Synthesis and antiviral evaluation of polyhalogenated imidazole nucleosides: Dimensional analogues of 2,5,6-trichloro-1-(β-D-ribofuranosyl) benzimidazole

Article abstract of DOI:10.1021/jm040016q

A series of polyhalogenated imidazole nucleosides were designed and synthesized as ring-contracted analogues of 2,5,6-trichloro-1-(β-D- ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analogue (BDCRB) in an effort to explore the spatial limitation of t

Full text of DOI:10.1021/jm040016q

J. Med. Chem. 2004, 47, 5743-5752
5743
Synthesis and Antiviral Evaluation of Polyhalogenated Imidazole Nucleosides:
Dimensional Analogues of 2,5,6-Trichloro-1-(â-D-ribofuranosyl)benzimidazole
Tun-Cheng Chien,† Sunita S. Saluja,‡,§ John C. Drach,§,|| and Leroy B. Townsend*,†,§
Department of Chemistry, College of Literature, Science and Arts, Department of Medicinal Chemistry, College of Pharmacy,
and Department of Biologic and Materials Sciences, School of Dentistry, The University of Michigan,
Ann Arbor, Michigan 48109-1065
Received January 23, 2004
A series of polyhalogenated imidazole nucleosides were designed and synthesized as ring-
contracted analogues of 2,5,6-trichloro-1-(â-D-ribofuranosyl)benzimidazole (TCRB) and its
2-bromo analogue (BDCRB) in an effort to explore the spatial limitation of the active pocket(s)
in the target protein(s). 2,4,5-Trichloro-, 2-bromo-4,5-dichloro-, and 2,4,5-tribromoimidazole
nucleosides were prepared by a condensation of the preformed heterocycles with the appropriate
sugar precursors. The ribofuranosyl and xylofuranosyl analogues were prepared by a direct
glycosylation using the Vorbruggen’s silylation method and provided exclusively the â-anomers.
The arabinofuranosyl analogues were prepared by the sodium salt method to give both the R-
and â-anomers. The absolute configurations were established by 1H NMR spectroscopy.
Alkylation of the polyhalogenated imidazoles with the appropriate bromomethyl ethers gave
the acyclic acyclovir and ganciclovir analogues. In general, the parent polyhalogenated
imidazoles showed some activity against human cytomegalovirus (HCMV) (IC50 ∼ 35 µM).
However, with the exception of two tribromo analogues (7c, 13c-â), most of their nucleoside
derivatives were inactive against both HCMV and herpes simplex virus type-1 (HSV-1) and
were not cytotoxic. The results suggest that the ring-contracted nucleoside analogues of TCRB
and BDCRB interacted weakly or not at all with viral and cellular targets.
Introduction
Human cytomegalovirus (HCMV) is a significant
pathogen for immunocompromised individuals such as
bone marrow and organ transplant as well as AIDS
patients.1,2 Current FDA approved drugs including
ganciclovir,3 valganciclovir (a prodrug of ganciclovir),4
foscarnet,5 cidofovir,6 and the antisense oligonucleotide
fomivirsen7 have been used clinically for the treatment
of HCMV patients. Although they are effective, several
disadvantages exist. These drugs have poor oral bio-
Figure 1. Benzimidazole nucleosides (1b-c), tricyclic nucleo-
availability (except the prodrug valganciclovir) and
produce certain toxicities.8,9 Drug-resistant strains of
the virus have been identified,10 and the mechanism of
action for the first four drugs is similar in that they
target viral DNA synthesis. These deficiencies have
limited the clinical uses of these drugs and provide a
strong rationale for the development of new drugs
effective against HCMV infections.
2,5,6-Trichloro-1-(â-D-ribofuranosyl)benzimidazole (1a,
TCRB) and 2-bromo-5,6-dichloro-1-(â-D-ribofuranosyl)-
benzimidazole (1b, BDCRB) were previously synthe-
sized in our laboratory and both compounds are active
against HCMV at noncytotoxic concentrations.11 TCRB
and BDCRB possess a unique mechanism of action that
involves inhibition of viral DNA processing, but not viral
DNA synthesis.12 In an effort to investigate the mech-
anism of action and interaction with their putative
sides (2a-c), and imidazole nucleosides synthesized and
evaluated as potential antiviral agents.
protein target(s), as well as optimizing their antiviral
activities vs cytotoxicities, intensive structure-activity
relationship (SAR) studies have been conducted in our
research group.13 In an effort to explore the spatial
requirements of the binding site(s) of the putative target
protein(s) to the heterocyclic aglycons, we have synthe-
sized and evaluated a series of polyhalogenated tricyclic
nucleosides (2a-c). These compounds were designed as
dimensional extended analogues14-17 of the polyhalo-
genated benzimidazole nucleosides (TCRB and BDCRB)
to probe the possible binding sites.These studies re-
vealed that the extended analogues retained some
antiviral activity but also were somewhat cytotoxic. To
continue our SAR studies involving dimensional ana-
logues, a series of polyhalogenated imidazole nucleosides
have been designed as ring-contracted analogues of
TCRB and BDCRB. 2,4,5-Trichloro-, 2-bromo-4,5-dichlo-
ro-, and 2,4,5-tribromoimidazoles (3a-c)18 were selected
as the polyhalogenated imidazole precursors for this
study. We now report the synthesis and the antiviral
* Corresponding author. Phone: (734) 764-7547. Fax: (734) 763-
5633. E-mail: ltownsen@umich.edu.
† Department of Chemistry, College of Literature, Science and Arts.
‡ Current address: Allied Signal, Morristown, NJ 07962.
§ Department of Medicinal Chemistry, College of Pharmacy.
|| Department of Biologic and Materials Sciences, School of Dentistry.
10.1021/jm040016q CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/13/2004

5744 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Chien et al.
Scheme 1
evaluation of a series of polyhalogenated imidazole
nucleosides with different moieties at the N-1 position.
reoisomers, which were separated by flash column
chromatography. 2-Bromo-4,5-dichloro-1-(2,3,5-tri-O-
benzyl-D-arabinofuranosyl)imidazole (12b) was obtained
in about a 1:1 ratio of the R- and â-isomers (12b-R: 35%;
12b-â: 35%) as determined by subsequent NMR studies.
However, the â-isomer of 2,4,5-tribromo-1-(2,3,5-tri-O-
benzyl-D-arabinofuranosyl)imidazole was found to be
predominant (12c-R: 5%; 12c-â: 81%). Removal of the
benzyl groups was accomplished with boron trichloride
at -78 °C in methylene chloride instead of the usual
hydrogenation methods to avoid a removal of halogen
substituents on the heterocycles.25 Debenzylation of
12b-c afforded the corresponding products 13b-c
(Scheme 2).
Results and Discussion
Chemistry. The most straightforward approach for
the synthesis of these polyhalogenated imidazole nu-
cleosides appeared to be a direct glycosylation or alky-
lation of the preformed heterocycles with the appropri-
ate protected sugars. The polyhalogenated imidazole
precursors, 2,4,5-trichloro-, 2-bromo-4,5-dichloro-, and
2,4,5-tribromoimidazoles (3a-c), were prepared from
commercially available imidazole by the literature
procedures.18 Compounds 3a-c were coupled with
1,2,3,5-tetra-O-acetyl-â-D-ribofuranose (4b) or 1-O-acetyl-
2,3,5-tri-O-benzoyl-â-D-ribofuranose (4a) under modified
Vorbruggen’s conditions.19,20 This gave exclusively the
â-anomers of the sugar-protected ribonucleoside ana-
logues 5a-c and 6b-c in good yields. Similarly, glyco-
sylation of compounds 3a-c with 1,2-di-O-acetyl-3,5-
di-O-(4-nitrobenzoyl)-1-â-D-xylofuranose21 (8) under
Vobruggen’s conditions afforded the protected xylosyl
nucleoside analogues 9a-c. Deprotection of the nucleo-
sides 5a-c, 6b-c, and 9a-c with methanolic ammonia
or aqueous sodium carbonate gave the desired ribosyl
and xylosyl analogues (7a-c, 10a-c) (Scheme 1).
Preparation of the â-D-arabinofuranosyl derivatives
13b-c was accomplished by a condensation of the
halogenated imidazoles (3b-c) with the appropriate
sugar derivatives by the sodium salt method.22 The
sodium salts of 3b-c were obtained in situ by adding
sodium hydride into the acetonitrile solution of the
imidazoles 3b-c, and these sodium salts were then
condensed with freshly prepared 2,3,5-tri-O-benzyl-R-
D-arabinofuranosyl chloride23,24 (11). These glycosylation
reactions gave a mixture of R- and â-anomeric diaste-
The anomeric configuration of the arabinonucleosides
13b-c was established by
1H, proton-decoupling, and
1-D NOE differential NMR spectroscopy data. A doublet
at δ 6.06 (J ) 6.3 Hz) was assigned as the anomeric
hydrogen (1′-H) of 13b-â. Since the 4′-H and 5′-H
protons of 13b-â appear as a multiplet at δ 3.71-3.63,
it is difficult to identify individual chemical shifts and
study the NOE between 1′-H and 4′-H. A proton-
decoupling experiment with irradiation at δ 6.06 only
affected the chemical shift at δ 4.18, which was then
assigned as 2′-H. Subsequent decoupling experiments
with irradiation at δ 4.18 and 5.69 (2′-OH) confirmed
the previous assignment and the peak at δ 3.96 was
then assigned as 3′-H. The 1-D NOE irradiation of 13b-â
at the 1′-H (δ 6.06) shows a significant NOE enhance-
ment at the 2′-H but not the 3′-H. Therefore, on the
basis of the above studies, the absolute configuration
at the anomeric position of 13b-â was assigned as â.
The R-anomer (13b-R) exhibits a peak for the anomeric
proton at δ 5.65 (J ) 7.5 Hz). The proton-decoupling
and D2O exchange experiments suggested the following

Polyhalogenated Imidazole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5745
Scheme 2
Scheme 3
assignments: 2′-H at δ 4.51, 2′-OH at δ 5.85, 3′-OH at
δ 5.66, 3′-H at δ 4.07, and 4′-H at δ 4.02. The 1-D NOE
study of 13b-R revealed that on irradiation of the 1′-H
only NOE enhancement of the 3′-H was observed, which
supports the assignment of the R-configuration to this
compound. The anomeric coupling constant of 13b-â is
6.3 Hz, while 13b-R is 7.5 Hz. A similar trend (Jâ < JR)
was also observed for compounds 12b,c. Furthermore,
the chemical shifts of the â-anomeric protons are about
0.4 ppm more downfield than the chemical shifts of the
R-anomeric protons. This result is consistent with our
previous observation that the peaks assigned to the
anomeric protons of the H1′-H2′-cis nucleosides (i.e.
â-arabinonucleosides) appear at lower field than the
peaks for the anomeric protons of the corresponding
H1′-H2′-trans nucleosides (i.e. R-arabinonucleosides).26
The absolute configuration of 13c-â was determined in
the same manner and was confirmed to be the â-ano-
mer.
Preparation of the acyclic analogues was accom-
plished by using the sodium salt method.22 The sodium
salt of 3a-c obtained in situ was condensed with
(1,3-diacetoxy-2-propoxy)methyl bromide27 (14) and
(2-acetoxyethoxy)methyl bromide28 (17), prepared by the
literature procedures, to furnish the protected acyclic
analogues 15a-c and 18a-c. Deprotection of 15a-c
and 18a-c using either methanolic ammonia or aque-
ous sodium carbonate afforded the desired acyclic
nucleosides 16a-c and 19a-c (Scheme 3).
The 4,5-dihalogenated imidazole nucleosides can be
viewed as ring-contracted analogues of DRB29,30 (1c).
The synthesis of 4,5-dihalogenated imidazole nucleo-
sides31-33 was approached by two different routes: (1)
debromination at the 2-position of a previously prepared
imidazole nucleoside and (2) the direct glycosidation of
certain 4,5-dihalogenated imidazoles. 2-Bromo-4,5-
dichloro-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)imida-
zole (6b) was treated with triphenylphosphine in a
mixture of toluene and methanol and the mixture was
heated at reflux temperature for 24 h. This reaction
afforded the debrominated product 21b, which was
identical to the product obtained from the direct gly-
cosidation of 4,5-dichloroimidazole18 (20b) by Vor-
bruggen’s method. However, a selective debromination
of 2,4,5-tribromo-1-(2,3,5-tri-O-benzoyl-â-D-ribofurano-
syl)imidazole (6c) under the above conditions was
unsuccessful.
Although a model reaction had demonstrated that
1-methyl-2,4,5-tribromoimidazole could be selectively
debrominated under the same conditions to give 1-meth-
yl-4,5-dibromoimidazole, the tribromoimidazole nucleo-
side 6c remained resistant toward a selective debromi-
nation at the 2-position when it was treated under the
same conditions. Therefore, 4,5-dibromoimidazole (20c),
prepared from 3c via the same debromination condi-
tions,34 was coupled with 1,2,3,5-tetra-O-acetyl-â-D-
ribofuranose (4b) to give the protected nucleoside
21c.31,32 Deprotection of the peracylated nucleosides

5746 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Chien et al.
Scheme 4
Table 1. Antiviral Activity and Cytotoxicity of Polyhalogenated Imidazole Nucleoside Analogues
50% inhibitory concentration (µM)
antiviral activity
substituent
R1
cytotoxicityc
compd
no.
HCMVa
plaque
HSV-1b
ELISA
R2
R4
R5
visual
growth
3a
3b
3c
7a
7b
7c
H
H
H
Cl
Br
Br
Cl
Br
Br
Cl
Br
Br
Br
Br
Cl
Br
Br
Cl
Br
Br
H
Cl
Cl
Br
Cl
Cl
Br
Cl
Cl
Br
Cl
Br
Cl
Cl
Br
Cl
Cl
Br
Cl
Br
Cl
Cl
Br
Cl
Cl
Br
Cl
Cl
Br
Cl
Br
Cl
Cl
Br
Cl
Cl
Br
Cl
Br
30
41
75
>100d
75
>100
>100
100
>100
>100
>100e
>100
>100
>100
>100
>100
45
>100
>100
>100
>100
238
118
24
37
ribose
>100
>100
30
>100
>100
>100e
>100
32
>100
>100
60
>100
>100
65
ribose
ribose
10a
xylose
xylose
xylose
arabinose
arabinose
DHPMf
DHPM
DHPM
HEMf
50
>100
>100
>100e
>100
90
10b
10c
>100
>100e
>100
>100
30
>100
>100
35
13b-â
13c-â
16a
>100
>100
>100
>100
>100
>100
>100
>100
2.9
25e
16b
>100
>100
25
16c
19a
19b
HEM
>100
>100
>100
>100
102
>100
>100
>100
>100
>100
>100
36
19c
HEM
22b
ribose
22c
ribose
H
1a (TCRB)g
1b (BDCRB)g
1c (DRB)g
2ah
0.7
130
42
30
2.0
26
3.2
18
21
19
2bh
18
2
90
8
2ch
70
11
GCVg
7.4
3.5
>100
>100
a Plaque reduction assays were performed in duplicate as described in the text. b Compounds were assayed by ELISA in quadruplicate
wells. c Visual cytotoxicity was scored on HFF cells at time of HCMV plaque enumeration; results of duplicate experiments presented.
Inhibition of KB cell growth was determined as described in the text in triplicate assays. d >100 indicates IC50 greater than the noted
(highest) concentration tested. e Average derived from two experiments. f Abbreviations used: DHPM, (1,3-dihydroxy-2-propoxy)methyl;
HEM, (2-hydroxyethoxy)methyl. g Data presented previously in ref 11. h Data presented previously in ref 17.
21b-c with methanolic ammonia gave the desired 4,5-
dihalogenated imidazole ribonucleosides 22b-c (Scheme
4) as ring-contracted analogues of DRB.
Biological Evaluations. All new perhalogenated
imidazole nucleosides were screened for antiviral activi-
ties against HCMV and herpes simplex virus type-1
(HSV-1) and for cytotoxicity. The results (Table 1) show
that only the parent perhalogenated imidazoles 3a-c
were slightly active against the HCMV, but only 3b was
free of cytotoxicity. Overall, the perhalogenated imida-

Polyhalogenated Imidazole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5747
equiv) and trimethylsilyl triflate (TMSOTf) (0.54 mL, 0.613
g, 2.76 mmol, 1.2 equiv) and stirred at 60 °C for an additional
2 h. The reaction mixture was cooled to room temperature and
CH3CN was removed under reduced pressure to dryness. The
residue was dissolved in EtOAc (150 mL). The organic layer
was successively washed with a saturated solution of NaHCO3
(2 × 50 mL), NaCl (2 × 50 mL), and H2O (50 mL) and then
dried over anhydrous Na2SO4 and evaporated to dryness. The
resulting oil was purified by column chromatography (Hex/
EtOAc ) 7:3) to give 5a (0.69 g, 1.61 mmol, oil, 70%): 1H NMR
(CDCl3, 360 MHz) δ 5.82 (d, 1 H, J ) 5.8 Hz, 1′-H), 5.64 (t, 1
H, J ) 6.2 Hz), 5.35 (t, 1 H, J ) 6.2 Hz), 4.43-4.45 (m, 1 H),
4.24-4.16 (m, 2 H, 5′-H), 2.08 (s, 3 H, CH3), 2.05 (s, 3 H, CH3),
1.98 (s, 3 H, CH3).
2-Bromo-4,5-dichloro-1-(2,3,5-tri-O-acetyl-â-D-ribofura-
nosyl)imidazole (5b). Compound 5b (oil, 0.43 g, 0.91 mmol,
51%) was prepared from 2-bromo-4,5-dichloroimidazole18 (3b,
0.40 g, 1.8 mmol) using BSA (0.67 mL, 0.549 g, 2.7 mmol, 1.5
equiv), CH3CN (40 mL), and then 4b (0.707 g, 2.2 mmol, 1.2
equiv) and TMSOTf (0.42 mL, 0.48 g, 2.16 mmol, 1.2 equiv)
by the method described for 5a and purified by column
chromatography (Hex/EtOAc ) 85:15-8:2): 1H NMR (DMSO-
d6, 360 MHz) δ 5.93 (d, 1 H, J ) 5.4 Hz, 1′-H), 5.57 (dd, 1 H,
J ) 5.4 & 7.3 Hz), 5.32 (t, 1 H, J ) 6.9 Hz), 4.42-4.36 (m, 2
H), 4.24-4.20 (m, 1 H), 2.09 (s, 3 H, CH3), 2.06 (s, 3 H, CH3),
2.03 (s, 3 H, CH3). Anal. (C14H15BrCl2N2O7) C, H, N.
2,4,5-Tribromo-1-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-
imidazole (5c). Compound 5c (oil, 2.34 g, 4.18 mmol, 64%)
was prepared from 2,4,5-tribromoimidazole18 (3c, 2.0 g, 6.56
mmol) using BSA (2.41 mL, 1.98 g, 9.71 mmol, 1.48 equiv),
CH3CN (100 mL), and then 4b (2.7 g, 8.48 mmol, 1.3 equiv)
and TMSOTf (1.27 mL, 1.46 g, 6.56 mmol, 1 equiv) by the
method described for 5a and purified by column chromatog-
raphy (Hex/EtOAc ) 8:2): 1H NMR (DMSO-d6, 360 MHz) δ
5.94 (d, 1 H, J ) 5.4 Hz, 1′-H), 5.57 (dd, 1 H, J ) 5.5 & 7.3
Hz), 5.33 (t, 1 H, J ) 6.8 Hz), 4.42-4.36 (m, 2 H), 4.22 (dd, 1
H, J ) 6.5 & 13.2 Hz), 2.10 (s, 3 H, CH3), 2.06 (s, 3 H, CH3),
2.03 (s, 3 H, CH3). Anal. (C14H15Br3N2O7) C, H, N.
2-Bromo-4,5-dichloro-1-(2,3,5-tri-O-benzoyl-â-D-ribo-
furanosyl)imidazole (6b). Compound 6b (foam, 1.41 g, 2.14
mmol, 92%) was prepared from 2-bromo-4,5-dichloroimida-
zole18 (3b, 0.50 g, 2.32 mmol) using BSA (0.69 mL, 0.566 g,
2.78 mmol, 1.2 equiv), CH3CN (20 mL), and then 1-O-acetyl-
2,3,5-tri-O-benzoyl-â-D-ribofuranose (4a, 1.40 g, 2.78 mmol, 1.2
equiv) and TMSOTf (0.49 mL, 0.567 g, 2.55 mmol, 1.1 equiv)
by the method described for 5a and purified by column
chromatography (CHCl3/MeOH ) 99.5:0.5, Rf ) 0.36): 1H
NMR (CDCl3, 300 MHz) δ 7.37-8.12 (m, 15 H, Ph), 6.22 (d, 1
H, J ) 5.7 Hz), 6.08 (dd, 1 H, J ) 5.5 & 6.8 Hz), 5.94 (dd, 1 H,
J ) 5.7 & 6.8 Hz), 4.90-4.97 (m, 1 H), 4.67-4.73 (m, 2 H,
5′-H). Anal. (C29H21BrCl2N2O7) C, H, N.
2,4,5-Tribromo-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-
imidazole (6c). Compound 6c (foam, 0.736 g, 0.98 mmol, 60%)
was prepared from 2,4,5-tribromoimidazole18 (3c, 0.50 g, 1.64
mmol) using BSA (0.53 mL, 0.434 g, 2.13 mmol, 1.3 equiv),
CH3CN (20 mL), and then 4a (0.99 g, 1.97 mmol, 1.2 equiv)
and TMSOTf (0.38 mL, 0.437 g, 1.97 mmol, 1.2 equiv) by the
method described for 5a and purified by column chromatog-
raphy [Hex/CH2Cl2 ) 3:7, Rf ) 0.36 (Hex/CHCl3 ) 3:7)]: 1H
NMR (CDCl3, 300 MHz) δ 8.11-7.38 (m, 15 H, Ph), 6.30-6.26
(m, 2 H), 6.20 (dd, 1 H, J ) 5.3 & 6.6 Hz), 4.87-4.82 (m, 1 H),
4.76 (dd, 1 H, J ) 3.6 & 12.2 Hz, 5′-H), 4.61 (dd, 1 H, J ) 4.5
& 12.2 Hz, 5′-H); MS (FAB) m/z 154 (100), 201 (22), 307 (25),
445 (40), 747 (5) (M+). Anal. (C29H21Br3N2O7) C, H, N.
2,4,5-Trichloro-1-(â-D-ribofuranosyl)imidazole (7a). A
mixture of 5a (1.14 g, 2.6 mmol), Na2CO3 (1.7 g, 16.0 mmol),
and 10% aqueous EtOH (100 mL) was stirred at room
temperature for 12 h. The excess ethanol was evaporated
under reduced pressure to dryness. The mixture was diluted
with H2O (100 mL), extracted with EtOAc (250 mL), washed
with H2O (50 mL), and dried (anhydrous Na2SO4), and the
solvent was removed under reduced pressure. The resulting
mixture was purified by column chromatography (CHCl3/
MeOH ) 9:1) to give 7a (0.32 g, 42%). The product (7a) was
zole nucleoside derivatives showed little significant
cytotoxicity and were mostly inactive against both
HCMV and HSV-1. Interestingly, the 1-ribosyl and
1-arabinosyl 2,4,5-tribromoimidazoles (7c, 13c) had
some activity against HCMV at noncytotoxic concentra-
tions but both were less active than the polyhalogenated
benzimidazoles 1a-b11 and the tricyclic analogues 2a-
c.17 This result implies that only 7c and 13c can fit into
a putative benzimidazole binding site. Since the bromine
atom is considerably bigger than the chlorine atom, the
bispherical bromo atoms may substitute for the o-
dichlorobenzene ring in TCRB (1a) or BDCRB (1b) in
space and contributed certain binding affinity to the
binding pocket to retain the activity. Furthermore, the
4,5-dibromoimidazole nucleoside 22c showed no activity
against either virus, which revealed that the removal
of the 2-bromo group from 7c eliminated the antiviral
activity. This trend is consistent with our previous
observation that DRB30 (1c) is less active than TCRB
(1a) and BDCRB (1b). The acyclic nucleoside analogues
16a-c and 19a-c were nearly inactive and noncyto-
toxic. This result also parallels our early conclusion for
acyclic nucleoside analogues of polyhalogenated benz-
imidazoles.35 In general, low activity and no cytotoxicity
suggested that the ring-contracted analogues lost their
binding affinity for the HCMV target and did not affect
cellular enzymes. Therefore, the ring extension linearly
along the C2 axis of the imidazole ring is critical for
activity against HCMV.
Experimental Section
General Chemical Procedures. Melting points were
taken on a Thomas-Hoover Unimelt capillary melting point
apparatus and are uncorrected. Nuclear magnetic resonance
(NMR) spectra were obtained at 300, 360, or 500 MHz with
Bruker DPX300, WP360SY, or DRX500 instruments. The
chemical shift values are reported in δ values (parts per
million, ppm) relative to the standard chemical shift of
tetramethylsilane (TMS). The coupling constant values are
expressed in hertz (Hz). Elemental analyses were performed
by M-H-W Laboratories, Phoenix, AZ, or by the Analytical
Laboratory, Department of Chemistry, University of Michigan,
Ann Arbor, MI. Mass spectrometry was performed by the
Analytical Laboratory, Department of Chemistry, University
of Michigan. Thin-layer chromatography (TLC) was performed
on silica gel GHLF-254 plates (Merck Reagents). Compounds
on thin-layer chromatography were visualized by illumination
under UV light (254 nm) or dipped into 10% methanolic
sulfuric acid followed by charring on a hot plate. Solvent
systems are expressed as a percentage of the more polar
component with respect to total volume (v/v%). E. Merck silica
gel (230-400 mesh) was used for flash column chromatogra-
phy, and this technique has been described by Still et al (J.
Org. Chem. 1978, 43, 2923-2925). The reported yields have
not been optimized. Sodium hydride of 60% oil dispersion
(Aldrich) was used for all the sodium salt glycosylation
reactions unless specified otherwise. A 1 M solution of boron
trichloride in dichloromethane (Aldrich) was used for the
debenzylation process. Evaporations were carried out under
reduced pressure (water aspirator) with the bath temperature
below 50 °C unless specified otherwise. Materials obtained
from commercial suppliers were used without further purifica-
tion.
2,4,5-Trichloro-1-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-
imidazole (5a). To a solution of 2,4,5-trichloroimidazole18 (3a,
0.4 g, 2.3 mmol) in CH3CN (100 mL) was added N,O-bis-
(trimethylsilyl)acetamide (BSA) (0.86 mL, 0.702 g, 3.45 mmol,
1.5 equiv) and the reaction mixture was stirred at room
temperature for 20 min. This solution was treated with 1,2,3,5-
tetra-O-acetyl-â-D-ribofuranose (4b, 0.89 g, 2.7 mmol, 1.2

5748 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
recrystallized from Hex/EtOAc (0.198 g, 26%): mp 110-112
Chien et al.
Hz), 5.82 (dd, 1 H, J ) 2.0 & 5.8 Hz), 4.84-4.73 (m, 3 H), 2.07
(s, 3 H, CH3); 13C & DEPT135 NMR (CDCl3, 125 MHz) δ 169.8,
164.5, 164.0, 151.6, 151.3, 134.8, 134.1, 131.3 (2 × CH), 129.4,
124.4 (CH), 124.1 (CH), 117.3, 114.5, 90.3 (CH), 79.8 (CH), 77.6
(CH), 77.3 (CH), 62.5 (CH2), 20.8 (CH3).
1
°C (Hex/EtOAc); H NMR (DMSO-d6, 360 MHz) δ 5.64 (d, 1
H, J ) 6.7 Hz, 1′-H), 5.63 (d, 1 H, J ) 6.2 Hz, 2′-OH), 5.30 (d,
1 H, J ) 5.2 Hz, 3′-OH), 4.90 (t, 1 H, J ) 5.7 Hz, 5′-OH), 4.51
(dd, 1 H, J ) 6.2 & 12.5 Hz), 4.02 (dt, 1 H, J ) 3.6 & 5.4 Hz),
3.84 (dt, 1 H, J ) 3.6 & 5.5 Hz), 3.59 (dd, 1 H, J ) 5.5 & 11.7
Hz, 5′-H), 3.52 (dd, 1 H, J ) 5.8 & 11.7 Hz, 5′-H). Anal. (C8H9-
Cl3N2O4) C, H, N.
2-Bromo-4,5-dichloro-1-(â-D-ribofuranosyl)imidazole
(7b). A mixture of 5b (0.39 g, 0.82 mmol) and methanolic
ammonia (40 mL) was stirred at room temperature for 12 h.
The excess MeOH and NH3 were removed under reduced
pressure. The resulting residue was purified by column chro-
matography (CHCl3/MeOH ) 95:5) followed by crystallization
from Hex/EtOAc to give 7b (0.17 g, 60%): mp 135-138 °C
(Hex/EtOAc); 1H NMR (DMSO-d6, 360 MHz) δ 5.64 (d, 1 H, J
) 6.7 Hz, 1′-H), 5.61 (d, 1 H, J ) 6.1 Hz, OH), 5.28 (d, 1 H, J
) 5.1 Hz, OH), 4.89 (t, 1 H, J ) 5.6 Hz, 5′-OH), 4.51 (dd, 1 H,
J ) 6.2 & 12.4 Hz), 4.01 (dd, 1 H, J ) 4.8 & 9.8 Hz), 3.82 (dd,
1 H, J ) 5.6 & 9.3 Hz), 3.62-3.49 (m, 2 H, 5′-H). Anal. (C8H9-
BrCl2N2O4) C, H, N.
2,4,5-Tribromo-1-(â-D-ribofuranosyl)imidazole (7c).
Method A. Compound 7c (0.293 g, 0.67 mmol, 61%) was
obtained from the deprotection of 5c (0.62 g, 1.10 mmol) with
Na2CO3 (0.35 g, 3.30 mmol, 3 equiv) in EtOH/H2O [50 mL, 9:1
(v/v)] as described for 7a and then purified by column
chromatography (CHCl3/MeOH ) 9:1): mp 162-165 °C (dec)
(Hex/EtOAc).
Method B. Compound 7c (1.54 g, 3.53 mmol, 76%) was
obtained from the deprotection of 6c (3.48 g, 4.64 mmol) with
28% NH4OH/MeOH/acetone [100 mL, 2:2:1 (v/v/v)] as de-
scribed for 7b, and then purified by column chromatography
[CHCl3/MeOH ) 95:5, Rf ) 0.3 (CHCl3/MeOH ) 9:1)]. An
analytical sample of 7c was obtained by recrystallization from
CH3CN: mp 165-167 °C (CH3CN).
2,4,5-Tribromo-1-[2-O-acetyl-3,5-di-O-(4-nitrobenzoyl)-
1-â-D-xylofuranosyl]imidazole (9c). Compound 9c (foam,
2.44 g, 3.14 mmol, 53%) was prepared from 2,4,5-tribromoimi-
dazole18 (3c, 2.00 g, 6.55 mmol, 1.1 equiv) using BSA (1.92
mL, 1.58 g, 7.75 mmol, 1.3 equiv), CH3CN (60 mL), and then
8 (3.17 g, 5.96 mmol) and TMSOTf (1.61 mL, 1.86 g, 8.34 mmol,
1.4 equiv) by the method described for 5a and purified by
column chromatography [Hex/EtOAc ) 6:4-5:5, Rf ) 0.40
(Hex/EtOAc ) 6:4)]. An analytical sample was obtained by
recrystallization from Hex/EtOAc: mp 111-114 °C (dec) (Hex/
EtOAc); 1H NMR (CDCl3, 500 MHz) δ 8.36 (d, 2 H, J ) 8.9
Hz, Ph), 8.30 (d, 2 H, J ) 8.9 Hz, Ph), 8.23 (d, 2 H, J ) 8.9 Hz,
Ph), 8.19 (d, 2 H, J ) 8.9 Hz, Ph), 6.01 (d, 1 H, J ) 6.1 Hz,
1′-H), 5.90 (dd, 1 H, J ) 2.3 & 5.5 Hz, 3′-H), 5.86 (dd, 1 H, J
) 2.3 & 6.1 Hz, 2′-H), 4.86-4.82 (m, 1 H, 4′-H), 4.81-4.77 (m,
2 H, 5′-H), 2.20 (s, 3 H, CH3); 13C & DEPT135 NMR (CDCl3,
125 MHz) δ 169.8, 164.5, 164.0, 151.5, 151.3, 134.8, 134.1,
131.3 (2 × CH), 124.4 (CH), 124.1 (CH), 120.4, 119.0, 104.1,
90.4 (CH), 79.9 (CH), 77.8 (CH), 77.2 (CH), 62.5 (CH2), 20.8
(CH3). Anal. (C24H17Br3N4O11·1/2EtOAc) C, H, N.
2,4,5-Trichloro-1-(â-D-xylofuranosyl)imidazole (10a).
Compound 10a (0.488 g, 1.61 mmol, 71%) was obtained from
the deprotection of 9a (1.45 g, 2.27 mmol) with methanolic
ammonia (23 mL) as described for 7b and then purified by
column chromatography [CHCl3/MeOH ) 95:5-93:7, Rf ) 0.33
(CHCl3/MeOH ) 9:1)]. An analytical sample of 10a was
obtained by recrystallization from EtOAc/MeOH: mp 187-
190 °C (dec) (EtOAc/MeOH); 1H NMR (DMSO-d6, 500 MHz) δ
5.87 (d, 1 H, J ) 5.4 Hz, OH), 5.53 (d, 1 H, J ) 6.4 Hz, 1′-H),
5.39 (d, 1 H, J ) 4.7 Hz, OH), 4.56 (t, 1 H, J ) 5.7 Hz, OH),
4.45 (dd, 1 H, J ) 5.4 & 10.7 Hz), 4.17 (dd, 1 H, J ) 4.6 &
10.7 Hz), 4.11 (dt, 1 H, J ) 4.3 & 6.7 Hz), 3.73-3.68 (m, 1 H,
5′-H), 3.66-3.61 (m, 1 H, 5′-H); 13C & DEPT135 NMR (DMSO-
d6, 500 MHz) δ 130.5, 125.8, 114.5, 91.3 (CH), 82.4 (CH), 79.6
(CH), 76.1 (CH), 60.9 (CH2). Anal. (C8H9Cl3N2O4) C, H, N.
Compound 7c: 1H NMR (DMSO-d6, 300 MHz) δ 5.71 (d, 1
H, J ) 4.7 Hz, 1′-H), 5.53 (d, 1 H, J ) 5.9 Hz, OH), 5.26 (d, 1
H, J ) 5.3 Hz, OH), 4.78 (t, 1 H, J ) 5.6 Hz, OH), 4.52 (dd, 1
H, J ) 5.0 & 10.2 Hz), 4.11 (dd, 1 H, J ) 4.8 & 9.5 Hz), 3.89
(dd, 1 H, J ) 4.9 & 9.7 Hz), 3.37-3.50 (m, 2 H, 5′-H); 13C NMR
(DMSO-d6, 75 MHz) δ 129.9, 120.0, 101.0, 92.4, 86.8, 74.0, 71.3,
62.7; MS (CI, CH4) m/z 133 (100), 226 (30), 306 (45), 408 (15),
435 (5) (M+ + 1); HRMS calcd for C8H10Br3N2O4 (M + 1)
434.8191, found 434.8176. Anal. (C8H9Br3N2O4) C, H, N.
2,4,5-Trichloro-1-[2-O-acetyl-3,5-di-O-(4-nitrobenzoyl)-
1-â-D-xylofuranosyl]imidazole (9a). Compound 9a (oil, 1.46
g, 2.27 mmol, 61%) was prepared from 2,4,5-trichloroimida-
zole18 (3a, 0.634 g, 3.70 mmol) using BSA (1.19 mL, 0.978 g,
4.81 mmol, 1.3 equiv), CH3CN (40 mL), and then 1,2-di-O-
acetyl-3,5-di-O-(4-nitrobenzoyl)-1-â-D-xylofuranose21 (8, 1.97 g,
3.70 mmol, 1.0 equiv) and TMSOTf (1.07 mL, 1.23 g, 5.55
mmol, 1.5 equiv) by the method described for 5a and purified
by column chromatography [Hex/EtOAc ) 7:3-5:5, Rf ) 0.37
(Hex/EtOAc ) 65:35)]: 1H NMR (CDCl3, 500 MHz) δ 8.37 (d,
2 H, J ) 8.8 Hz, Ph), 8.30 (d, 2 H, J ) 8.8 Hz, Ph), 8.22 (d, 2
H, J ) 8.9 Hz, Ph), 8.19 (d, 2 H, J ) 8.9 Hz, Ph), 5.91 (d, 1 H,
J ) 5.6 Hz, 1′-H), 5.88 (dd, 1 H, J ) 3.4 & 6.3 Hz), 5.81 (dd,
1 H, J ) 1.9 & 5.6 Hz), 4.83-4.72 (m, 3 H), 2.21 (s, 3 H, CH3);
13C & DEPT135 NMR (CDCl3, 125 MHz) δ 169.8, 164.5, 163.9,
151.6, 151.3, 134.8, 134.0, 131.3 (2 × CH), 130.1, 127.8, 124.4
(CH), 124.1 (CH), 113.9, 89.3 (CH), 79.8 (CH), 77.8 (CH), 77.4
(CH), 62.5 (CH2), 20.8 (CH3).
2-Bromo-4,5-dichloro-1-[2-O-acetyl-3,5-di-O-(4-nitroben-
zoyl)-1-â-D-xylofuranosyl]imidazole (9b). Compound 9b
(oil, 4.81 g, 6.99 mmol, 89%) was prepared from 2-bromo-4,5-
dichloroimidazole18 (3b, 1.70 g, 7.89 mmol) using BSA (2.55
mL, 2.09 g, 10.25 mmol, 1.3 equiv), CH3CN (100 mL), and then
8 (5.45 g, 10.25 mmol, 1.3 equiv) and TMSOTf (2.44 mL, 2.81
g, 12.62 mmol, 1.6 equiv) by the method described for 5a and
purified by column chromatography [Hex/EtOAc ) 7:3-5:5,
Rf ) 0.23 (Hex/EtOAc ) 7:3)]: 1H NMR (CDCl3, 500 MHz) δ
8.37 (d, 2 H, J ) 8.8 Hz, Ph), 8.31 (d, 2 H, J ) 8.8 Hz, Ph),
8.22 (d, 2 H, J ) 8.7 Hz, Ph), 8.19 (d, 2 H, J ) 8.7 Hz, Ph),
5.95 (d, 1 H, J ) 5.8 Hz, 1′-H), 5.89 (dd, 1 H, J ) 1.8 & 5.0
2-Bromo-4,5-dichloro-1-(â-d-xylofuranosyl)imidazole
(10b). Compound 10b (0.49 g, 1.41 mmol, 63%) was obtained
from the deprotection of 9b (1.53 g, 2.22 mmol) with metha-
nolic ammonia (30 mL) as described for 7b, and then purified
by column chromatography (CHCl3/MeOH ) 95:5-93:7, Rf )
0.20 (CHCl3/MeOH ) 93:7)). An analytical sample of 10b was
obtained by recrystallization from MeOH. mp 185-187 °C (dec)
1
(MeOH). H NMR (DMSO-d6, 500 MHz) δ 5.86 (d, 1 H, J )
5.6 Hz, OH), 5.53 (d, 1 H, J ) 6.7 Hz, 1′-H), 5.39 (d, 1 H, J )
4.8 Hz, OH), 4.55 (t, 1 H, J ) 5.6 Hz, 5′-OH), 4.48 (dd, 1 H, J
) 5.6 Hz, 11.0 Hz), 4.18 (dt, 1 H, J ) 4.7 & 6.4 Hz), 4.11 (dt,
1 H, J ) 4.1 & 6.9 Hz), 3.73-3.68 (m, 1 H, 5′-H), 3.66-3.62
(m, 1 H, 5′-H); 13C & DEPT135 NMR (DMSO-d6, 75 MHz) δ
127.3, 119.0, 114.8, 92.1 (CH), 82.2 (CH), 79.5 (CH), 76.2 (CH),
61.0 (CH2). Anal. (C8H9BrCl2N2O4) C, H, N.
2,4,5-Tribromo-1-(â-D-xylofuranosyl)imidazole (10c).
Compound 10c (0.794 g, 1.82 mmol, 58%) was obtained from
the deprotection of 9c (2.44 g, 3.14 mmol) with methanolic
ammonia (31 mL) as described for 7b and then purified by
column chromatography [CHCl3/MeOH ) 93:7-9:1, Rf ) 0.19
(CHCl3/MeOH ) 9:1)]. An analytical sample of 10c was
obtained by recrystallization from MeOH: mp 186-188 °C
(dec) (MeOH); 1H NMR (DMSO-d6, 500 MHz) δ 5.84 (d, 1 H, J
) 5.6 Hz, OH), 5.55 (d, 1 H, J ) 7.0 Hz, 1′-H), 5.41 (d, 1 H, J
) 4.7 Hz, OH), 4.56-4.52 (m, 2 H, OH & CH), 4.20 (dd, 1 H,
J ) 4.7 & 11.2 Hz), 4.12 (dt, 1 H, J ) 4.5 & 6.8 Hz), 3.74-3.69
(m, 2 H, 5′-H); 13C & DEPT135 NMR (DMSO-d6, 125 MHz) δ
120.5, 119.1, 105.5, 92.1 (CH), 82.0 (CH), 79.6 (CH), 76.2 (CH),
61.1 (CH2); MS (EI, 70 eV) m/z 302 (37), 304 (100), 306 (97),
308 (35), 434 (5) (M+), 436 (25) (M + 2), 438 (24) (M + 4), 440
(4) (M + 6); HRMS calcd for C8H9Br3N2O4 (M+): 433.8112,
found 433.8114. Anal. (C8H9Br3N2O4) C, H, N.
2-Bromo-4,5-dichloro-1-(2,3,5-tri-O-benzyl-r-D-arabino-
furanosyl)imidazole (12b-r) and 2-Bromo-4,5-dichloro-

Polyhalogenated Imidazole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5749
1-(2,3,5-tri-O-benzyl-â-D-arabinofuranosyl)imidazole (12b-
â). NaH (0.39 g, 13.2 mmol, 80% oil dispersion) was added to
a stirred suspension of 2-bromo-4,5-dichloroimidazole18 (3b,
2.4 g, 11.1 mmol) in dry CH3CN (120 mL) under a nitrogen
atmosphere. The solution was stirred until hydrogen evolution
has ceased and a clear solution was obtained (30 min). A
solution of 2,3,5-tri-O-benzyl-R-D-arabinofuranosyl chloride24
[11, prepared from 5.9 g (10.3 mmol) of 2,3,5-tri-O-benzyl-1-
O-(p-nitrobenoyl)-D-arabinose] in CH3CN (20 mL) was then
added dropwise. The reaction mixture was stirred for an
additional 16 h. The resulting mixture was concentrated under
reduced pressure to dryness. The residue was dissolved in
EtOAc (200 mL). The organic layer was washed with H2O (100
mL) and dried over anhydrous Na2SO4. The resulting syrup
was purified by flash column chromatography (Hex/EtOAc )
9:1) to give the following products: 1.58 g (2.56 mol) of 12b-R,
2.14 g of a mixture of 12b-R and 12b-â, and 1.46 g (2.36 mmol)
of 12b-â. The mixture was rechromatographed (Hex/EtOAc )
9:1) to give 0.82 g (1.33 mmol) of 12b-R and 0.98 g (1.58 mmol)
of 12b-â.
12b-r: foam, 2.40 g (3.90 mmol), 35% from 3b; 1H NMR
(DMSO-d6, 360 MHz) δ 7.37-7.11 (m, 15 H, 3 × Ph), 5.88 (d,
1 H, J ) 6.7 Hz, 1′-H), 4.70 (t, 1 H, J ) 6.4 Hz, CH), 4.62-
4.42 (m, 7 H, 3 × CH2 and 1 × CH), 4.32 (t, 1 H, J ) 6.4 Hz,
CH), 3.65 (dd, 1 H, J ) 2.9 & 11.0 Hz, 5′-H), 3.60 (dd, 1 H, J
) 4.3 & 11.0 Hz, 5′-H).
12b-â: foam, 2.44 g (3.94 mmol), 35% from 3b; 1H NMR
(DMSO-d6, 360 MHz) δ 7.39-6.84 (m, 15 H, 3 × Ph), 6.36 (d,
1 H, J ) 6.4 Hz, 1′-H), 4.62-3.74 (m, 11 H, 3 × CH2, 2′-H,
3′-H, 2 × 5′-H).
3′-H), 3.71-3.63 (m, 3 H, 4′-H, and 2 × 5′-H); 13C NMR (DMSO-
d6, 90 MHz) δ 125.5, 117.0, 115.0, 88.1, 83.2, 76.3, 75.8, 61.2.
Anal. (C8H9BrCl2N2O4) C, H, N.
The r-isomer, 2-bromo-4,5-dichloro-1-(r-D-arabino-
furanosyl)imidazole (13b-r), was obtained by debenzylation
of 12b-R (1.54 g, 2.5 mmol) with 1 M BCl3 in CH2Cl2 (20 mL)
in dry CH2Cl2 (70 mL) by the method described for 13b-â to
give 13b-R (0.7 g, 2.01 mmol, 80%). An analytical sample was
1
recrystallized from EtOH: mp 186-189 °C (dec) (EtOH); H
NMR (DMSO-d6, 360 MHz) δ 5.85 (d, 1 H, exchanges with D2O,
J ) 5.7 Hz, 2′-OH), 5.66 (d, 1 H, exchanges with D2O, J ) 5.6
Hz, 3′-OH), 5.65 (d, 1 H, J ) 7.5 Hz, 1′-H), 4.90 (dd, 1 H,
exchanges with D2O, J ) 5.0 & 6.4 Hz, 5′-OH), 4.51 (dd, 1 H,
J ) 7.8 & 13.5 Hz, 2′-H), 4.06-4.01 (m, 2 H, 3′-H & 4′-H),
3.61 (dd, 1 H, J ) 4.3 & 11.9 Hz, 5′-H), 3.46 (ddd, 1 H, J )
3.4, 6.5, 12.2 Hz, 5′-H); 13C NMR (DMSO-d6, 90 MHz) δ 126.3,
117.6, 113.4, 91.2, 84.0, 77.7, 73.0, 60.3. Anal. (C8H9BrCl2N2O4)
C, H, N.
2,4,5-Tribromo-1-(â-D-arabinofuranosyl)imidazole (13c-
â). Compound 13c-â (0.70 g, 1.60 mmol, 64%) was obtained
by debenzylation of 12c-â (1.77 g, 2.5 mmol) with 1 M BCl3 in
CH2Cl2 (25 mL) in dry CH2Cl2 (60 mL) by the method described
for 13b-â. An analytical sample was recrystallized from
1
EtOAc: mp 173-175 °C (dec) (EtOAc); H NMR (DMSO-d6,
360 MHz) δ 6.06 (d, 1 H, J ) 6.2 Hz, 1′-H), 5.63 (d, 1 H,
exchanges with D2O, J ) 6.0 Hz, 2′-OH), 5.53 (bs, 1 H,
exchanges with D2O, OH), 4.84 (bs, 1 H, exchanges with D2O,
OH), 4.14 (dd, 1 H, J ) 4.9 & 10.8 Hz, 2′-H), 4.08-4.03 (m, 1
H, 3′-H), 3.71-3.67 (m, 3 H, 4′-H and 2 × 5′-H); 13C NMR
(DMSO-d6, 90 MHz) δ 118.8, 117.1, 105.3, 88.5, 83.4, 76.4, 76.2,
61.3. Anal. (C8H9Br3N2O4) C, H, N.
2,4,5-Tribromo-1-(2,3,5-tri-O-benzyl-R-D-arabinofura-
nosyl)imidazole (12c-R) and 2,4,5-Tribromo-1-(2,3,5-tri-
O-benzyl-â-D-arabinofuranosyl)imidazole (12c-â). Com-
pound 12c-R and 12c-â were prepared from 2,4,5-tri-
bromoimidazole18 (3c, 2.4 g, 7.9 mmol) using NaH (60% in
mineral oil, 0.426 g, 10.7 mmol, 1.35 equiv), CH3CN (120 mL),
then 11 (prepared from 5.5 g (9.8 mmol) of 2,3,5-tri-O-benzyl-
1-O-(p-nitrobenoyl)-D-arabinose)24 as described for 12b-R and
12b-â, and purified by flash column chromatography (Hex/
EtOAc ) 9:1) to give the following products: 0.30 g (0.42 mmol)
of 12c-R, 0.37 g (0.52 mmol) of a mixture of 12c-R and 12c-â
(analyzed by 1H NMR showing 1:1 ratio), and 4.45 g (6.29
mmol) of 12c-â.
12c-r: foam, 0.30 g (0.42 mmol), 5% from 3c; 1H NMR
(DMSO-d6, 360 MHz) δ7.35-7.08 (m, 15 H, 3 × Ph), 5.92 (d,
1 H, J ) 7.1 Hz, 1′-H), 4.73 (t, 1 H, J ) 6.7 Hz), 4.65-4.43 (m,
7 H, 3 × CH2, and CH), 4.34 (t, 1 H, J ) 6.7 Hz), 3.65 (dd, 1
H, J ) 3.0 & 11.1 Hz, 5′-H), 3.62 (dd, 1 H, J ) 4.4 & 11.0 Hz,
5′-H). 12c-â: foam, 4.45 g (6.29 mmol), 81% from 3c; 1H NMR
(DMSO-d6, 360 MHz) δ 7.35-7.00 (m, 15 H, 3 × Ph), 6.36 (d,
1 H, J ) 6.3 Hz, 1′-H), 4.61-3.98 (m, 9 H, 3 × CH2, 2′-H, 3′-H
and 4′-H), 3.84 (dd, 1 H, J ) 7.0 & 10.8 Hz, 5′-H), 3.75 (dd, 1
H, J ) 3.5 & 10.8 Hz, 5′-H).
2,4,5-Trichloro-1-[(1,3-diacetoxy-2-propoxy)methyl]im-
idazole (15a). NaH (60% in mineral oil, 0.12 g, 3.0 mmol) was
added to a stirred solution of 2,4,5-trichloroimidazole18 (3a,
0.35 g, 2.0 mmol) in dry CH3CN (30 mL) under a N2
atmosphere. The solution was stirred until hydrogen evolution
has ceased and a clear solution was obtained (20 min). (1,3-
Diacetoxy-2-propoxy)methyl bromide27 (14, 0.65 g, 2.4 mmol)
in CH3CN (10 mL) was then added dropwise. The reaction
mixture was stirred for an additional 3 h at room temperature.
The resulting mixture was concentrated under reduced pres-
sure to dryness, diluted with H2O (50 mL), extracted with
EtOAc (150 mL), washed with H2O (100 mL), and dried
(anhydrous Na2SO4(s)), and the solvent was removed under
reduced pressure to yield an oil. The resulting oil was purified
by flash column chromatography (Hex/EtOAc ) 65:35) to give
15a (oil, 0.57 g, 78%): 1H NMR (CDCl3, 360 MHz) δ 5.35 (s, 2
H, 1′-H), 4.24-3.87 (m, 5 H, 2 × CH2 & CH), 1.83 (s, 6 H, 2 ×
CH3).
2-Bromo-4,5-dichloro-1-[(1,3-diacetoxy-2-propoxy)-
methyl]imidazole (15b). Compound 15b (oil, 1.0 g, 2.49
mmol, 54%) was prepared from 2-bromo-4,5-dichloroimida-
zole18 (3b, 1.0 g, 4.6 mmol) using NaH (60% in mineral oil,
0.22 g, 5.5 mmol, 1.2 equiv), CH3CN (125 mL), and then 14
(1.49 g, 5.5 mmol, 1.2 equiv) by the method described for 15a
and purified by column chromatography (Hex/EtOAc ) 85:15).
An analytical sample of 15b was obtained by recrystallization
from Hex/EtOAc: mp 76-78 °C (Hex/EtOAc); 1H NMR (DMSO-
d6, 360 MHz) δ 5.42 (s, 2 H, 1′-H), 4.12 (dd, 2 H, J ) 2.9 &
11.7 Hz, CH2), 4.01 (dd, 2 H, J ) 6.7 & 11.7 Hz, CH2), 3.95-
3.93 (m, 1 H, CH), 1.96 (s, 6 H, 2 × CH3). Anal. (C11H13-
BrCl2N2O5) C, H, N.
2,4,5-Tribromo-1-[(1,3-diacetoxy-2-propoxy)methyl]im-
idazole (15c). Compound 15c (oil, 0.541 g, 1.10 mmol, 85%)
was prepared from 2,4,5-tribromoimidazole18 (3c, 0.4 g, 1.3
mmol) using NaH (60% in mineral oil, 0.61 g, 1.5 mmol, 1.15
equiv), CH3CN (40 mL), and then 14 (0.422 g, 1.5 mmol, 1.15
equiv) by the method described for 15a and purified by column
chromatography (Hex/EtOAc ) 95:5-8:2). An analytical sample
of 15c was obtained by recrystallization from Hex/EtOAc: mp
76-77 °C (Hex/EtOAc); 1H NMR (DMSO-d6, 360 MHz) δ 5.43
(s, 2 H, 1′-H), 4.12 (dd, 2 H, J ) 3.3 & 11.7 Hz, CH2), 4.01 (dd,
2 H, J ) 6.8 & 11.7 Hz, CH2), 3.96-3.92 (m, 1 H, CH), 1.96 (s,
6 H, 2 × CH3). Anal. (C11H13Br3N2O5) C, H, N.
2-Bromo-4,5-dichloro-1-(â-D-arabinofuranosyl)imid-
azole (13b-â). To a solution of 12b-â (1.49 g, 2.4 mmol) in
dry CH2Cl2 (75 mL) at -78 °C under a nitrogen atmosphere
was added dropwise 1 M BCl3 in CH2Cl2 (25 mL) while the
bath temperature was maintained at -78 °C. The reaction
mixture was stirred for an additional 2 h. The resulting
mixture was brought to 0 °C and stirred for an additional 2 h
and kept in a 0 °C freezer for 16 h. MeOH (20 mL) was added
at -78 °C and the reaction mixture was brought to room
temperature, neutralized (pH ) 7) with 28% NH4OH, and then
concentrated to yield an oil. The resulting oil was diluted with
H2O (100 mL), extracted with EtOAc (200 mL), washed with
H2O (100 mL), dried (anhydrous Na2SO4), and concentrated
in vacuo. The resulting oil was purified by column chroma-
tography (CHCl3/MeOH ) 8:2) to give 13b-â (0.47 g, 1.35
mmol, 56%). An analytical sample was recrystallized from
1
MeOH: mp 190-192 °C (dec) (MeOH); H NMR (DMSO-d6,
360 MHz) δ 6.06 (d, 1 H, J ) 6.3 Hz, 1′-H), 5.69 (d, 1 H,
exchanges with D2O, J ) 5.2 Hz, 2′-OH), 5.54 (m, 1 H,
exchanges with D2O, OH), 4.85 (bs, 1 H, exchanges with D2O,
OH), 4.18 (dd, 1 H, J ) 5.2 & 11.2 Hz, 2′-H), 3.96 (m, 1 H,

5750 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Chien et al.
2,4,5-Trichloro-1-[(1,3-dihydroxy-2-propoxy)methyl]-
imidazole (16a). Compound 16a (0.34 g, 1.24 mmol, 93%) was
obtained from the deprotection of 15a (0.480 g, 1.34 mmol)
with methanolic ammonia (25 mL) as described for 7b and
then purified by column chromatography (CHCl3/MeOH ) 93:
obtained from the deprotection of 18c (0.31 g, 0.73 mmol) with
Na2CO3 (0.67 g, 0.63 mmol, 0.86 equiv) in MeOH/EtOH/H2O
(5 mL:5 mL:1 mL) as described for 7a and purified by column
chromatography (CHCl3/MeOH ) 9:1). An analytical sample
of 19c was obtained by recrystallization from Hex/EtOAc: mp
1
1
7): mp 79-80 °C; H NMR (DMSO-d6, 360 MHz) δ 5.36 (s, 2
97 °C (Hex/EtOAc); H NMR (DMSO-d6, 360 MHz) δ 5.37 (s,
H, 1′-H), 4.72 (t, 2 H, J ) 7.1 Hz, 2 × OH), 3.55-3.14 (m, 5 H,
2 H, 1′-H), 4.75 (t, 1 H, J ) 5.3 Hz, OH), 3.52-3.41 (m, 4 H,
2 × CH2). Anal. (C6H7Br3N2O2) C, H, N.
2 × CH2 & CH). Anal. (C7H9Cl3N2O3) C, H, N.
2-Bromo-4,5-dichloro-1-[(1,3-dihydroxy-2-propoxy)-
methyl]imidazole (16b). Compound 16b (0.219 g, 0.69 mmol,
92%) was obtained from the deprotection of 15b (0.30 g, 0.74
mmol) with methanolic ammonia (15 mL) as described for 7b
and recrystallized from Hex/EtOAc: mp 119-121 °C (Hex/
4,5-Dichloro-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-
imidazole (21b). Method A. A mixture of 6b (1.32 g, 2.0
mmol) and triphenylphosphine (1.05 g, 4 mmol, 2 equiv) was
suspended in a mixture of toluene (15 mL) and methanol (5
mL). The reaction mixture was heated at reflux for 24 h. The
solvents were evaporated under vacuum. To the resulting
mixture ethyl acetate (30 mL) was added and the precipitate
was collected by filtration (identified as methyl triphenylphos-
phinium bromide). The solution was concentrated and the
resulting oil was purified by column chromatography (Hex/
EtOAc ) 75:25) to give 21b [oil, 0.83 g, 1.43 mmol, 71%, Rf )
0.38 (Hex/EtOAc ) 7:3)].
1
EtOAc); H NMR (DMSO-d6, 360 MHz) δ 5.43 (s, 2 H, 1′-H),
4.70 (t, 2 H, J ) 5.4 Hz, 2 × OH), 3.57-3.51 (m, 1 H, CH),
3.46-3.40 (m, 2 H, CH2), 3.34-3.27 (m, 2 H, CH2). Anal. (C7H9-
BrCl2N2O3) C, H, N.
2,4,5-Tribromo-1-[(1,3-dihydroxy-2-propoxy)methyl]-
imidazole (16c). Compound 16c (0.167 g, 0.41 mmol, 97%)
was obtained from the deprotection of 15c (0.20 g, 0.42 mmol)
with methanolic ammonia (30 mL) as described for 7b and
recrystallized from Hex/EtOAc (97%): mp 113-115 °C (Hex/
Method B. To a solution of 4,5-dichloroimidazole18 (20b,
0.55 g, 4 mmol) in dry CH3CN (40 mL) was added BSA (1.19
mL, 0.976 g, 4.8 mmol, 1.2 equiv) and the reaction mixture
was stirred at 35 °C for 20 min. To this solution was added 4a
(1.77 g, 3.51 mmol) and TMSOTf (1.01 mL, 1.16 g, 5.2 mmol,
1.3 equiv) and the reaction was stirred at 65 °C for 90 min.
The reaction mixture was cooled to room temperature and the
acetonitrile was removed under reduced pressure. To the
residue was added EtOAc (80 mL), and the organic layer was
successively washed with solutions of saturated NaHCO3 (40
mL), H2O (40 mL), and saturated NaCl (40 mL) and then dried
over anhydrous MgSO4 and evaporated to dryness. The result-
ing oil was chromatographed on a silica gel flash column (Hex/
EtOAc ) 6:4) to give 21b [foam, 1.76 g, 3.03 mmol, 86% from
sugar 4a, Rf ) 0.70 (Hex/EtOAc ) 5:5)]. 1H NMR (CDCl3, 500
MHz) δ 8.12-7.96 (m, 6 H, Ph), 7.69 (1 H, s, 2-H), 7.65-7.29
(m, 9 H, Ph), 6.13 (d, 1 H, J ) 5.1 Hz), 5.96-5.93 (m, 2 H),
4.88 (dd, 1 H, J ) 2.9 & 12.4 Hz), 4.81 (dd, 1 H, J ) 3.4 & 6.6
Hz), 4.71 (dd, 1 H, J ) 3.6 & 12.3 Hz); 13C NMR (CDCl3, 75
MHz) δ 166.5, 165.6, 165.2, 134.4, 134.3, 134.1, 133.1, 130.3,
130.2, 130.1, 129.44, 129.24, 129.04, 129.02, 128.87, 128.54,
128.07, 113.2, 87.6, 81.3, 74.8, 71.4, 63.8; MS (FAB) m/z 105
(100), 445 (64), 581 (1.53) (M + 1); HRMS calcd for C29H23-
Cl2N2O7 (M + 1) 581.0882, found 581.0904. Anal. (C29H22-
Cl2N2O7) C, H, N.
1
EtOAc); H NMR (DMSO-d6, 360 MHz) δ 5.44 (s, 2 H, 1′-H),
4.66 (bs, 2 H, 2 × OH), 3.58 -3.52 (m, 1 H, CH), 3.44 (dd, 2 H,
J ) 4.7 & 11.4 Hz, CH2), 3.31 (dd, 2 H, J ) 6.1 & 11.4 Hz,
CH2). Anal. (C7H9Br3N2O3) C, H, N.
2,4,5-Trichloro-1-[(2-acetoxyethoxy)methyl]imid-
azole (18a). Compound 18a (oil, 0.41 g, 1.43 mmol, 84%) was
prepared from 2,4,5-trichloroimidazole18 (3a, 0.30 g, 1.7 mmol)
using NaH (60% in mineral oil, 0.104 g, 2.6 mmol, 1.5 equiv),
CH3CN (25 mL), and then (2-acetoxyethoxy)methylbromide28
(17, 0.336 g, 1.7 mmol, 1.0 equiv) by the method described for
15a and purified by column chromatography (Hex/EtOAc )
8:2): 1H NMR (DMSO-d6, 360 MHz) δ 5.37 (s, 2 H, 1′-H), 4.07
(t, 2 H, J ) 4.5 Hz, CH2), 3.70 (t, 2 H, J ) 4.5 Hz, CH2), 1.95
(s, 3 H, CH3).
2-Bromo-4,5-dichloro-1-[(2-acetoxyethoxy)methyl]im-
idazole (18b). Compound 18b (0.626 g, 1.90 mmol, 82%) was
prepared from 2-bromo-4,5-dichloroimidazole18 (3b, 0.50 g, 2.3
mmol) using NaH (60% in mineral oil, 0.12 g, 3.0 mmol, 1.3
equiv), CH3CN (10 mL), and then 17 (0.57 g, 3.0 mmol, 1.3
equiv) by the method described for 15a and purified by column
chromatography (Hex/EtOAc ) 8:2): mp 68-71 °C (Hex/
EtOAc); 1H NMR (CDCl3, 300 MHz) δ 5.38 (s, 2 H, 1′-H), 4.23
(t, 2 H, J ) 4.6 Hz, CH2), 3.76 (t, 2 H, J ) 4.6 Hz, CH2), 2.08
(s, 3 H, CH3). Anal. (C8H9BrCl2N2O3) C, H, N.
4,5-Dibromo-1-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-
imidazole31,32 (21c). To a solution of 4,5-dibromoimidazole34
(20c, 0.45 g, 2.0 mmol) in dry CH3CN (30 mL) was added BSA
(0.60 mL, 0.488 g, 2.4 mmol, 1.2 equiv) and the reaction
mixture was stirred at 35 °C for 20 min. To this solution was
added 4b (0.76 g, 2.4 mmol, 1.2 equiv) and TMSOTf (0.46 mL,
0.533 g, 2.4 mmol, 1.2 equiv) and the mixture stirred at 65 °C
for 90 min. The reaction mixture was cooled to room temper-
ature and the acetonitrile was removed under reduced pres-
sure. To the residue was added EtOAc (40 mL), and the organic
layer was successively washed with solutions of saturated
NaHCO3 (20 mL), H2O (20 mL), and saturated NaCl (20 mL)
and then dried over anhydrous MgSO4 and evaporated to
dryness. The resulting oil was purified by column chromatog-
raphy (Hex/EtOAc ) 5:5) to give 21c (oil, 0.91 g, 1.89 mmol,
95%, Rf ) 0.29): 1H NMR (CDCl3, 500 MHz) δ 7.80 (s, 1 H,
2-H), 5.81 (d, 1 H, J ) 5.1 Hz, 1′-H), 5.50 (t, 1 H, J ) 5.2 Hz),
5.36 (t, 1 H, J ) 5.2 Hz), 4.41-4.38 (m, 1 H), 4.33-4.32 (m, 2
H), 2.10 (s, 3 H, CH3), 2.09 (s, 3 H, CH3), 2.07 (s, 3 H, CH3);
13C NMR (CDCl3, 75 MHz) δ 170.5, 169.9, 169.4, 135.8, 118.8,
103.0, 88.6, 74.1, 70.2, 62.9, 50.6, 21.1, 20.9, 20.8; MS (CI, CH4)
m/z 259 (100), 405 (16), 483 (9.62) (M + 1), 485 (18.29) (M +
3), 487 (9.50) (M + 5); HRMS calcd for C14H17Br2N2O7 (M + 1)
482.9402, found 482.9405.
2,4,5-Tribromo-1-[(2-acetoxyethoxy)methyl]imid-
azole (18c). Compound 18c (0.411 g, 0.98 mmol, 61%) was
prepared from 2,4,5-tribromoimidazole18 (3b, 0.50 g, 1.6 mmol)
using NaH (60% in mineral oil, 0.088 g, 2.2 mmol, 1.38 equiv),
CH3CN (40 mL), and then 17 (0.40 g, 2.1 mmol, 1.3 equiv) by
the method described for 15a and purified by column chro-
matography (Hex/EtOAc ) 8:2, oil, 59%): 1H NMR (DMSO-
d6, 360 MHz) δ 5.37 (s, 2 H, 1′-H), 4.09 (t, 2 H, J ) 4.5 Hz,
CH2), 3.69 (t, 2 H, J ) 4.5 Hz, CH2), 1.98 (s, 3 H, CH3).
2,4,5-Trichloro-1-[(2-hydroxyethoxy)methyl]imid-
azole (19a). Compound 19a (0.26 g, 1.07 mmol, 83%) was
obtained from the deprotection of 18a (0.37 g, 1.29 mmol) with
Na2CO3 (0.137 g, 1.29 mmol, 1 equiv) in EtOH/H2O [30 mL,
9:1 (v/v)] as described for 7a and then purified by column
chromatography (CHCl3/MeOH ) 97:3): mp 57-58 °C; 1H
NMR (DMSO-d6, 360 MHz) δ 5.37 (s, 2 H, 1′-H), 4.73 (t, 1 H,
J ) 5.2 Hz, OH), 3.53-3.46 (m, 4 H, 2 × CH2). Anal. (C6H7-
Cl3N2O2) C, H, N.
2-Bromo-4,5-dichloro-1-[(2-hydroxyethoxy)methyl]im-
idazole (19b). Compound 19b (0.372 g, 1.28 mmol, 68%) was
obtained from the deprotection of 18b (0.623 g, 1.88 mmol)
with Na2CO3 (0.416 g, 3.92 mmol, 2.1 equiv) in EtOH/H2O [50
mL, 9:1 (v/v)] as described for 7a and purified by column
1
chromatography (Hex/EtOAc ) 1:1): mp 89-91 °C; H NMR
4,5-Dichloro-1-(â-D-ribofuranosyl)imidazole (22b). A
mixture of 21b (0.87 g, 1.50 mmol) and 28% NH4OH/MeOH/
acetone [30 mL, 2:2:1 (v/v/v)] was stirred at room temperature
for 12 h. The solvents and NH3 were removed under reduced
pressure. The resulting oil was chromatographed on a silica
(DMSO-d6, 360 MHz) δ 5.35 (s, 2 H, 1′-H), 4.74 (bs, 1 H, OH),
3.51-3.44 (m, 4 H, 2 × CH2). Anal. (C6H7BrCl2N2O2) C, H, N.
2,4,5-Tribromo-1-[(2-hydroxyethoxy)methyl]imid-
azole (19c). Compound 19c (0.21 g, 0.55 mmol, 75%) was

Polyhalogenated Imidazole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5751
gel flash column (EtOAc/MeOH ) 98:2) to give the crude
product. The product was recrystallized from Hex/EtOAc to
give 22b [0.359 g, 1.33 mol, 89%, Rf ) 0.33 (EtOAc/MeOH )
95:5)]: mp 103-104 °C (Hex/EtOAc); 1H NMR (DMSO-d6, 500
MHz) δ 8.14 (s, 1 H, 2-H), 5.63 (d, 1 H, J ) 6.0 Hz, OH), 5.52
(d, 1 H, J ) 5.4 Hz, 1′-H), 5.28 (d, 1 H, J ) 5.2 Hz, OH), 5.08
(t, 1 H, J ) 5.3 Hz, OH), 4.32 (dd, 1 H, J ) 5.3 & 10.6 Hz),
4.06 (dd, 1 H, J ) 4.8 & 9.2 Hz), 3.92 (dd, 1 H, J ) 3.8 & 7.7
Hz), 3.63-3.51 (m, 2 H, 5′-H); 13C NMR (DMSO-d6, 125 MHz)
δ 135.3, 125.8, 112.9, 89.7, 86.4, 75.1, 70.6, 61.6; MS (CI, CH4)
m/z 137 (71), 139 (49), 141 (10), 269 (100) (M + 1), 270 (14)
(M + 2), 271 (64) (M + 3), 273 (14) (M + 5); HRMS calcd for
C8H11Cl2N2O4 (M + 1) 269.0096, found 269.0084. Anal. (C8H10-
Cl2N2O4) C, H, N.
Following virus adsorption, the compounds, prepared as 10
mg/mL stock solutions in DMSO, were diluted with growth
medium and were added to duplicate wells in four to eight
selected concentrations. After incubation at 37 °C for 7-10
days, cell sheets were fixed and stained with crystal violet,
and microscopic plaques were 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.
HSV-1 ELISA. An ELISA was employed39 to detect HSV-
1. Ninety-six-well cluster dishes were planted with 10000
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 horseradish peroxidase conjugated rabbit anti-HSV-1
antibody was added. Following removal of the antibody
containing solution, plates were rinsed and then developed by
adding 150 µL per well of a solution of tetramethylbenzidine
as substrate. The reaction was stopped with H2SO4 and
absorbance was read at 450 and 570 nm. Drug effects were
calculated as a percentage of the reduction in absorbance in
the presence of each drug concentration compared to absor-
bance obtained with virus in the absence of drug.
Cytotoxicity Assays. Two different assays were used for
routine cytotoxicity testing. (i) Cytotoxicity produced in sta-
tionary HFF cells was determined by microscopic inspection
of cells not affected by the virus used in plaque assays.37 (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.40 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 were read at 570 nm
in a spectrophotometer designed to read 96-well ELISA assay
plates.
4,5-Dibromo-1-(â-D-ribofuranosyl)imidazole (22c). A
mixture of 21c (0.91 g, 1.89 mmol) and MeONa (0.41 g, 7.55
mmol, 4 equiv) in MeOH (40 mL) was stirred at room
temperature for 1 h. The methanol was evaporated under
reduced pressure. H2O (20 mL) was added to the residue and
then extracted with EtOAc (40 mL), washed with H2O (50 mL),
and dried (anhydrous Na2SO4), and the solvent was removed
under reduced pressure. The resulting mixture was purified
by column chromatography (EtOAc/MeOH ) 99:1) to give the
crude product [oil, 0.295 g, 0.824 mmol, 44%, Rf ) 0.27 (EtOAc/
MeOH ) 97:3)]. The oil was crystallized from a mixture of
CHCl3/EtOAc to give 22c (0.143 g, 0.40 mmol, 21%): mp 105-
1
107 °C (CHCl3/EtOAc); H NMR (DMSO-d6, 300 MHz) δ 8.21
(s, 2-H), 5.59 (d, 1 H, J ) 6.1 Hz, OH), 5.51 (d, 1 H, J ) 5.3
Hz, 1′-H), 5.26 (d, 1 H, J ) 5.1 Hz, OH), 5.07 (t, 1 H, J ) 5.3
Hz, OH), 4.3 (t, 1 H, J ) 5.0 Hz), 4.05 (t, 1 H, J ) 4.3 Hz),
3.91 (dd, 1 H, J ) 3.7 & 7.6 Hz), 3.61 (dd, 1 H, J ) 3.7 & 12.0
Hz, 5′-H), 3.52 (dd, 1 H, J ) 3.6 & 12.0 Hz, 5′-H); 13C NMR
(DMSO-d6, 75 MHz) δ 137.1, 116.5, 103.4, 89.9, 85.5, 74.4, 69.8,
60.7; MS (CI, CH4) m/z 225 (51), 227 (100), 229 (49), 357 (6)
(M + 1), 359 (11) (M + 3), 361 (6) (M + 5); HRMS calcd for
C8H11Br2N2O4 (M + 1) 356.9086, found 356.9088. Anal. (C8H10-
Br2N2O4) C, H, N.
Biological Evaluation. Cell Culture Procedures. The
routine growth and passage of KB, BSC-1, and HFF cells were
performed in monolayer cultures using minimal essential
medium (MEM) with either Hanks salts [MEM(H)] or Earle
salts [MEM(E)] supplemented with 10% calf serum 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.36
Virological Procedures. 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.37 High titer HSV-1 stocks were prepared by infecting
KB cells at an moi of <0.1 also as detailed previously.37 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.38 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 and 2 or 3 days for HSV-1.
Plaques were enumerated under 20-fold magnification in wells
having the dilution that 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.
Data Analysis. Dose-response relationships were used to
quantitate drug effects by linear regression of the percent
inhibition of parameters derived in the preceding assays
against log drug concentrations. Fifty percent inhibitory
concentrations (IC50’s) were calculated from the linear portions
of the regression lines. Samples containing positive controls
(acyclovir for HSV-1, GCV for HCMV, and 2-acetylpyridine
thiosemicarbazone for cytotoxicity) were used in all assays.
Acknowledgment. We thank Julie M. Breitenbach,
Kathy Z. Borysko, and Roger G. Ptak for expert perfor-
mance of antiviral and cytotoxicity assays. These studies
were supported by research grants U19-AI31718 and
P01-AI46390 from the National Institutes of Health.
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