Synthesis, antiviral activity, and mode of action of some 3-substituted 2,5,6-trichloroindole 2′- and 5″-deoxyribonucleosides

Article abstract of DOI:10.1021/jm0400606

A series of chlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The 2′- and 5′-deoxy derivatives of the reported 3-formyl-2,5,6-tric

Full text of DOI:10.1021/jm0400606

J. Med. Chem. 2004, 47, 5773-5782
5773
Synthesis, Antiviral Activity, and Mode of Action of Some 3-Substituted
2,5,6-Trichloroindole 2′- and 5′-Deoxyribonucleosides
John D. Williams,† Roger G. Ptak,‡,§ John C. Drach,‡ and Leroy B. Townsend*,†,#
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature,
Sciences and the Arts, and Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan 48019
Received March 4, 2004
A series of chlorinated indole nucleosides has been synthesized and tested for activity against
human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity.
The 2′- and 5′-deoxy derivatives of the reported 3-formyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)-
indole (FTCRI) and 3-cyano-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole (CTCRI) were synthesized
by either a modification of the appropriate 3-unsubstituted sugar-modified nucleoside analogues
or by a glycosylation of 3-substituted heterocycles with a protected R-chlorosugar. The
modifications were guided in part by structural similarity to the corresponding series of
chlorinated benzimidazole ribonucleosides and the fact that 5′-deoxy analogues of 2,5,6-trichloro-
1-(â-D-ribofuranosyl)benzimidazole (TCRB) are very active against HCMV. The 5′-deoxy
analogues of FTCRI and CTCRI were nearly as active as FTCRI and CTCRI, suggesting that
the chlorinated benzimidazole nucleosides and the chlorinated indole nucleosides act in a similar
manner. This hypothesis was supported by time-of-addition studies using FTCRI and by the
resistance of TCRB-resistant strains of HCMV to four different 3-substituted indole ribonucleo-
sides. The 2′-deoxy analogues of the trichlorinated indole nucleosides also had potent antiviral
activity, in contrast to decreased activity and selectivity observed for 2′-deoxy TCRB compared
to TCRB. In addition, 3-acetyl-2,5,6-trichloro-1-(2-deoxy-â-D-ribofuranosyl)indole was also active
and much less cytotoxic (HCMV IC50 ) 0.30 µM, HFF CC50 >100 µM) than previous analogues.
None of the analogues had significant activity against HSV-1.
Introduction
a wide range of nucleoside analogues, including 2,5,6-
trichloro-1-(â-D-ribofuranosyl)benzimidazole (TCRB, 1,
Figure 1).9 Although this compound demonstrated ex-
cellent antiviral activity and selectivity in vitro, it is
degraded (via glycosidic bond cleavage) too rapidly in
vivo to be of interest as a clinical candidate.10 Further
investigations have led to the syntheses of numerous
TCRB analogues with stabilized glycosidic bonds,11-13
including the clinical candidate maribavir.14
We recently reported that certain indole nucleosides
including 3-formyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)-
indole (FTCRI, 2, Figure 1) and 3-cyano-2,5,6-trichloro-
1-(â-D-ribofuranosyl)indole (CTCRI, 3, Figure 1) are
potent and selective inhibitors of HCMV replication in
vitro.15 On the basis of the structural similarity between
TCRB and FTCRI, we wanted to determine whether
FTCRI shares the unique mode of action demonstrated
by TCRB.16
Human cytomegalovirus (HCMV) is an opportunistic
pathogen that is endemic in both industrialized and
developing nations.1 It is estimated that 50% of the
American public is seropositive for HCMV.2 Although
HCMV poses little risk to healthy individuals, a variety
of immunocompromised populations are susceptible to
HCMV-related pathologies. AIDS patients, for example,
are susceptible to retinitis and gastritis, transplant
recipients are susceptible to organ rejection, and neo-
nates are at risk for a host of birth defects and
developmental disorders.1,3
There are currently five FDA-approved drugs avail-
able for the treatment of HCMV, namely, ganciclovir
(CGV),4 valganciclovir,5 cidofovir,6 foscarnet,7 and fo-
mivirsen.8 All of these compounds suffer limitations,
however, including poor bioavailability and toxicity.
Furthermore, all of the licensed compounds (with the
exception of fomivirsen) act upon the viral DNA poly-
merase, making the emergence of new drug-resistant
viral strains more likely.
Two virological lines of evidence can be used to
support our hypothesis that TCRB, FTCRI, and CTCRI
share a common mode of action. First, because TCRB
exerts its inhibitory effect late in the viral lifecycle,16
a
The search for new compounds with fewer or less
time-of-addition study would be useful in determining
whether this feature is shared with FCTRI and CTCRI.
Second, several viral strains that are resistant to TCRB
have been isolated and characterized in our laborato-
ries.17 If the analogues of FTCRI and CTCRI show
reduced efficacy against these mutant virus strains,
then the specific mutations found in the viruses would
be implicated in the mode of action for the indole
nucleosides as well.
severe limitations has led our laboratory to synthesize
* To whom correspondence should be addressed. Address: Depart-
ment of Medicinal Chemistry, College of Pharmacy, University of
Michigan, 428 Church Street, Ann Arbor, MI, 48109-1065. Phone:
(734) 764-7547. Fax: (734) 763-5633. E-mail: ltownsen@umich.edu.
† Department of Medicinal Chemistry.
‡ Department of Biologic and Materials Sciences.
§ Present Address: Southern Research Institute, 431 Aviation Way,
Frederick, MD 21701.
# Department of Chemistry.
10.1021/jm0400606 CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/13/2004

5774 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
Formylation and cyanation of 9 were performed as
above, and the intermediates 10a and 10b were depro-
tected with 90% aqueous trifluoroacetic acid to provide
the 5′-deoxy-5′-azido analogues 3-formyl-2,5,6-trichloro-
1-(5-deoxy-5-azido-â-D-ribofuranosyl)indole (11a) and
3-cyano-2,5,6-trichloro-1-(5-deoxy-5-azido-â-D-ribofura-
nosyl)indole (11b).
Synthesis of the 5′-deoxy-5′-fluoro derivatives pro-
ceeded once again from the partially protected indole
nucleoside analogue 4. The unprotected hydroxyl group
at the 5′-position of 4 was fluorinated using Deoxofluor
reagent26 to provide 2,5,6-trichloro-1-(5-deoxy-5-fluoro-
â-D-ribofuranosyl)indole (12, Scheme 3) in good yield.
As above, formylation and cyanation of intermediate 12
was followed by deprotection with 90% aqueous trifluo-
roacetic acid to provide 3-formyl-2,5,6-trichloro-1-(5-
deoxy-5-fluoro-â-D-ribofuranosyl)indole (14a) and 3-cy-
ano-2,5,6-trichloro-1-(5-deoxy-5-fluoro-â-D-ribofurano-
syl)indole (14b).
We initially elected to use a similar strategy for the
synthesis of our target 2′-deoxy nucleoside analogues
(i.e., modification of preformed 2′-deoxy nucleosides).
Unfortunately, modification of the intermediate 1513
proved to be difficult in the case of formylation and
acetylation. Formylation of 15 under the Vilsmeier-
Haack conditions used previously did not produce the
desired nucleoside analogue 16. Instead, the formylated
heterocycle was the only material isolated in any
significant quantity. Acetylation of 15 also resulted in
a glycosidic bond cleavage of the nucleoside instead of
providing the intermediate 17 (Scheme 4).
Although the strategy is less efficient, we then elected
to synthesize the target nucleosides by glycosylation of
the preformed heterocycles with the 2-deoxy R-chloro-
sugar 20.27 The formylated heterocycle 19 and acety-
lated heterocycle 23 could be easily synthesized by a
Vilsmeier-Haack formylation and a modified Friedel-
Crafts acylation,28 respectively (Scheme 5). Compounds
19 and 23 were glycosylated with the R-chlorosugar 20
to provide the protected nucleoside analogues 21 and
24, respectively. However, deprotection of these inter-
mediates produced two very different outcomes. The
3-acetyl derivative 24 was deprotected and provided the
expected nucleoside 3-acetyl-2,5,6-trichloro-1-(2-deoxy-
â-D-ribofuranosyl)indole (25). When identical conditions
were applied to the 3-formyl analogue 21, they produced
only the 2-methoxy derivative 22. We were unable to
find conditions that would afford 21 without a concomi-
tant displacement of the 2-chloro substituent. Interest-
ingly, the protected 3-nitrile derivative 26, synthesized
from 15 without glycosidic bond cleavage, was depro-
tected with methanolic sodium methoxide to provide
both the 2-chloro analogue 3-cyano-2,5,6-trichloro-1-(2-
deoxy-â-D-ribofuranosyl)indole (27a) and the 2-methoxy
analogue 3-cyano-2-methoxy-5,6-dichloro-1-(2-deoxy-â-
D-ribofuranosyl)indole (27b) in a 2.5:1 ratio.
Figure 1. TCRB and analogous indole nucleosides.
We would also like to establish a structural line of
evidence that the benzimidazole and indole nucleosides
share a common pathway. It has been established that
phosphorylation of TCRB at the 5′-position is not
required for antiviral activity because the 5′-deoxy, 5′-
deoxy-5′-fluoro, and 5′-deoxy-5′-azido analogues of TCRB
are all as active as TCRB itself.18-20 These hypothesized
similarities between the benzimidazole and indole nu-
cleosides prompted us to synthesize a series of 5′-
deoxyindole nucleosides. If these 5′-deoxy FTCRI and
CTCRI analogues retained their antiviral activity, this
would strongly suggest that (as is the case for TCRB21)
5′-phosphorylation is not required for antiviral activity
and would further strengthen our hypothesis that both
chlorinated indole and benzimidazole nucleosides share
a common mechanism of action.
In contrast to the 5′-deoxy analogue of TCRB, its 2′-
deoxy analogue was not as active as the ribofuranosyl
analogues.22 This difference in antiviral activity prompted
us to also synthesize the 2′-deoxy analogues of FTCRI
and CTCRI in order to determine whether the same
trend observed for 2′-deoxy TCRB would hold for these
2′-deoxyindole nucleosides.
Results and Discussion
Chemistry. For the desired series of 5′-deoxyindole
nucleosides, two methodologies could be pursued: (1)
modification of an appropriate sugar precursor followed
by glycosylation and (2) modification of existing nucleo-
side precursors (with the glycosidic bond already estab-
lished). The latter strategy was chosen for the synthesis
of the target nucleoside analogues because it had the
advantage of requiring fewer overall synthetic trans-
formations.
To synthesize the desired 5′-deoxy analogues, the
partially protected intermediate 2,5,6-trichloro-1-(2,3-
O-isopropylidene-â-D-ribofuranosyl)indole13 (4, Scheme
1) was tosylated at the 5′-position and this compound
(5) was then reduced with sodium borohydride in
DMSO23 to provide the deoxygenated intermediate 6.
This nucleoside was either formylated under Viles-
meier-Haack24 conditions using phosphorus oxychloride
in DMF or cyanated with chlorosulfonyl isocyanate
(CSI) followed by DMF25 to provide the 3-substituted
analogues 7a and 7b, respectively. These compounds
were then deprotected with 90% aqueous trifluoroacetic
acid to yield the desired 5′-deoxyindole nucleosides
3-formyl-2,5,6-trichloro-1-(5-deoxy-â-D-ribofuranosyl)in-
dole (8a) and 3-cyano-2,5,6-trichloro-1-(5-deoxy-â-D-ri-
bofuranosyl)indole (8b).
We also pursued the synthesis of some other selected
2′-deoxyindole nucleosides. The 3-iodinated nucleoside
analogue 28 was available from a previous investigation
in our lab.15 This prompted us to use 28 in the synthesis
of some selected 3-heteroaryl derivatives via palladium-
catalyzed coupling reactions. Compound 28 was sub-
jected to a Suzuki-type coupling with either 2-furylbo-
ronic acid or 3-thienylboronic acid according to the
The 5′-deoxy-5′-azido analogues of FTCRI and CTCRI
were also synthesized from the 5′-tosyloxy intermediate
5 (Scheme 1). The nucleoside 5 was treated with sodium
azide in DMSO to provide compound 9 (Scheme 2).

Trichloroindole Deoxyribonucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5775
Scheme 1a
a Reagents and conditions: (a) TsCl, pyridine, 20 °C, 16 h; (b) NaBH4, DMSO, 75 °C, 16 h; (c) POCl3, DMF, 70 °C, 16 h or CSI, CH2Cl2,
20 °C, 16 h, then DMF, 20 °C, 1 h; (d) 90% TFA, 20 °C, 2 min.
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) Deoxofluor, CH2Cl2, 20 °C, 16
h; (b) POCl3, DMF, 70 °C, 16 h or CSI, CH2Cl2, 20 °C, 16 h, then
DMF, 20 °C, 1 h; (c) 90% TFA, 20 °C, 2 min.
a Reagents and conditions: (a) NaN3, DMF, 75 °C, 16 h; (b)
POCl3, DMF, 70 °C, 16 h or CSI, CH2Cl2, 20 °C, 16 h, then DMF,
20 °C, 1 h; (c) 90% TFA, 20 °C, 2 min.
Scheme 4a
procedure of Huff29 to provide 29a and 29b, respectively
(Scheme 6). These 3-heteroaryl analogues were then
deprotected with methanolic sodium methoxide to pro-
vide 3-(2-furyl)-2,5,6-trichloro-1-(2-deoxy-â-D-ribofura-
nosyl)indole (30a) and 3-(3-thienyl)-2,5,6-trichloro-1-(2-
deoxy-â-D-ribofuranosyl)indole (30b) in good yields.
It has been established that the 2-bromo analogue of
TCRB [i.e., 2-bromo-5,6-dichloro-1-(â-D-ribofuranosyl)-
benzimidazole, BDCRB] has greater antiviral activity
and selectivity than TCRB itself.9 Our preliminary
evaluation of the chlorinated 2′-deoxy nucleosides (vide
supra) revealed that 3-acetyl-2,5,6-trichloro-1-(2-deoxy-
â-D-ribofuranosyl)indole (25) was a particularly selective
compound. This prompted us to synthesize the 2-bromo
analogue of 25 to determine whether this modification
at the 2-position of the heterocycle would increase the
activity or selectivity as was the case in the benzimi-
dazole series.
However, our initial attempts to synthesize the
requisite 3-acetyl-2-bromo-5,6-dichloroindole (32, Scheme
7) were complicated by a halogen exchange at the
2-position. Acetylation of 3113 using the same proce-
dure28 as that used for the 2-chloro congener 18 provided
a mixture of the desired 2-bromo heterocycle 32 along
with a substantial amount of the 2-chloro analogue 23.
Replacing the tin(IV) chloride with tin(IV) bromide did
a Reagents and conditions: (a) POCl3, DMF, 60 °C, 16 h; (b)
AcCl, AlCl4, CH2Cl2, 20 °C, 15 min.
not improve the outcome but instead resulted in the
exclusive production of the undesired 2-chloro analogue
23. When tin(IV) chloride and acetyl chloride were
replaced by tin(IV) bromide and acetyl bromide, the
desired product 32 was produced in good yield without
the concomitant halogen exchange. The requisite het-
erocycle was then glycosylated as above with R-chloro-
sugar 20 and the intermediate (33) was deprotected
with sodium methoxide to yield the desired nucleoside
3-acetyl-2-bromo-5,6-dichloro-1-(2-deoxy-â-D-ribofurano-
syl)indole (34).
Biological Evaluation. The compounds were tested
for antiviral activity against HCMV and HSV-1 and for
cytotoxicity in HFF and KB cell lines. Among the series

5776 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
Scheme 5a
a Reagents and conditions: (a) POCl3, DMF, 60 °C, 16 h; (b) NaH, THF, 0 °C, 10 min, then 20, THF/toluene, 20 °C, 16 h; (c) NaOMe,
MeOH, 20 °C, 16 h; (d) AcCl, SnCl4, CH2Cl2, MeNO2, 0-20 °C, 45 min; (e) CSI, CH2Cl2, 20 °C, 16 h, then DMF, 20 °C, 1 h.
Scheme 6a
analogues with 2-methoxy substituents, 22 and 27b,
were substantially less active than their 2-chloro coun-
terparts. However, we were surprised by the potent anti-
HCMV activity of the corresponding 2-chloro analogues
25 and 27a. Neither 25 nor 27a were substantially less
active than FTCRI and CTCRI. In fact, the 3-acetyl
analogue 25 was also much less toxic than any previous
chlorinated indole nucleosides in this series. The selec-
tivity of 25 is quite remarkable, with the concentration
required for toxicity approximately 1000-fold more than
that needed for antiviral activity.
The 3-heteroaryl analogues of 2′-deoxyindole nucleo-
sides (30a and 30b) were again less active than the
either 2′-deoxy nucleoside analogues 25 and 27a or
ribofuranosyl analogues FTCRI (2) or CTCRI (3). In
contrast, the 2-bromo analogue of the very selective
compound 25 (i.e., 34) was as active and no more
cytotoxic than 25 (see Table 1), although we had
expected an increase in activity and selectivity.
Mechanism of Action. In our studies to determine
the mechanism of action for the chlorinated indole
nucleosides, we have established several lines of evi-
dence. Because TCRB has a unique mechanism of action
and acts very late in the viral replication cycle,16 the
time-of-addition study is especially diagnostic. Data in
Figure 2 demonstrate that as expected, TCRB acted late
in the viral replication cycle, at a time later than the
DNA polymerase inhibitor ganciclovir (GCV). The indole
nucleoside FTCRI (2) also acted very late in the replica-
tion cycle, losing its effectiveness later than GCV, and
at a time that was virtually indistinguishable from
TCRB. Thus, FTCRI acted very late in the viral replica-
tion cycle at a time when DNA synthesis had been
mostly concluded.
a Reagents and conditions: (a) Ar-B(OH)2, Pd(OAc)2, (o-tol)3P,
Na2CO3, DMF/n-PrOH/H2O, 120 °C, 5-15 min; (b) NaOMe,
MeOH, 20 °C, 45-90 min.
of 5′-deoxy nucleoside analogues, most were as potent
or more potent than their ribofuranosyl counterparts
(Table 1). Both 5′-deoxy analogues 8a and 8b were as
potent as their congeners 2 and 3, respectively. This
strongly suggests that, as is the case for TCRB and its
5′-deoxy analogue, no phosphorylation is required for
antiviral activity in these chlorinated indole nucleosides.
The 5′-deoxy-5′-fluoro and 5′-deoxy-5′-azido analogues
of FTCRI (i.e., 11a and 14a) were also nearly as active
as FTCRI itself, but the corresponding analogues of
CTCRI (i.e., 11b and 14b) were substantially less active.
The reason for this difference in activity among the
different 3-substituted indole nucleosides is unknown.
As expected from our experiences with 2-substituted
indole nucleosides in a previous study,30 the 2′-deoxy
As a second line of evidence, the activity of four
chlorinated indole nucleosides was determined in viral

Trichloroindole Deoxyribonucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5777
Scheme 7a
a Reagents and conditions: (a) AcCl, SnCl4, CH2Cl2, MeNO2, 0-20 °C, 45 min; (b) AcCl, SnBr4, CH2Cl2, MeNO2, 0-20 °C, 45 min; (c)
AcBr, SnBr4, CH2Cl2, MeNO2, 0-20 °C, 45 min; (d) NaH, THF, 0 °C, 10 min, then 20, THF/toluene, 20 °C, 16 h; (e) NaOMe, MeOH, 20
°C, 16 h.
Table 1. Antiviral Activity and Cytotoxicity of 2′- and 5′-Deoxyribosylindole Nucleosides
50% inhibitory concentration (µM)
antiviral cytotoxicity
compd
R2
R3
R2′
R5′
HCMV plaquea
HSV-1 ELISAb
HFF visualc
KB growthc
8a
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-OMe
-Cl
-Cl
-OMe
-Cl
-Cl
-Br
-Cl
-Cl
-CHO
-CN
-OH
-OH
-OH
-OH
-OH
-OH
-H
-H
0.30
0.19
0.32
3.0
20
32
25
8b
-H
32
32
40
11a
11b
14a
14b
22
-CHO
-CN
-N3
-N3
-F
10
32
25
15
>100d
32
60
-CHO
-CN
0.60
2.7
20
20
-F
40
32
50
-CHO
-COCH3
-CN
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
>100
0.30
0.30
68
3.6
38
>100
8.0
35
100
>100
32
20
25
-H
400
60
27a
27b
30a
30b
34
-H
-CN
-H
90
>100
3.2
>100
50
-(2-furyl)
-(3-thienyl)
-COCH3
-CHO
-CN
-H
20
-H
20
32
50
-H
0.34
0.23
0.55
2.9
>100
40
>100
45
>100
45
2e
-OH
-OH
3e
>100
102
32
35
TCRBf
238
100
>100
118
>100
210
5′-deoxy TCRBg
2′-deoxy TCRBh
BDCRBf
0.36
20
41
130
3.5
100
>100
>100
0.7
GCVi
7.4
a Plaque reduction assays were performed in duplicate wells as described in the text. b Compounds were assayed by ELISA in
quadruplicate wells. c Visual cytotoxicity was scored on HFF cells at the time of HCMV plaque enumeration in duplicate wells; inhibition
of KB cell growth was determined in triplicate wells as described in the text. d >100 indicates an IC50 greater than the highest concentration
tested. e Data for compounds 2 and 3 published previously as compounds 9a and 9b, respectively, in ref 15. f Data for TCRB and BDCRB
published previously as compounds 9 and 11, respectively, in ref 9. g Data for 5′-deoxy TCRB published previously as compound 210 in
ref 19. h Data for 2′-deoxy TCRB published previously as compound 3b in ref 22. i Averages from 108, 33, and 3 experiments, respectively,
using GCV.
strains that are resistant to TCRB. Data in Table 2
compare the activity of these compounds to that of
BDCRB and GCV in wild-type HCMV and three viral
strains that are resistant to TCRB and BDCRB. The
data readily establish that all of the indole nucleosides
were less active against the TCRB-resistant viruses

5778 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
that the chlorinated benzimidazole nucleosides (i.e.,
TCRB and BDCRB) share a common viral target with
the chlorinated indole nucleosides (i.e., FTCRI and its
analogues).
A third line of evidence for a common mechanism
between chlorinated indole and benzimidazole nucleo-
sides is our observation that both 5′-deoxy TCRB
analogues and 5′-deoxy FTCRI analogues are potent
inhibitors of HCMV replication (Table 1). These data
strongly suggest that as in the chlorinated benzimida-
zole series21 5′-phosphorylation is not required for
activity in the chlorinated indole series. This parallel
further strengthens the similarities between chlorinated
indole nucleosides and chlorinated benzimidazole nu-
cleosides.
Figure 2. HCMV time-of-addition study comparing the
activity of the indole nucleoside FTCRI (2) to that of the
benzimidazole nucleoside TCRB (1) and the DNA synthesis
inhibitor GCV. Drugs were added to cells infected with HCMV
at time zero and at the indicated time points. Inhibition of
viral replication was measured by yield reduction assays on
samples taken at 96 h at the end of one viral replication cycle.
Data are presented as the mean of duplicate assays.
Experimental Section
General Procedures. All solvents were dried prior to use
according to known procedures. All reagents were obtained
from commercial sources or were synthesized from literature
procedures and were used without further purification unless
otherwise noted. Air-sensitive reactions were performed under
slight positive pressure of argon. Room temperature is as-
sumed to be between 20 and 25 °C. Evaporation of solvents
was accomplished under reduced pressure (water aspirator,
12 mmHg) at less than 40 °C unless otherwise noted. Chro-
matography solvent systems are expressed in v/v ratios or as
% vol. Melting points were taken on a Mel-Temp apparatus
and are uncorrected. Thin-layer chromatography was per-
formed on silica gel GHLF plates from Analtech (Newark, DE).
Chromatograms were visualized under UV light at 254 nm.
Spectra for all compounds are presented in Supporting Infor-
mation. 1H NMR spectra were obtained at 500 MHz on a
Bruker DRX500 spectrometer. 13C NMR spectra were obtained
at 125 MHz on a Bruker DRX500 spectrometer. 19F NMR
spectra were obtained at 300 MHz on a Bruker DPX300
Table 2. Cross-Resistance between BDCRB and Selected
Indole Nucleosides in BDCRB-Resistant Viruses
1
spectrometer. Chemical shift values for H were determined
relative to an internal tetramethylsilane standard (0.00 ppm).
Chemical shift values for 13C were determined relative to the
solvent used (39.52 ppm for DMSO-d6 and 77.23 ppm for
CDCl3). Chemical shift values for 19F were determined relative
to an external TFA standard (-76.50 ppm). Mass spectrometry
and elemental analysis for selected compounds (listed in
Supporting Information) were performed at the University of
Michigan Department of Chemistry mass spectrometry facility
and elemental analysis facility, respectively.
50% inhibitory concentration (µM)a
viral strain
(mutant gene)
C4
r56
D10
(UL89)
(UL56 +
UL89)
compd
wild type
(UL56)
2,5,6-Trichloro-1-(2,3-O-isopropylidene-5-O-p-toluene-
sulfonyl-â-D-ribofuranosyl)indole (5). 2,5,6-Trichloro-1-
(2,3-O-isopropylidene-â-D-ribofuranosyl)indole13 (4, 0.79 g, 2.0
mmol) was dissolved in dry pyridine (20 mL) to which was
added p-toluenesulfonyl chloride (0.77 g, 4.0 mmol). The
resulting solution was stirred at room temperature for 16 h,
then the solvent was evaporated and the residual oil diluted
with 5% aqueous Na2CO3 (50 mL) and brine (50 mL). This
mixture was extracted with EtOAc (2 × 50 mL) and the
combined organic extracts were dried over MgSO4, filtered, and
evaporated to yield a pale-yellow oil. The oil was dissolved in
CHCl3 (2 mL) and subjected to column chromatography (50
mm × 450 mm) on silica gel with 3:1 hexane/EtOAc. Fractions
containing product were pooled and evaporated to yield 0.83
g (75%) of 5 as a colorless foam: mp 65-70 °C; Rf ) 0.4 (3:1
hexane/EtOAc).
2,5,6-Trichloro-1-(2,3-O-isopropylidene-5-deoxy-â-D-ri-
bofuranosyl)indole (6). Compound 5 (693 mg, 1.3 mmol) was
dissolved in dry DMSO (15 mL) to which was added sodium
borohydride (0.24 g, 6.3 mmol). The resulting solution was
heated on a 45 °C oil bath for 16 h, then cooled to room
temperature and poured into brine (200 mL). The suspension
was extracted with EtOAc (2 × 200 mL). The organic extracts
were washed successively with water (200 mL) and brine (200
mL), then combined and dried over MgSO4, filtered, and
BDCRBb
GCV
0.99 ( 0.12 6.7 ( 0
1.2 1.2
0.46 ( 0.24 1.8 ( 0
12 ( 5
1.2
29 ( 0
2.5
FTCRIb
0.88 ( 0.08 4.8 ( 1.7
UMJD1844b,c 0.26 ( 0.04 1.2 ( 0.1 0.72 ( 0.09 6.2 ( 0.3
UMJD1847b,c 0.22 ( 0
25b
0.22 ( 0
1.6 ( 0.7 0.88 ( 0.03 3.3 ( 1.7
1.8 ( 0.6 0.76 ( 0.07 2.1 ( 0.5
a Plaque reduction assays were performed in duplicate wells as
described in the text. Wild-type virus is Towne strain; UL56, UL89,
and UL56 + UL89 viral strains contain single mutations in the
specified ORF and have been characterized as found in ref 17.b IC50
values were calculated from the average of two experiments.
c Compounds UMJD1844 and UMJD1847 published previously as
compounds 9c and 7a, respectively, in ref 15.
than wild-type HCMV. Resistance to TCRB and BDCRB
has been traced to point mutations in both UL56 and
UL89 genes of HCMV.17 Although the exact nature of
inhibition of these gene products has not been defini-
tively proven,31 both genes are involved in the cleavage
of concatemeric viral DNA into genome-sized pieces
before packaging. Because mutations causing resistance
to TCRB and BDCRB also caused resistance to several
chlorinated indole nucleosides, this is further evidence

Trichloroindole Deoxyribonucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5779
evaporated to yield a clear residue. The residue was dissolved
in CHCl3 (2 mL) and subjected to column chromatography (50
mm × 450 mm) on silica gel with 5:1 hexane/EtOAc. Fractions
containing product were pooled and evaporated to yield 355
mg (74%) of 6 as a colorless oil that solidifies upon standing:
mp 69-71 °C; Rf ) 0.6 (5:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-
deoxy-5-azido-â-D-ribofuranosyl)indole (10a). Compound
9 (283 mg, 0.68 mmol) was treated as per 7a above with dry
DMF (10 mL) and phosphorus oxychloride (1.5 mL, 2.5 g, 16
mmol), then recrystallized from boiling CHCl3/hexane to yield
187 mg (62%) of 10a as a pale-yellow crystalline solid: mp
142-143 °C; Rf ) 0.4 (2:1 hexane/EtOAc).
3-Cyano-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-deoxy-
5-azido-â-D-ribofuranosyl)indole (10b). Compound 9 (426
mg, 1.0 mmol) was treated as per 7b above with chlorosulfonyl
isocyanate (135 µL, 220 mg, 1.5 mmol) and recrystallized from
boiling hexane to yield 240 mg (53%) of 10b as a white
crystalline solid: mp 144-145 °C; Rf ) 0.5 (2:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(5-deoxy-5-azido-â-D-ribo-
furanosyl)indole (11a). Compound 10a (168 mg, 0.38 mmol)
was treated as per 8a above with 90% aqueous TFA (10 mL)
and recrystallized from boiling CHCl3 to yield 117 mg (76%)
of 11a as a pale-yellow crystalline solid: mp 160-161 °C; Rf
) 0.5 (10% MeOH/CHCl3). Anal. (C14H11Cl3N4O4‚1/20CHCl3) C,
H, N.
3-Cyano-2,5,6-trichloro-1-(5-deoxy-5-azido-â-D-ribofura-
nosyl)indole (11b). Compound 10b (216 mg, 0.49 mmol) was
treated as per 8a above with 90% aqueous TFA (10 mL) and
recrystallized from boiling CHCl3 to yield 165 mg (84%) of 11b
as a white powder: mp 161-162 °C; Rf ) 0.5 (10% MeOH/
CHCl3). Anal. (C14H10Cl3N5O3‚1/20CHCl3) C, H, N.
2,5,6-Trichloro-1-(2,3-O-isopropylidene-5-deoxy-5-fluoro-
â-D-ribofuranosyl)indole (12). Compound 413 (0.98 g, 2.5
mmol) was dissolved in dry CH2Cl2 (5 mL) to which was added
bis(2-methoxyethyl)aminosulfur trifluoride (Deoxofluor,26 0.70
mL, 0.84 g, 3.8 mmol). The resulting solution was stirred at
room temperature for 16 h, and then the reaction was
quenched with 5% Na2CO3 (10 mL). The biphasic mixture was
poured into brine (40 mL) and extracted with EtOAc (2 × 50
mL). The combined organic extracts were dried over MgSO4,
filtered, and evaporated to yield a yellow oil. This oil was
dissolved in CHCl3 (1 mL) and subjected to column chroma-
tography (40 mm × 350 mm) on silica gel with 5:1 hexane/
EtOAc. Fractions containing product were pooled and evapo-
rated to yield 0.46 mg (47%) of 12 as a pale-yellow oil. Rf )
0.4 (5:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-
deoxy-5-fluoro-â-D-ribofuranosyl)indole (13a). Compound
12 (232 mg, 0.59 mmol) was treated as per 7a above with dry
DMF (5 mL) and phosphorus oxychloride (0.30 mL, 0.49 g, 3.2
mmol) to yield 126 mg (51%) of 13a as a white crystalline
solid: mp 147-148 °C; Rf ) 0.5 (2:1 hexane/EtOAc).
3-Cyano-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-deoxy-
5-fluoro-â-D-ribofuranosyl)indole (13b). Compound 12 (412
mg, 1.0 mmol) was treated as per 7b above with chlorosulfonyl
isocyanate (0.14 mL, 0.23 g, 1.6 mmol) to yield 198 mg (45%)
of 13b as a white crystalline solid: mp 162-163 °C; Rf ) 0.5
(2:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(5-deoxy-5-fluoro-â-D-ribo-
furanosyl)indole (14a). Compound 13a (133 mg, 0.31 mmol)
was treated as per 8a above with 90% aqueous TFA (5 mL)
and recrystallized from 10% MeOH/CHCl3 and hexane to yield
82 mg (68%) of 14a as a white crystalline solid: mp 207-208
°C; Rf ) 0.5 (10% MeOH/CHCl3). Anal. (C14H11Cl3FNO4) C, H,
N.
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-
deoxy-â-D-ribofuranosyl)indole (7a). Compound 6 (192 mg,
0.51 mmol) was dissolved in dry DMF (5 mL) to which was
added phosphorus oxychloride (0.25 mL, 0.41 g, 2.7 mmol). The
resulting yellow solution was heated on a 60 °C oil bath for
16 h, then cooled to room temperature and poured into 10%
NaHCO3 (200 mL). The aqueous suspension was extracted
with EtOAc (2 × 50 mL), and the combined organic extracts
were washed with brine (50 mL), dried over MgSO4, filtered,
and evaporated to yield a yellow oil. The oil was dissolved in
CHCl3 (1 mL) and subjected to column chromatography (40
mm × 350 mm) on silica gel with 3:1 hexane/EtOAc. Fractions
containing product were pooled and evaporated to yield 180
mg (87%) of 7a as a white solid. A sample was recrystallized
from CHCl3/hexane to yield a white crystalline solid: mp 153-
154 °C; Rf ) 0.3 (5:1 hexane/EtOAc).
3-Cyano-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-deoxy-
â-D-ribofuranosyl)indole (7b). Compound 6 (450 mg, 1.2
mmol) was dissolved in dry CH2Cl2 (10 mL) to which was
added chlorosulfonyl isocyanate (0.20 mL, 0.32 g, 2.2 mmol).
The resulting solution was stirred at room temperature for
16 h, then dry DMF (1 mL) was added, and the solution was
stirred an additional 1 h at room temperature. To the resulting
solution were added 5% Na2CO3 (20 mL) and brine (30 mL).
This aqueous suspension was extracted with EtOAc (2 × 50
mL). The combined organic extracts were dried over MgSO4,
filtered, and evaporated to yield a yellow-orange oil. The oil
was dissolved in CHCl3 (1 mL) and subjected to column
chromatography (40 mm × 350 mm) on silica gel with 5:1
hexane/EtOAc. Fractions containing product were pooled and
evaporated to yield 153 mg (32%) of 7b as a white crystalline
solid: mp 165-166 °C; Rf ) 0.4 (5:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(5-deoxy-â-D-ribofuranosyl)-
indole (8a). Compound 7a (139 mg, 0.34 mmol) was dissolved
in 90% aqueous trifluoroacetic acid (5 mL). The solution was
stirred at room temperature for 5 min, then evaporated to
approximately 1 mL. The remainder was poured into 5%
aqueous Na2CO3 (10 mL) and brine (40 mL). The aqueous
suspension was extracted with EtOAc (2 × 100 mL), and the
combined organic extracts were dried over MgSO4, filtered, and
evaporated to yield a pale-yellow solid. The solid was dissolved
in DMF (0.5 mL) and subjected to column chromatography (40
mm × 350 mm) on silica gel with 10% MeOH/CHCl3. Fractions
containing product were pooled and evaporated to yield a white
solid. This solid was recrystallized from boiling hexane to yield
86 mg (70%) of 8a as a white crystalline solid: mp 217-218
°C; Rf ) 0.6 (10% MeOH/CHCl3). Anal. (C14H12Cl3NO4) C, H,
N.
3-Cyano-2,5,6-trichloro-1-(5-deoxy-â-D-ribofuranosyl)-
indole (8b). Compound 7b (207 mg, 0.52 mmol) was treated
as per 8a above with 90% aqueous TFA (5 mL) and recrystal-
lized from 10% MeOH/CHCl3 and hexane to yield 152 mg (82%)
of 8b as a white crystalline solid: mp 227-228 °C; Rf ) 0.6
(10% MeOH/CHCl3). Anal. (C14H11Cl3N2O3‚1/5H2O) C, H, N.
3-Cyano-2,5,6-trichloro-1-(5-deoxy-5-fluoro-â-D-ribo-
furanosyl)indole (14b). Compound 13b (133 mg, 0.31 mmol)
was treated as per 8a above with 90% aqueous TFA (5 mL)
and recrystallized from EtOAc/hexane to yield 150 mg (88%)
of 14b as a white crystalline solid: mp 246-247 °C; Rf ) 0.6
(10% MeOH/CHCl3). Anal. (C14H10Cl3FN2O3) C, H, N.
3-Formyl-2,5,6-trichloroindole (19). 2,5,6-Trichloroin-
dole13 (18, 2.19 g, 9.9 mmol) was dissolved in dry DMF (25
mL) to which was added phosphorus oxychloride (2.8 mL, 4.6
g, 30 mmol). The solution was heated on a 60 °C oil bath for
16 h, and then the solvent was removed under vacuum (0.5
mmHg, 40 °C). The residual orange oil was poured into cold
water (400 mL), and the pale-yellow suspension was stirred
at 4 °C for 2 h. The suspension was filtered, and the solids
2,5,6-Trichloro-1-(2,3-O-isopropylidene-5-deoxy-5-azido-
â-D-ribofuranosyl)indole (9). Compound 5 (0.77 g, 1.4 mmol)
was dissolved in dry DMF (20 mL) to which was added sodium
azide (1.5 g, 23.0 mmol), and the mixture was heated on a 75
°C oil bath for 16 h. The resulting suspension was cooled to
room temperature and evaporated under high vacuum (0.5
mmHg, 35 °C), and the residue was dissolved in EtOAc (150
mL). The organic solution was washed with brine (50 mL), then
dried over MgSO4, filtered, and evaporated to yield a clear oil.
This oil was dissolved in CHCl3 (2 mL) and subjected to column
chromatography (50 mm × 450 mm) on silica gel with 3:1
hexane/EtOAc. Product-containing fractions were pooled and
evaporated to yield 0.56 mg (95%) of 9 as a clear oil: Rf ) 0.6
(3:1 hexane/EtOAc).

5780 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
were rinsed with cold water (100 mL). The solids were
suspended in boiling MeOH (250 mL), and the suspension was
evaporated to approximately 150 mL and then allowed to stand
at 4 °C for 8 h. The resulting suspension was filtered, and the
solids were rinsed with cold MeOH (25 mL). The solids were
dried in a vacuum oven (60 °C, 12 mmHg) for 12 h to yield
1.80 g (73%) of 19 as a pale-yellow powder: mp >280 °C (dec);
Rf ) 0.5 (5% MeOH/CHCl3).
(220 mg, 4.1 mmol). The suspension was stirred at room
temperature for 16 h, after which time the solution first
clarified and a precipitate then formed. The suspension was
allowed to stand at 4 °C for 4 h and was then filtered, and the
solids were rinsed with cold MeOH (10 mL). The solid was
recrystallized from warm MeOH to yield 0.28 g (74%) of 25 as
a white crystalline solid: mp >200 °C (dec); Rf ) 0.4 (10%
MeOH/CHCl3). Anal. (C15H14Cl3NO4) C, H, N.
3-Cyano-2,5,6-trichloro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-
â-D-ribofuranosyl]indole (26). 2,5,6-Trichloro-1-[3,5-di-O-(p-
toluoyl)-2-deoxy-â-D-ribofuranosyl]indole13 (15, 1.15 g, 2.0
mmol) was treated as per 7b above with chlorosulfonyl
isocyanate (0.35 mL, 0.57 g, 4.0 mmol) and recrystallized from
CHCl3/MeOH to yield 0.64 g (55%) of 26 as a white crystalline
solid: mp 192-193 °C; Rf ) 0.5 (3:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-
â-D-ribofuranosyl]indole (21). Compound 19 (0.63 g, 2.5
mmol) was suspended in dry THF (75 mL) to which was added
60% sodium hydride in mineral oil (0.20 g, 5.0 mmol). The
suspension was stirred at room temperature for 15 min until
gas evolution had ceased, then filtered into a suspension of
3,5-di-(O-p-toluoyl)-2-deoxy-R-D-ribofuranosyl chloride27 (20,
1.00 g, 2.6 mmol) in toluene (25 mL). The resulting solution
was stirred at room temperature for 16 h, then evaporated to
dryness. The residue was suspended in brine (20 mL) and
extracted with EtOAc (2 × 40 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to
yield a pale-yellow solid. The solid was suspended in CHCl3
(10 mL), and the residual solids were filtered off. The filtrate
was evaporated, then dissolved in CHCl3 (2 mL) and subjected
to column chromatography (50 mm × 450 mm) on silica gel
with 3:1 hexane/EtOAc. Fractions containing product were
pooled and evaporated to yield 0.75 g (49%) of 21 as a white
solid. An analytical sample was prepared by recrystallization
from MeOH/CHCl3: mp 153-154 °C; Rf ) 0.3 (3:1 hexane/
EtOAc).
3-Formyl-2-methoxy-5,6-dichloro-1-(2-deoxy-â-D-ribo-
furanosyl)indole (22). Compound 21 (198 mg, 0.33 mmol)
was suspended in dry MeOH (10 mL) to which was added
sodium methoxide (75 mg, 1.4 mmol). The suspension was
stirred at room temperature for 16 h, after which time the
solution clarified. The solvent was then removed under
vacuum, and the residue was suspended in water (50 mL) and
extracted with EtOAc (2 × 100 mL). The combined organic
extracts were washed with brine (25 mL), dried over MgSO4,
filtered, and evaporated to yield a damp solid. The crude
material was dissolved in MeOH (1 mL) and subjected to
column chromatography (40 mm × 350 mm) on silica gel with
10% MeOH/CHCl3. Fractions containing product were pooled
and evaporated to yield a white solid. The solid was recrystal-
lized from warm MeOH to yield 82 mg (68%) of 22 as a white
crystalline solid: mp 186-187 °C; Rf ) 0.3 (10% MeOH/
CHCl3). Anal. (C15H15Cl2NO5‚1/2H2O) C, H, N.
3-Acetyl-2,5,6-trichloroindole (23). Compound 1813 (2.21
g, 10.0 mmol) was dissolved in CH2Cl2 (50 mL) and cooled to
0 °C. Tin tetrachloride (1.4 mL, 12 mmol) was added over 5
min, then the resulting suspension was warmed to room
temperature and stirred for 30 min at that temperature. Acetyl
chloride (0.7 mL, 0.77 g, 10 mmol) was added, followed by
nitromethane (50 mL), and the resulting suspension was
stirred for 15 min at room temperature. The reaction was
quenched with 50 mL of cold water, and the biphasic suspen-
sion was stirred vigorously for 30 min. The organic phase was
separated, and the organic phase was extracted with THF (2
× 50 mL). The combined organic extracts were dried over
MgSO4, filtered, and evaporated to dryness. The red solid was
recrystallized from boiling THF to yield 0.98 g (37%) of 23 as
a pale-pink powder: mp >320 °C (dec); Rf ) 0.6 (5% MeOH/
CHCl3).
3-Acetyl-2,5,6-trichloro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-
â-D-ribofuranosyl]indole (24). Compound 23 (0.63 g, 2.5
mmol) was treated as per 21 above with 60% sodium hydride
in mineral oil (0.25 g, 6.3 mmol) and 3,5-di-(O-p-toluoyl)-2-
deoxy-R-D-ribofuranosyl chloride27 (20, 1.00 g, 2.6 mmol) to
yield 1.12 g (61%) of 24 as a white solid. An analytical sample
was prepared by recrystallization from MeOH/CHCl3: mp
100-103 °C; Rf ) 0.4 (3:1 hexane/EtOAc).
3-Cyano-2,5,6-trichloro-1-(2-deoxy-â-D-ribofuranosyl)-
indole (27a) and 3-Cyano-2-methoxy-5,6-dichloro-1-(2-
deoxy-â-D-ribofuranosyl)indole (27b). Compound 26 (366
mg, 0.63 mmol) was suspended in dry MeOH (30 mL) to which
was added sodium methoxide (82 mg, 1.5 mmol). The suspen-
sion was stirred at room temperature for 16 h, then the solvent
was removed under vacuum, and the residue was suspended
in brine (100 mL) and water (10 mL). The aqueous mixture
was extracted with EtOAc (2 × 100 mL), and the combined
organic extracts were dried over MgSO4, filtered, and evapo-
rated to yield a viscous residue. The residue was dissolved in
DMF (0.5 mL) and subjected to column chromatography (40
mm × 350 mm) on silica gel with 10% MeOH/CHCl3. Fractions
containing product were pooled and evaporated to yield a white
solid. The solid was dissolved in DMF (0.5 mL) and subjected
to column chromatography (40 mm × 350 mm) on C18 reverse-
phase silica gel with 75% MeOH/H2O. Fractions containing
the more rapidly eluting material were pooled and evaporated
to yield 47 mg (21%) of 27b as a white powder. Fractions
containing the more slowly eluting material were pooled and
evaporated to yield 120 mg (53%) of 27a as a white powder.
27b: mp 222-223 °C; Rf ) 0.4 (10% MeOH/CHCl3). Anal.
(C15H14Cl2N2O4‚1/5H2O) C, H, N. 27a: mp 187-188 °C; Rf )
0.4 (10% MeOH/CHCl3). Anal. (C14H11Cl3N2O3‚1/5H2O) C, H,
N.
3-(2-Furyl)-2,5,6-trichloro-1-(3,5-di-O-toluoyl-2-deoxy-
â-D-ribofuranosyl)indole (29a). 3-Iodo-2,5,6-trichloro-1-(3,5-
di-O-p-toluoyl-2-deoxy-â-D-ribofuranosyl)indole15 (28, 300 mg,
0.43 mmol) and 2-furanboronic acid (55 mg, 0.49 mmol) were
suspended in n-propanol (2 mL) and dry DMF (0.5 mL). The
suspension was stirred at room temperature for 10 min, then
palladium(II) acetate (10 mg, 0.045 mmol) and tri-o-tolylphos-
phine (42 mg, 0.14 mmol) were added, followed by 2.0 M
aqueous Na2CO3 (0.25 mL, 0.52 mmol) and H2O (0.5 mL). The
resulting suspension was heated on a 120 °C oil bath for 20
min. The resulting dark solution was cooled and poured into
EtOAc (50 mL). The organic solution was washed with brine
(50 mL), dried over MgSO4, filtered, and evaporated to yield
an orange solid. The solid was dissolved in CHCl3 (1 mL) and
subjected to column chromatography (40 mm × 350 mm) on
silica gel with 5:1 hexane/EtOAc. Fractions containing product
were pooled and evaporated to yield 158 mg (58%) of 29a as a
white crystalline solid: mp 85-87 °C; Rf ) 0.5 (3:1 hexane/
EtOAc).
3-(3-Thienyl)-2,5,6-trichloro-1-(3,5-di-O-toluoyl-2-deoxy-
â-D-ribofuranosyl)indole (29b). Compound 2815 (280 mg,
0.40 mmol) was treated as per 29a above with 3-thiophenebo-
ronic acid (58 mg, 0.45 mmol), palladium(II) acetate (10 mg,
0.045 mmol), tri-o-tolylphosphine (42 mg, 0.14 mmol), 2.0 M
aqueous Na2CO3 (0.25 mL, 0.50 mmol), and H2O (0.5 mL) to
yield 184 mg (70%) of 29b as a white crystalline solid: mp
94-96 °C; Rf ) 0.3 (5:1 hexane/acetone).
3-(2-Furyl)-2,5,6-trichloro-1-(2-deoxy-â-D-ribofuranosyl)-
indole (30a). Compound 29a (142 mg, 0.22 mmol) was treated
as per 22 above with sodium methoxide (30 mg, 0.56 mmol)
and recrystallized from 10% MeOH/CHCl3 and hexane to yield
67 mg (75%) of 30a as a white crystalline solid: mp 140-141
°C; Rf ) 0.5 (10% MeOH/CHCl3). Anal. (C17H14Cl3NO4‚1/4H2O)
C, H, N.
3-Acetyl-2,5,6-trichloro-1-(2-deoxy-â-D-ribofuranosyl)-
indole (25). Compound 24 (0.62 g, 1.0 mmol) was suspended
in dry MeOH (50 mL) to which was added sodium methoxide

Trichloroindole Deoxyribonucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5781
3-(3-Thienyl)-2,5,6-trichloro-1-(2-deoxy-â-D-ribofurano-
syl)indole (30b). Compound 29b (159 mg, 0.24 mmol) was
treated as per 22 above with sodium methoxide (30 mg, 0.56
mmol) and recrystallized from 10% MeOH/CHCl3 and hexane
to yield 84 mg (82%) of 30b as a white crystalline solid: mp
144-145 °C; Rf ) 0.5 (10% MeOH/CHCl3). Anal. (C17H14Cl3-
NO3S‚1/4H2O) C, H, N.
KB cells at a moi of <0.1 also as detailed previously.33 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.34 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 for 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.
HCMV Plaque Reduction Assay. 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, 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 employed35 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, the 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.33 (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.36 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.
3-Acetyl-2-bromo-5,6-dichloroindole (32). 2-Bromo-5,6-
dichloroindole13 (31, 1.25 g, 4.7 mol) was dissolved in dry CH2-
Cl2 (25 mL) to which was added tin tetrabromide (2.50 g, 5.7
mmol). The suspension was stirred for 10 min at room
temperature until the solids had completely dissolved, then
acetyl bromide (0.37 mL, 0.62 g, 5.0 mmol) was added in one
portion, followed by nitromethane (25 mL). The resulting dark
solution was stirred at room temperature for 20 min. Water
(25 mL) was then added, and the biphasic mixture was stirred
vigorously for 10 min. The mixture was poured into brine (200
mL) and EtOAc (550 mL), and the mixture was heated on a
steam bath until the solids had mostly dissolved. The aqueous
layer was separated and discarded, and the organic layer was
heated to reflux on a steam bath. The organic suspension was
filtered, and the filtrate was evaporated to approximately 50
mL. The resulting solids were filtered and rinsed with cold
EtOAc to yield 1.05 g (72%) of 32 as a pale-pink powder: mp
>285 °C (dec); Rf ) 0.6 (1:1 hexane/EtOAc).
3-Acetyl-2-bromo-5,6-dichloro-1-[3,5-di-O-(p-toluoyl)-2-
deoxy-â-D-ribofuranosyl]indole (33). Compound 32 (0.92
g, 3.0 mmol) was suspended in dry THF (150 mL) to which
was added sodium hydride (60% in mineral oil, 200 mg, 5.0
mmol). The suspension was stirred at room temperature for
15 min until gas evolution had ceased, then filtered into a
suspension of 3,5-di-(O-p-toluoyl)-2-deoxy-R-D-ribofuranosyl
chloride27 (20, 1.22 g, 3.1 mmol) in dry toluene (150 mL). The
resulting solution was stirred at room temperature for 16 h,
then evaporated to dryness. The residue was suspended in
brine (90 mL) and water (10 mL) and extracted with EtOAc
(2 × 50 mL). The combined organic extracts were dried over
MgSO4, filtered, and evaporated to yield an orange oil. The
oil was dissolved in CHCl3 (3 mL) and subjected to column
chromatography (50 mm × 450 mm) on silica gel with 2:1
hexane/EtOAc. Fractions containing product were pooled and
evaporated to yield a pale-yellow solid. This solid was recrys-
tallized from EtOAc/hexane to yield 1.13 g (57%) of 33 as a
white crystalline solid: mp 100-101 °C; Rf ) 0.4 (2:1 hexane/
EtOAc).
3-Acetyl-2-bromo-5,6-dichloro-1-(2-deoxy-â-D-ribofura-
nosyl)indole (34). Compound 33 (1.24 g, 1.7 mmol) was
suspended in absolute MeOH (75 mL) to which was added
sodium methoxide (250 mg, 4.6 mmol). The suspension was
stirred at room temperature for 2 h, after which time the
solution first clarified and then a precipitate formed. Water
(100 mL) was added, and the suspension was evaporated until
no more MeOH remained. The remaining solids were filtered
and recrystallized twice from MeOH/H2O to yield 475 mg (68%)
of 34 as a white crystalline solid: mp slow >235 °C (dec); Rf
) 0.5 (10% MeOH/CHCl3). Anal. (C15H14BrCl2NO4) 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.32
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 log10 drug concentrations. Fifty percent inhibitory
concentrations (IC50 values) were calculated from the linear
portions of the regression lines. Samples containing positive
controls (acyclovir for HSV-1, GCV for HCMV, and 2-acetylpy-
ridine thiosemicarbazone for cytotoxicity) were used in all
assays.
HCMV Time-of-Addition Study. As described above, HFF
cells at 10000 cells per well in 96-well cluster plates were
infected with HCMV (Towne strain) at 0.5 pfu/cell. At in-
fection and the indicated times postinfection, media were
replaced with either fresh media or media containing selected
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.33 High-titer HSV-1 stocks were prepared by infecting

5782 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
Ribonucleoside Is Mediated through the UL89 Gene Product.
J. Virol. 1998, 72, 717-725.
virus inhibitory but nontoxic drug concentrations plus 0.5%
methylcellulose and 5% FBS. After incubation at 37 °C for 96
h in an atmosphere of 5% CO2, plates were frozen at -80 °C.
They were subsequently thawed, and the titer in each well
was determined by plaque assay in 96-well plates as we have
described previously.34 Drug effects on HCMV titers were
calculated as a percentage of reduction in titer in the presence
of each drug compared to the titer observed in the absence of
drug at each time point. Data are presented as the mean
duplicate results for each drug at each time point.
(17) Krosky, P. M.; Underwood, M. R.; Turk, S. R.; Feng, K. W.-H.;
Jain, R. G.; Ptak, R. G.; Westerman, A. C.; Biron, K. K.;
Townsend, L. B.; Drach, J. C. Resistance of Human Cytomega-
lovirus to Benzimidazole Ribonucleosides Maps to Two Open
Reading Frames: UL89 and UL56. J. Virol. 1998, 72, 4721-
4728.
(18) Gudmundsson, K. S.; Drach, J. C.; Wotring, L. L.; Townsend, L.
B. Synthesis and Antiviral Activity of Certain 5′-Modified
Analogs of 2,5,6-Trichloro-1-(â-D-ribofuranosyl)benziminazole. J.
Med. Chem. 1997, 40, 785-793.
(19) Townsend, L. B.; Drach, J. C. Polysubstituted Benzimidazoles
as Antiviral Agents. U.S. Patent 5,360,795, 1994.
(20) Migawa, M. T.; Girardet, J.-L.; Walker, J. A., II; Koszalka, G.
W.; Chamberlain, S. D.; Drach, J. C.; Townsend, L. B. Design,
Synthesis, and Antiviral Activity of R-Nucleosides: D- and
L-Isomers of Lyxofuranosyl- and (5-Deoxylyxofuranosyl)benz-
imidazoles. J. Med. Chem. 1998, 41, 1242-1251.
(21) Krosky, P. M.; Borysko, K. Z.; Nassiri, M. R.; Devivar, R. V.;
Ptak, R. G.; Davis, M. G.; Biron, K. K.; Townsend, L. B.; Drach,
J. C. Phosphorylation of â-D-Ribosyl-benzimidazoles Is Not
Required for Activity against Human Cytomegalovirus. Anti-
microb. Agents Chemother. 2002, 46, 478-486.
Acknowledgment. We thank Kathy Z. Borysko and
Julie M. Breitenbach for expert performance of antiviral
and cytotoxicity assays. These studies were supported
by Training Grant T32-GM07767 and Research Grants
U19-AI31718 and P01-AI46390 from the National In-
stitutes of Health.
Supporting Information Available: 1H NMR, 13C NMR,
and 19F NMR for all new compounds and HRMS and elemental
analysis results for all target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(22) Zou, R.; Kawashima, E.; Freeman, G. A.; Koszalka, G. W.; Drach,
J. C.; Townsend, L. B. Design, Synthesis, and Antiviral Evalu-
ation of 2-Deoxy-D-ribosides of Substituted Benzimidazoles as
Potential Agents for Human Cytomegalovirus Infections. Nu-
cleosides Nucleotides 2000, 19, 125-154.
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