation occurred) and was extracted
again with EtOAc (70 mL × 3). The EtOAc solution was dried
(Na2SO4) and evaporated to dryness. The residue was sus-
pended in 50 mL of CHCl3 and filtered. The filter cake was
washed with portions of CHCl3 to give 3.40 g of 7 as a yellowish
solid. The filtrate and washings were evaporated to dryness.
The residue was again suspended in a small amount of CHCl3
and filtered to give an additional 0.355 g of 7. The total yield
of 7 was 3.755 g (91%): mp 152-153 °C; HRMS: (EI) m/ z
2-Ch lor o-5(6)-iod o-6(5)-n itr oben zim id a zole (13). The
procedure for the preparation of 12 was followed to give a
brown solid. This solid was dissolved in a mixture of concen-
trated H2SO4/ice-H2O (14 mL/20 mL), and a solution of
NaNO2/H2O (0.994 g, 13.688 mmol/5 mL) was added dropwise
at 0 °C. After the addition was complete, stirring was
continued at 0 °C for 1 h. The excess NaNO2 was destroyed
by the addition of an aqueous urea solution (0.411 g/3 mL),
and the reaction mixture was treated with an aqueous KI
solution (2.272 g/5 mL) at room temperature for 18 h. The
contents were transferred to a separatory funnel and extracted
with EtOAc (100 mL × 2). The EtOAc solution was washed
with H2O (100 mL), a saturated NaHCO3 solution (100 mL)
and a saturated NaCl solution (100 mL), dried (Na2SO4), and
evaporated. The residue was chromatographed on a silica
column (2.2 × 25 cm, eluted successively with 1%, 2% MeOH/
CHCl3, v/v). Fractions 20-41 (20 mL per fraction) were
collected, washed with an aqueous Na2S2O3 solution (1 g/100
mL), dried (Na2SO4), and evaporated. The residue was
1
169.0455 (100, M+ ) 169.0452); H NMR (DMSO-d6) δ 10.79

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 815
recrystallized from MeOH to give 0.593 g (three crops, 37%)
13C NMR (DMSO-d6) δ 170.05, 169.27, 168.34 (3 COCH3),
of 13 as yellowish crystals: mp ∼213 °C dec; HRMS: (EI) m/ z
148.54, 148.37, 148.19, 145.88, 145.71, 145.54 (C5 and C6,
1
2
322.8968 (100, M+ ) 322.8957); H NMR (DMSO-d6) δ 13.98
1J F-C ) 241 Hz, J F-C ) 16 Hz), 139.68 (C2), 136.71, 136.58
3
3
(br s, 1, 1-NH), 8.24, 8.16 (2 s, 2, 4-H and 7-H). Anal. (C7H3-
ClIN3O2) C, H, N.
(C3a, J F-C ) 11 Hz), 129.45, 129.32 (C7a, J F-C ) 12 Hz),
2 2
106.66, 106.44 (C4, J F-C ) 20 Hz), 101.79, 101.52 (C7, J F-C
) 24 Hz), 86.43 (C1′), 78.16 (C4′), 70.97 (C2′), 70.39 (C3′), 62.69
(C5′), 20.49, 20.09, 19.48 (3 COCH3).
6(5)-Am in o-2-ch lor o-5(6)-iod oben zim id a zole (14).
A
mixture of 13 (1.04 g, 3.215 mmol) and Raney nickel (0.322 g,
wet weight) in 32 mL of EtOH was hydrogenated (50 psi of
H2) at room temperature for 6 h. The reaction mixture was
filtered, and the filtrate was evaporated. The residue was
chromatographed on a silica column (2 × 30 cm, eluted
successively with CHCl3, 1%, 2% MeOH/CHCl3, v/v). Evapora-
tion of fractions 24-37 (20 mL per fraction) gave 0.72 g (76%)
of 14 as a solid. An analytical sample was obtained by
recrystallization from MeCN: mp >200 °C dec; HRMS: (EI)
m/ z 292.9202 (100, M+ ) 292.9217). 1H NMR (DMSO-d6) δ
12.80 (br s, 1, 1-NH), 7.75 (s, 1, 4-H), 6.87 (s, 1, 7-H), 5.03 (s,
2, 6-NH2). Anal. (C7H5ClIN3) C, H, N.
2-Ch lor o-5,6-d ibr om o-1-(2,3,5-tr i-O-a cetyl-â-D-r ibofu r a -
n osyl)ben zim id a zole (16b) a n d 2-Ch lor o-5,6-d ibr om o-1-
(2,3,5-tr i-O-acetyl-r-D-r ibofu r an osyl)ben zim idazole (17b).
To a suspension of 10 (2.483 g, 8 mmol) in dry MeCN (40 mL)
was added 2 mL (8 mmol) of BSA. The reaction mixture was
stirred at 70 °C for 15 min to give a clear solution. This
solution was cooled to room temperature and then treated with
1,2,3,5-tetra-O-acetyl-â-D-ribofuranose (2.8 g, 8.8 mmol) and
TMSOTf (1.855 mL, 9.6 mmol) at room temperature for 30
min. The reaction mixture was diluted with 160 mL of EtOAc.
The EtOAc solution was washed with a saturated NaHCO3
solution (160 mL × 2) and a saturated NaCl solution (160 mL),
dried (Na2SO4), and evaporated. The residue was recrystal-
lized from MeOH to give 3.51 g of 16b as white crystals. The
mother liquor was evaporated, and the residue was chromato-
graphed on a silica column (2 × 10 cm, eluted with CHCl3).
Evaporation of the fractions 10-17 (5 mL per fraction) and
recrystallization from MeOH gave an additional 0.293 g of 16b
as white crystals. The total yield of 16b was 3.803 g (84%):
mp 142-143 °C; HRMS (EI) m/ z 565.9082 (1, M+ ) 565.9091);
1H NMR (DMSO-d6) δ 8.21 (s, 1, 7-H), 8.12 (s, 1, 4-H), 6.25 (d,
1, 1′-H, J 1′-2′ ) 7.0 Hz), 5.55 (t, 1, 2′-H, J 2′-3′ ) 7.0 Hz), 5.43
(m, 1, 3′-H, J 3′-4′ ) 4.5 Hz), 4.52-4.34 (m, 3, 4′-H and 5′-H),
2.15, 2.14, 2.02 (3 s, 9, 3 Ac); 13C NMR (DMSO-d6) δ 169.96,
169.47, 169.17 (3 COCH3), 141.65, 141.31 (C3a and C2), 133.16
(C7a), 123.53 (C4), 118.44, 118.08 (C5 and C6), 116.38 (C7),
86.67 (C1′), 79.59 (C4′), 70.56 (C2′), 68.68 (C3′), 62.59 (C5′),
20.69, 20.29, 20.00 (3 COCH3). Anal. (C18H17Br2ClN2O7) C,
H, N.
2-Ch lor o-5,6-d iiod oben zim id a zole (15). Compound 14
(0.72 g, 2.453 mmol) was dissolved in a mixture of concentrated
H2SO4/ice-H2O (6.6 mL/10 mL), and a solution of NaNO2/H2O
(0.508 g, 7.362 mmol/25 mL) was added dropwise at 0 °C. After
the addition was complete, the reaction mixture was stirred
at room temperature for 1 h. An aqueous KI solution (2.036
g, 12.265 mmol/15 mL) was added, and stirring was continued
at room temperature for 3 h and then at 100 °C for 15 min.
The reaction mixture was cooled to room temperature and
extracted with EtOAc (100 mL). The EtOAc solution was
washed with an aqueous Na2S2O3 solution (3 g/100 mL) and a
saturated NaCl solution (100 mL), dried (Na2SO4), and evapo-
rated. The residue was recrystallized from MeCN to give 0.501
g of 15 as a crystalline compound. The mother liquor was
evaporated, and the residue was chromatographed on a silica
column (2 × 20 cm, eluted successively with CHCl3, 1%,
MeOH/CHCl3, v/v). Evaporation of fractions 13-20 (15 mL
per fraction) gave an additional 0.363 g of 15. The total yield
of 15 was 0.864 g (87%): mp 228-229 °C dec; HRMS (EI) m/ z
Evaporation of fractions 21-33 (5 mL per fraction) gave 0.37
1
403.8064 (100, M+ ) 403.8072); H NMR (DMSO-d6) δ 13.50
g (8%) of 17b as a syrup. HRMS (EI) m/ z 565.9116 (5, M+
)
(br s, 1, 1-NH), 8.11 (s, 2, 4-H and 7-H). Anal. (C7H3ClI2N2)
C, H, N.
565.9091). 1H NMR (DMSO-d6) δ 8.07, 8.06 (2 s, 2, 7-H and
4-H), 6.70 (d, 1, 1′-H, J 1′-2′ ) 4.5 Hz), 5.71 (t, 1, 2′-H, J 2′-3′
)
5.0 Hz), 5.50 (dd, 1, 3′-H, J 3′-4′ ) 6.5 Hz), 4.81 (m, 1, 4′-H,
J 4′-5′ ) 3.5 Hz, J 4′-5′′ ) 5.5 Hz), 4.36 (dd, 1, 5′-H, J 5′-5′′ ) 12.0
Hz), 4.27 (dd, 1, 5′′-H), 2.09, 2.05, 1.57 (3 s, 9, 3 Ac); 13C NMR
(DMSO-d6) δ 170.08, 169.27, 168.33 (3 COCH3), 141.45 (C3a),
141.06 (C2), 133.95 (C7a), 123.22 (C4), 117.86, 117.39 (C5 and
C6), 117.39 (C7), 86.33 (C1′), 78.42 (C4′), 70.95 (C2′), 70.46
(C3′), 62.82 (C5′), 20.55, 20.17, 19.56 (3 COCH3).
2-Ch lor o-5,6-d iflu or o-1-(2,3,5-tr i-O-a cetyl-â-D-r ibofu r a -
n osyl)ben zim id a zole (16a ) a n d 2-ch lor o-5,6-d iflu or o-1-
(2,3,5-tr i-O-acetyl-r-D-r ibofu r an osyl)ben zim idazole (17a).
To a suspension of 8 (0.943 g, 5 mmol) in dry ClCH2CH2Cl (25
mL) was added 1.25 mL (5 mmol) of BSA. The reaction
mixture was stirred at 75 °C for 30 min to give a clear solution.
To this solution were added 1,2,3,5-tetra-O-acetyl-â-D-ribo-
furanose (1.75 g, 5.5 mmol) and TMSOTf (1.07 mL, 5.5 mmol),
and stirring was continued at 75 °C for 30 min. The reaction
mixture was cooled to room temperature, diluted with 100 mL
of CHCl3, and washed with a saturated NaHCO3 solution (100
mL × 2) and a saturated NaCl solution (100 mL). The CHCl3
phase was dried (Na2SO4) and evaporated. The residue was
chromatographed on a silica column (3 × 25 cm, eluted with
CHCl3). Evaporation of the appropriate fractions and recrys-
tallization of the residue from MeOH gave 1.257 g (3 crops,
56%) of 16a as crystalline needles: mp 90-91 °C; HRMS (EI)
m/ z 446.0694 (5, M+ ) 446.0692); 1H NMR (DMSO-d6) δ 7.93,
2-Ch lor o-5,6-d iiod o-1-(2,3,5-t r i-O-a cet yl-â-D-r ib ofu r a -
n osyl)b en zim id a zole (16c) a n d 2-ch lor o-5,6-d iiod o-1-
(2,3,5-tr i-O-a cetyl-r-D-r ibofu r a n osyl)ben zim id a zole (17c).
To a suspension of 15 (0.809 g, 2 mmol) in dry MeCN (20 mL)
was added 1 mL (4 mmol) of BSA. The reaction mixture was
stirred at 80 °C for 15 min to give a clear solution. This
solution was cooled to room temperature and then treated with
1,2,3,5-tetra-O-acetyl-â-D-ribofuranose (0.70 g, 2.2 mmol) and
TMSOTf (0.964 mL, 5 mmol) at room temperature for 1 h. The
reaction mixture was diluted with EtOAc (100 mL). The
EtOAc solution was washed with a 1:1 saturated NaHCO3/
saturated NaCl solution (100 mL × 2), dried (Na2SO4), and
evaporated. The residue was chromatographed on a silica
column (2 × 35 cm, eluted with CHCl3, 0.5% MeOH/CHCl3,
v/v). Evaporation of fractions 11-30 (15 mL per fraction) and
recrystallization from MeOH gave 1.023 g (2 crops, 77%) of
16c as white crystals: mp 123-125 °C; HRMS (EI) m/ z
661.8823 (14, M+ ) 661.8811); 1H NMR (DMSO-d6) δ 8.34 (s,
1, 7-H), 8.25 (s, 1, 4-H), 6.22 (d, 1, 1′-H, J 1′-2′ ) 7.0 Hz), 5.54
(t, 1, 2′-H, J 2′-3′ ) 7.0 Hz), 5.41 (m, 1, 3′-H, J 3′-4′ ) 4.5 Hz),
4.46, 4.36 (2 m, 3, 4′-H and 5′-H), 2.17, 2.14, 2.01 (3 s, 9, 3
Ac). 13C NMR (DMSO-d6) δ 170.05, 169.56, 169.26 (3 COCH3),
142.58 (C3a), 140.73 (C2), 134.10 (C7a), 128.93 (C4), 121.54
(C7), 102.17, 101.51 (C5 and C6), 86.53 (C1′), 79.53 (C4′), 70.54
(C2′), 68.74 (C3′), 62.67 (C5′), 21.01, 20.38, 20.09 (3 COCH3).
Anal. (C18H17ClI2N2O7) C, H, N.
3
4
7.82 (2 dd, 2, 7-H and 4-H, J F-H ) 10.5 Hz, J F-H ) 7.5 Hz),
6.23 (d, 1, 1′-H, J 1′-2′ ) 7.0 Hz), 5.55 (t, 1, 2′-H, J 2′-3′ ) 7.0
Hz), 5.44 (m, 1, 3′-H, J 3′-4′ ) 4.5 Hz), 4.45 (m, 3, 4′-H and 5′-
H), 2.13, 2.10, 2.02 (3 s, 9, 3 Ac); 13C NMR (DMSO-d6) δ 169.95,
169.50, 169.18 (3 COCH3), 148.76, 148.62, 146.10, 145.93 (C5
1
2
and C6, J F-C ) 242 Hz, J F-C ) 15 Hz), 140.16 (C2), 137.01,
3
3
136.88 (C3a, J F-C ) 12 Hz), 128.54, 128.40 (C7a, J F-C ) 12
2
Hz), 107.31, 107.09 (C4, J F-C ) 20 Hz), 100.93, 100.66 (C7,
2J F-C ) 24 Hz), 86.89 (C1′), 79.41 (C4′), 70.40 (C2′), 68.62 (C3′),
62.67 (C5′), 20.43, 20.28, 20.00 (3 COCH3). Anal. (C18H17
-
ClF2N2O7) C, H, N.
Further elution and evaporation of the appropriate fractions
gave 0.456 g (20%) of 17a as a syrup: HRMS (EI) m/ z
446.0680 (12, M+ ) 446.0692); 1H NMR (DMSO-d6) δ 7.75 (m,
2, 7-H and 4-H), 6.69 (d, 1, 1′-H, J 1′-2′ ) 4.0 Hz), 5.69 (t, 1,
2′-H, J 2′-3′ ) 5.0 Hz), 5.49 (dd, 1, 3′-H, J 3′-4′ ) 7.0 Hz), 4.90
(m, 1, 4′-H), 4.37 (dd, 1, 5′-H, J 5′-4′ ) 3.5 Hz, J 5′-5′′ ) 12.0 Hz),
4.26 (dd, 1, 5′′-H, J 5′′-4′ ) 5.5 Hz), 2.09, 2.03, 1.54 (3 s, 9, 3 Ac).
Evaporation of fractions 40-44 (20 mL per fraction) gave
0.108 g (8%) of 17c as a syrup: HRMS (EI) m/ z 661.8798 (21,
1
M+ ) 661.8811); H NMR (DMSO-d6) δ 8.24 (s, 1, 7-H), 8.21

816 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
(s, 1, 4-H), 6.67 (d, 1, 1′-H, J 1′-2′ ) 4.5 Hz), 5.69 (t, 1, 2′-H,
J 2′-3′ ) 5.0 Hz), 5.49 (dd, 1, 3′-H, J 3′-4′ ) 7.0 Hz), 4.74 (m, 1,
MeOH, decolorized with activated carbon, and filtered. The
filtrate was evaporated, and the residue was recrystallized
twice from MeOH/H2O to give 0.132 g (55%) of 18a as white
crystalline needles: mp 110-115 °C; HRMS (EI) m/ z 284.0610
4′-H, J 4′-5′ ) 3.5 Hz, J 4′-5′′ ) 5.5 Hz), 4.35 (dd, 1, 5′-H, J 5′-5′′
)
12.0 Hz), 4.26 (dd, 1, 5′′-H), 2.09, 2.06, 1.58 (3 s, 9, 3 Ac); 13C
NMR (DMSO-d6) δ 170.06, 169.28, 168.30 (3 COCH3), 142.37
(C3a), 140.43 (C2), 134.83 (C7a), 128.60 (C4), 122.70 (C7),
101.26, 100.46 (C5 and C6), 86.23 (C1′), 78.36 (C4′), 70.89 (C2′),
70.45 (C3′), 62.88 (C5′), 20.55, 20.24, 19.58 (3 COCH3).
2-Ch lor o-5,6-d iflu or o-1-(â-D-r ibofu r a n osyl)ben zim id a -
zole (2). Compound 16a (0.894 g, 2 mmol) was treated with
20 mL of NH3/MeOH (saturated at 0 °C) in a pressure bottle
at room temperature for 3 h. The reaction mixture was
evaporated and coevaporated with MeOH to give a solid. This
was recrystallized from MeOH to give 0.573 g (3 crops, 89%)
of 2 as a crystalline compound: mp ∼210 °C dec; HRMS (EI)
m/ z 320.0385 (20, M+ ) 320.0375); 1H NMR (DMSO-d6) δ 8.34
1
(25, M+ ) 284.0609); H NMR (DMSO-d6) δ 7.59 (dd, 1, 7-H,
4
3
3J F-H ) 10.5 Hz, J F-H ) 7.5 Hz), 7.50 (dd, 1, 4-H, J F-H
)
11.5 Hz, 4J F-H ) 7.5 Hz), 6.49 (d, 1, 1′-H, J 1′-2′ ) 5.0 Hz), 5.76
(d, 1, 3′-OH, J 3′-3′OH ) 7.0 Hz), 5.72 (t, 1, 2′-H, J 2′-3′ ) 5.5 Hz),
4.84 (t, 1, 5′-OH, J 5′-5′OH ) 5.5 Hz), 4.08 (m, 1, 3′-H, J 3′-4′
)
9.0 Hz), 3.69 (m, 1, 5′-H, J 5′-4′ ) 1.5 Hz, J 5′-5′′ ) 12.0 Hz), 3.54
(m, 1, 4′-H), 3.49 (m, 1, 5′′-H, J 4′-5′′ ) 5.0 Hz); 13C NMR (DMSO-
d6) δ 164.66 (C2), 147.81, 147.57, 145.12, 144.88 (C5 or C6,
1J F-C ) 244 Hz, 2J F-C ) 22 Hz), 146.71, 146.58, 144.13, 144.00
(C6 or C5, 1J F-C ) 234 Hz, 2J F-C ) 11 Hz), 142.00, 141.87 (C3a,
3
3J F-C ) 11 Hz), 124.84, 124.71 (C7a, J F-C ) 12 Hz), 106.30,
2
2
106.08 (C4, J F-C ) 21 Hz), 98.89, 98.63 (C7, J F-C ) 24 Hz),
91.11 (C2′), 84.64 (C1′), 80.93 (C4′), 69.61 (C3′), 59.50 (C5′).
Anal. (C12H10F2N2O4) C, H, N.
3
4
(dd, 1, 7-H, J F-H ) 11.5 Hz, J F-H ) 7.5 Hz), 7.77 (dd, 1, 4-H,
3J F-H ) 11.0 Hz, J F-H ) 7.5 Hz), 5.88 (d, 1, 1′-H, J 1′-2′ ) 8.0
4
Hz), 5.50 (d, 1, 2′-OH, J 2′-2′OH ) 6.5 Hz), 5.44 (t, 1, 5′-OH,
J 5′-5′OH ) 4.5 Hz), 5.29 (d, 1, 3′-OH, J 3′-3′OH ) 4.5 Hz), 4.40
(m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.14 (m, 1, 3′-H, J 3′-4′ ) 1.5 Hz),
5,6-Dibr om o-1-r-D-r ibofu r a n osylben zim id a zole 2,2′-O-
Cyclon u cleosid e (18b). A solution of 0.37 g (0.65 mmol) of
17b in 15 mL of NH3/MeOH (saturated at 0 °C) was stirred in
a pressure bottle at room temperature for 5 h. Volatile
materials were removed by evaporation, and the resulting solid
was recrystallized from MeOH to give 0.140 g of 18b as white
crystals. The mother liquor was evaporated to dryness and
the residue was triturated with H2O (10 mL × 3) and then
recrystallized from MeOH/Et2O (diffusion) to give an additional
0.084 g of 18b as white crystals. The total yield of 18b was
0.224 g (85%): mp 140-160 °C (melted slowly over a large
4.01 (m, 1, 4′-H), 3.72 (m, 2, 5′-H and 5′′-H, J 5′-4′ ) J 5′′-4′
)
2.5 Hz, J 5′-5′′ ) 12.0 Hz); 13C NMR (DMSO-d6) δ 148.53, 148.42,
148.37, 148.26, 145.88, 145.76, 145.72, 145.60 (C5 and C6,
2
1J F-C ) 241 Hz, J F-C ) 16 Hz), 140.84 (C2), 137.16, 137.04
3
3
(C3a, J F-C ) 11 Hz), 128.55, 128.42 (C7a, J F-C ) 12 Hz),
2
2
106.77, 106.54 (C4, J F-C ) 20 Hz), 102.12, 101.84 (C7, J F-C
) 25 Hz), 89.10 (C1′), 86.39 (C4′), 71.55 (C2′), 69.81 (C3′), 61.12
(C5′). Anal. (C12H11ClF2N2O4) C, H, N.
range of temperature); HRMS (EI) m/ z 403.8995 (26, M+
)
2-Ch lor o-5,6-d ib r om o-1-â-D-r ib ofu r a n osylb en zim id a -
zole (3). To a solution of Na2CO3 (0.212 g, 2 mmol) in H2O (4
mL) were added successively 18 mL of EtOH, 18 mL of MeOH,
and 1.137 g (2 mmol) of 16b. The reaction mixture was stirred
at room temperature for 2 h. AcOH (0.24 mL) was added, and
stirring was continued at room temperature for 15 min.
Volatile materials were removed by evaporation. The residue
was triturated with H2O (40 mL × 2) and recrystallized from
MeOH to give 0.719 g (2 crops, 76% based on C12H11Br2-
ClN2O4‚MeOH) of 3 as white crystals: mp 128-137 °C;
1
403.9007); H NMR (DMSO-d6) δ 7.88 (s, 1, 7-H), 7.80 (s, 1,
4-H), 6.52 (d, 1, 1′-H, J 1′-2′ ) 5.0 Hz), 5.78 (d, 1, 3′-OH, J 3′-3′OH
) 7.0 Hz), 5.74 (t, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.84 (t, 1, 5′-OH,
J 5′-5′OH ) 5.0 Hz), 4.08 (m, 1, 3′-H, J 3′-4′ ) 9.0 Hz), 3.69 (m, 1,
5′-H, J 4′-5′ ) 0.5 Hz, J 5′-5′′ ) 11.5 Hz), 3.49 (m, 2, 4′-H and
5′′-H, J 4′-5′′ ) 5.0 Hz); 13H NMR (DMSO-d6) δ 164.87 (C2),
147.18 (C3a), 129.70 (C7a), 122.19 (C4), 116.15, 114.34 (C5
and C6), 114.49 (C7), 91.41 (C2′), 84.64 (C1′), 80.91 (C4′), 69.60
(C3′), 59.50 (C5′). Anal. (C12H10Br2N2O4) C, H, N.
1
HRMS: (FAB) m/ z 440.8862 (3, MH+ ) 440.8852); H NMR
5,6-Diiod o-1-r-D-r ib ofu r a n osylb en zim id a zole 2,2′-O-
Cyclon u cleosid e (18c). A solution of 0.10 g (0.151 mmol) of
17c in 10 mL of NH3/MeOH (saturated at 0 °C) was stirred in
a pressure bottle at room temperature for 5 h. Volatile
materials were removed by evaporation and coevaporation
with MeOH (3×). The resulting solid was recrystallized from
MeOH to give 0.054 g (2 crops, 72%) of 18c as white crystals:
mp ∼155 °C (melted over a large range of temperature); HRMS
(EI) m/ z 499.8719 (100, M+ ) 499.8728); 1H NMR (DMSO-d6)
δ 8.01, 7.97 (2 s, 2, 7-H and 4-H), 6.51 (d, 1, 1′-H, J 1′-2′ ) 5.0
Hz), 5.73 (d, 1, 3′-OH, J 3′-3′OH ) 7.0 Hz), 5.725 (t, 1, 2′-H, J 2′-3′
) 5.0 Hz), 4.79 (t, 1, 5′-OH, J 5′-5′OH ) 5.0 Hz), 4.06 (m, 1, 3′-H,
J 3′-4′ ) 9.0 Hz), 3.69 (m, 1, 5′-H, J 4′-5′ ) 1.0 Hz, J 5′-5′′ ) 11.0
Hz), 3.50 (m, 2, 4′-H and 5′′-H, J 4′-5′′ ) 5.0 Hz); 13H NMR
(DMSO-d6) δ 164.30 (C2), 148.16 (C3a), 130.55 (C7a), 127.73
(C4), 119.63 (C7), 98.92, 96.97 (C5 and C6), 91.26 (C2′), 84.53
(C1′), 80.83 (C4′), 69.61 (C3′), 59.50 (C5′). Anal. (C12H10I2N2O4)
C, H, N.
2-Ch lor o-5,6-d ib r om o-1-â-D-r ib ofu r a n osylb en zim id a -
zole (3) fr om 19. To a stirred suspension of 2-chloro-1-â-D-
ribofuranosylbenzimidazole23 (19, 0.57 g, 2 mmol) in H2O (50
mL) was added dropwise 50 mL of Br2/H2O (saturated at 20
°C) over a period of 30 min. After the addition was complete,
stirring was continued at room temperature for 3 h. The
reaction mixture was allowed to stand in an ice-H2O bath for
30 min and then filtered. The filter cake was washed with
portions of cold H2O until the washings were neutral. The
resulting white solid was air-dried and recrystallized from
MeOH to give 0.757 g (3 crops, 80% as C12H11Br2ClN2O4‚-
MeOH) of 3 as white crystals. This material was identical to
the product obtained from the deprotection of 16b.
(DMSO-d6) δ 8.68 (s, 1, 7-H), 8.09 (s, 1, 4-H), 5.88 (d, 1, 1′-H,
J 1′-2′ ) 8.0 Hz), 5.51 (d, 1, 2′-OH, J 2′-2′OH ) 6.5 Hz), 5.40 (t, 1,
5′-OH, J 5′-5′OH ) 4.5 Hz), 5.30 (d, 1, 3′-OH, J 3′-3′OH ) 4.5 Hz),
4.42 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.13 (m, 1, 3′-H, J 3′-4′ ) 2.0
Hz), 4.01 (m, 1, 4′-H), 3.69 (m, 2, 5′-H and 5′′-H, J 5′-4′ ) J 5′′-4′
) 2.5 Hz, J 5′-5′′ ) 12.0 Hz); 13C NMR (DMSO-d6) δ 142.08 (C2),
141.84 (C3a), 133.14 (C7a), 123.14 (C4), 117.94 [C7 and C5
(or C6)], 117.58 {C6 (or C5)}, 89.14 (C1′), 86.48 (C4′), 71.68
(C2′), 69.83 (C3′), 61.09 (C5′). Anal. (C12H11Br2ClN2O4) C, H,
N.
2-Ch lor o-5,6-d iiod o-1-â-D-r ib ofu r a n osylb e n zim id a -
zole (4). To a solution of Na2CO3 (0.053 g, 0.5 mmol) in H2O
(1 mL) were added successively 4.5 mL of EtOH, 4.5 mL of
MeOH, and 0.331 g (0.5 mmol) of 16c. 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 was triturated with H2O (10 mL × 2) and recrystallized
from MeOH/H2O to give 0.237 g (3 crops, 88%) of 4 as white
crystals; mp 147-149 °C; HRMS (CI) m/ z 536.8571 (5, MH+
) 536.8573); 1H NMR (DMSO-d6) δ 8.78 (s, 1, 7-H), 8.23 (s, 1,
4-H), 5.85 (d, 1, 1′-H, J 1′-2′ ) 8.0 Hz), 5.49 (d, 1, 2′-OH, J 2′-2′OH
) 6.5 Hz), 5.33 (t, 1, 5′-OH, J 5′-5′OH ) 5.0 Hz), 5.28 (d, 1, 3′-
OH, J 3′-3′OH ) 4.5 Hz), 4.41 (m, 1, 2′-H, J 2′-3′ ) 5.5 Hz), 4.12
(m, 1, 3′-H, J 3′-4′ ) 2.0 Hz), 3.99 (m, 1, 4′-H), 3.68 (m, 2, 5′-H
and 5′′-H, J 5′-4′ ) J 5′′-4′ ) 3.0 Hz, J 5′-5′′ ) 12.0 Hz); 13C NMR
(DMSO-d6) δ 142.79 (C3a), 141.52 (C2), 133.04 (C7a), 128.58
(C4), 123.19 (C7), 101.34, 100.87 (C5 and C6), 89.02 (C1′), 86.42
(C4′), 71.53 (C2′), 69.87 (C3′), 61.13 (C5′). Anal. (C12H11
-
ClI2N2O4) C, H, N.
5,6-Diflu or o-1-r-D-r ibofu r a n osylben zim id a zole 2,2′-O-
Cyclon u cleosid e (18a ). A solution of compound 17a (0.376
g, 0.842 mmol) in 15 mL of NH3/MeOH (saturated at 0 °C)
was stirred in a pressure bottle at room temperature for 20 h.
Volatile materials were removed by evaporation and coevapo-
ration with MeOH. The residue was dissolved in 15 mL of
Cell Cu ltu r e P r oced u r es. The routine growth and pas-
sage of KB, BSC-1, and 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 10% calf serum or 10% fetal bovine serum (HFF cells).
The sodium bicarbonate concentration was varied to meet the

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 817
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-
fied isolate Po, of HCMV was kindly provided by Dr. Mark
Stinski, University of Iowa. The KOS strain of HSV-1 was
used in most experiments and was provided by Dr. Sandra K.
Weller, University of Connecticut. Stock HCMV was prepared
by infecting HFF cells at a multiplicity of infection (moi) of
<0.01 plaque-forming units (pfu) per cell as detailed previ-
ously.29 High titer HSV-1 stocks were prepared by infecting
KB cells at an moi of <0.1 also as detailed previously.29 Virus
titers were determined using monolayer cultures of HFF cells
for HCMV and monolayer cultures of BSC-1 cells for HSV-1
as described earlier.30 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 11 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 dilution which gave 5-20
plaques per well. Virus titers were calculated according to the
following formula: Titer (pfu/mL) ) number of plaques × 5 ×
3n; 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
HCMV per cm2 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
concentrations. After incubation at 37 °C, for 7 days, cell
sheets were fixed and stained with crystal violet and micro-
scopic plaques enumerated as described above. Drug effects
were calculated as a percentage of reduction in number of
plaques in the presence of each drug concentration compared
to the number observed in the absence of drug.
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 affected by the virus used in plaque assays.29 (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.32 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
in a CO2 incubator, rinsed, fixed with 95% ethanol, and stained
with 0.1% crystal violet. Acidified ethanol was added and
plates read at 570 nm in a spectrophotometer designed to read
96-well ELISA assay plates.
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
)
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.
Ack n ow led gm en t. This research work was sup-
ported by federal funds from the Department of Health
and Human Services research contracts NOl-AI42554
and NOl-AI7264, Research Grant UOl-AI31718 from the
National Institute of Allergy and Infectious Diseases,
and Research Agreement DRDA-942921 with Glaxo-
Wellcome. The authors thank members of Dr. Drach’s
research group, especially Roger G. Ptak and Drs.
Edward D. Kreske, Mary S. Ludwig, and Allison C.
Westerman, for the antiviral evaluations. We also
appreciate the contributions of J . Hinkley for the
preparation of chemical intermediates and Ms. Marina
Savic for preparation of the manuscript.
HCMV Yield Assa y. HFF cells were planted as described
above in 96-well cluster dishes and incubated overnight, the
medium was removed, and the cultures were inoculated with
HCMV at a moi of 0.5 to 1 pfu per cell as reported elsewhere.30
After virus adsorption, inoculum was replaced with 0.2 mL of
fresh medium containing test compounds. The first row of 12
wells was left undisturbed and served as virus controls. Each
well in the second row received an additional 0.1 mL of
medium with test compound at three times the desired final
concentration. The contents of the 12 wells were mixed by
repeated pipetting and then serially diluted 1:3 along the
remaining wells. In this manner, six compounds could be
tested in duplicate on a single plate with concentrations from
100 to 0.14 µM. Plates were incubated at 37 °C for 7 days
and subjected to one cycle of freezing and thawing; 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 11 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 calculated as described above.
HSV-1 ELISA. An ELISA was employed31 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
as substrate. The reaction was stopped with H2SO4 and
absorbence was read at 450 and 570 nm. Drug effects were
calculated as a percentage of the reduction in absorbence in
the presence of each drug concentration compared to absor-
bence obtained with virus in the absence of drug.
Refer en ces
(1) Zou, R.; Kreske, E. D.; Drach, J . C.; Townsend, L. B. Benzimi-
dazole Ribonucleosides: Design, Synthesis, and Evaluation of
2-Chloro-5,6-diiodo-1-(â-D-ribofuranosyl) benzimidazole, an ana-
log of TCRB. A portion of this work was presented at the Fifth
International Conference on Antiviral Research, Vancouver,
B.C., Canada, March 8, 1992.
(2) J acobson, M. A.; Mills, J . Serious Cytomegalovirus Disease in
the Acquired Immunodeficiency Syndrome (AIDS), Clinical
Findings, Diagnosis, and Treatment. Ann. Intern. Med. 1988,
108, 585-594.
(3) Alford, C. A.; Britt, W. J . Cytomegalovirus. In The Human
Herpesviruses; Roizman, B., Whitley, R. J ., Lopez, C., Eds.;
Raven Press: New York, 1993; pp 227-255.
(4) Meyers, J . D.; Flournoy, N.; Thomas, E. D. Risk Factors for
Cytomegalovirus Infection after Human Marrow Transplanta-
tion. J . Infect. Dis. 1986, 153, 478-488.
(5) Glenn, J . Cytomegalovirus Infections Following Renal Trans-
plantation. Rev. Infect. Dis. 1981, 3, 1151-1178.
(6) Rytel, M. W.; Kauffman, H. M. Clinical Efficacy of Adenine
Arabinoside in Therapy of Cytomegalovirus Infections in Renal
Allograft Recipients. J . Infect. Dis. 1976, 133, 202-205.
(7) Meyers, J . D.; McGuffin, R. W.; Neiman, P. E.; Singer, J . W.;
Thomas, E. D. Toxicity and Efficacy of Human Leukocyte
Interferon for Treatment of Cytomegalovirus Pneumonia after
Marrow Transplantation. J . Infect. Dis. 1980, 141, 555-562.
(8) Meyers, J . D.; McGuffin, R. W.; Bryson, Y. J .; Cantell, K.;
Thomas, E. D. Treatment of Cytomegalovirus Pneumonia after
Marrow Transplant with Combined Vidarabine and Human
Leukocyte Interferon. J . Infect. Dis. 1982, 146, 80-84.
(9) Wade, J . C.; McGuffin, R. W.; Springmeyer, S. C.; Newton, B.;
Singer, J . W.; Meyers, J . D. Treatment of Cytomegaloviral
Pneumonia with High-Dose Acyclovir and Human Leukocyte
Interferon. J . Infect. Dis. 1983, 148, 557-562.
(10) Faulds, D.; Heel, R. C. Ganciclovir. A Review of its Antiviral
Activity, Pharmacokinetic Properties and Therapeutic Efficacy
in Cytomegalovirus Infections. Drugs 1990, 39, 597-638.
(11) Chrisp, P.; Clissold, S. P. Foscarnet. A Review of its Antiviral
Activity, Pharmacokinetic Properties and Therapeutic use in
Immunocompromised Patients with Cytomegalovirus Retinitis.
Drugs 1991, 41, 104-129.

818 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Notes
(12) (a) Kirsch, L. S.; Arevalo, J . F.; De Clercq, E.; Chavez, de la Paz.
E.; Munguia, D.; Garcia, R.; Freeman, W. R. Phase I/II study of
intravitreal cidofovir for the treatment of cytomegalovirus
retinitis in patients with the acquired immunodeficiency syn-
drome. Am. J . Ophthalmol. 1995, 119, 466-476. (b) Polis, M.
A.; Spooner, K. M.; Baird, B. F.; Manischewitz, J . F.; J affe, H.
S.; Fisher, P. E.; Falloon, J .; Davey, R. T., J r.; Kovacs, J . A.;
Walker, R. E.; et al. Anticytomegaloviral activity and safety of
cidofovir in patients with human immunodeficiency virus infec-
tion and cytomegalovirus viruria. Antimicrob. Agents Chemother.
1995, 39, 882-886.
(13) Morris, D. J . Antiviral Chemotherapy for Cytomegalovirus
Disease. J . Antimicrob. Chemother. 1988, 21, 519-522.
(14) Field, A. K.; Biron, K. K. “The End of Innocence” Revisited:
Resistance of Herpesviruses to Antiviral Drugs. Clin. Microbiol.
Rev. 1994, 7, 1-13.
(15) Townsend, L. B.; Revankar, G. R. Benzimidazole Nucleosides,
Nucleotides, and Related Derivatives. Chem. Rev. 1970, 70, 389-
438.
(21) Uchida, M.; Morita, S.; Chihiro, M.; Kanbe, T.; Yamasaki, K.;
Yabuuchi, Y.; Nakagawa, K. Studies on Proton Pump Inhibitors.
I. Synthesis of 8-[(2-Benzimidazolyl)sulfinyl]-5,6,7,8-tetrahyd-
roquinolines and Related Compounds. Chem. Pharm. Bull. 1989,
37, 1517-1523.
(22) Harrison, D.; Ralph, J . T.; Smith, A. B. C. Some 1- and
2-Halogenobenzimidazoles. J . Chem. Soc. 1963, 2930-2937.
(23) Chernova, E. Y.; Mokrushina, G. A.; Chupakhin, O. N.; Ko-
tovskaya, S. K.; Il’enko, V. I.; Andreeva, O. T.; Boreko, E. I.;
Vladyko, G. V.; Korobchenko, L. V.; et al. Synthesis and
Biological Activity of 5,6-Dinitro Derivatives of Benzimidazole.
Khim.-Farm. Zh. 1991, 25, 50-52; Chem. Abstr. 1991, 115,
29203x.
(24) Baker, B. R.; J oseph, J . P.; Schaub, R. E.; Williams, J . H.
Puromycin Synthetic Studies. V. 6-Dimethylamino-9-(2′-ac-
etamino-â-D-glucopyranosyl)purine. J . Org. Chem. 1954, 19,
1786-1792.
(16) Tamm, I; Folkers, K.; Shunk, C. H.; Horsfall, F. L. Inhibition of
Influenza Virus Multiplication by N-Glycosides of Benzimida-
zoles. J . Exp. Med. 1954, 99, 227-250.
(25) Townsend, L. B. Nuclear Magnetic Resonance Spectroscopy in
the Study of Nucleic Acid Components and Certain Related
Derivatives. In Synthetic Procedures in Nucleic Acid Chemistry
Vol. 2, Physical and Physicochemical Aids in Characterization
and in Determination of Structure; Zorbach, W. W., Tipson, R.
S., Eds.; Wiley-Interscience: New York, 1973; pp 267-398.
(26) Revankar, G. R.; Townsend, L. B. The Synthesis of 2-Chloro-1-
(â-D-ribofuranosyl)-benzimidazole and Certain Related Deriva-
tives. J . Heterocycl. Chem. 1968, 5, 477-483.
(27) Kazimierczuk, Z.; Dudycz, L.; Stolarski, R.; Shugar, D. Prepara-
tion of 1-R-D-Arabinofuranosylbenzimidazole and its 5,6-Dichloro
Derivative, and the Direct Bromination of Benzimidazole Nu-
cleosides. Z. Naturforsch. 1980, 35c, 30-35.
(28) Revankar, G. R.; Townsend, L. B. The Synthesis of 2-Chloro-1-
(â-D-ribofuranosyl)-5,6-dimethylbenzimidazole and Certain Re-
lated Derivatives. J . Heterocycl. Chem. 1968, 5, 615-620.
(29) Turk, S. R.; Shipman, C., J r.; Nassiri, M. R.; Genzingler, G.;
Krawczyk, S. H.; Townsend, L. B.; Drach, J . C. Pyrrolo[2,3-d]-
pyrimidine Nucleosides as Inhibitors of Human Cytomegalovi-
rus, Antimicrob. Agents Chemother. 1987, 31, 544-550.
(30) Prichard, M. N.; Turk, S. R.; Coleman, L. A.; Engelhardt, S. L.;
Shipman, C., J r.; Drach, J . C. A Microtiter Virus Yield Reduction
Assay for The Evaluation of Antiviral Compounds Against
Human Cytomegalovirus and Herpes Simplex Virus. J . Virol.
Methods 1990, 28, 101-106.
(17) Tamm, I.; Sehgal, P. B. Halobenzimidazole Ribosides and RNA
Synthesis of Cells and Viruses. Adv. Virus Res. 1978, 22, 187-
258.
(18) (a) Townsend, L. B.; Devivar, R. V.; Turk, S. R.; Nassiri, M. R.;
Drach, J . C. Design, Synthesis and Antiviral Activity of Certain
2,5,6-trihalo-1-(â-D-ribofuranosyl)benzimidazoles. J . Med. Chem.
1995, 38, 4098-4105. (b) Townsend, L. B. Benzimidazole Ribo-
nucleosides: Design, Synthesis, and Evaluation of 2,5,6-Trichloro-
1-(â-D-ribofuranosyl) benzimidazole (TCRB), 2-Bromo-5,6-di-
chloro-1-(â-D-ribofuranosyl) benzimidazole (BDCRB), and Some
Structurally Related Analogs as Agents for Human Cytomega-
lovirus Infections. Fifth International Conference on Antiviral
Research, Vancouver, B.C., Canada, 8-13 March, 1992. (c)
Drach J . C.; Townsend, L. B.; Nassiri, M. R.; Turk, S. R.;
Coleman, L. A.; Devivar, R. V.; Genzlinger, G; Kreske, E. D.;
Renau, T. E.; Westerman, A. C.; Shipman, C. J r.; Biron, K. K.;
Dornsife, R.; Kern, E. R. Benzimidazole Ribonucleosides: A New
Class of Antivirals with Potent and Selective Activity Against
Human Cytomegalovirus. Fifth International Conference on
Antiviral Research, Vancouver, B.C., Canada, 8-13 March,
1992.
(19) Nassiri, M. R.;. Emerson, S. G.; Townsend, L. B.; Drach, J . C.;
Taichman, R. S. Comparison of benzimidazole nucleosides and
ganciclovir on the in vitro proliferation and colony formation of
human bone marrow progenitor cells. Br. J . Haematol. 1996,
93, 273-279.
(20) (a) Underwood, M. R.; Biron, K. K.; Hemphill, M. L.; Miller, T.
J .; Stanat, S. C.; Dornsife, R. E.; Drach, J . C.; Townsend, L. B.;
Edwards, C. A.; Harvey, R. J . High Molecular Weight HCMV
DNA Does Not Properly Mature in the Presence of 2-Bromo-
5,6-dichloro-1-â-D-ribofuranosylbenzimidazole (BDCRB). Herp-
esvirus Workshop, Pittsburgh, PA, J uly 1993. (b) Underwood,
M. R.; Stanat, S. C.; Drach, J . C.;Harvey R. J .; Biron, K. K.
Inhibition of HCMV DNA Processing by a New Class of Anti-
HCMV Compounds is Mediated Through the UL89 Gene Prod-
uct. Herpesvirus Workshop, Vancouver, B.C., August 1994.
(31) Prichard, M. N.; Shipman, C., J r. A Three-Dimensional Model
To Analyze Drug-Drug Interactions. Antiviral Res. 1990, 14,
181-206.
(32) Prichard, M. N.; Prichard, L. E.; Baguley, W. A.; Nassiri, M. R.;
Shipman, C., J r. Three-Dimensional Analysis of the Synergistic
Cytotoxicity of Ganciclovir and Zidovudine. Antimicrob. Agents
Chemother. 1991, 35, 1060-1065.
J M960462G