Design, synthesis, and antiviral activity of certain 3-substituted 2,5,6-trichloroindole nucleosides

Article abstract of DOI:10.1021/jm0400146

A series of trichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. Modifications of the previously reported 2,5,6-trichloro-1-(β-D-r

Full text of DOI:10.1021/jm0400146

J. Med. Chem. 2004, 47, 5753-5765
5753
Design, Synthesis, and Antiviral Activity of Certain 3-Substituted
2,5,6-Trichloroindole Nucleosides
John D. Williams,† Jiong J. Chen,‡ 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 January 20, 2004
A series of trichlorinated indole nucleosides has been synthesized and tested for activity against
human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity.
Modifications of the previously reported 2,5,6-trichloro-1-(â-D-ribofuranosyl)indole at the
3-position of the heterocycle were designed in part to test our hypothesis that hydrogen bonding
is required at that position for antiviral activity. Analogues were synthesized using electrophilic
addition at the 3-position or by synthesis of modified indole heterocycles followed by
glycosylation and modification of the sugar. Among the modifications at the 3-position, only
those analogues with hydrogen-bond-accepting character were active against HCMV (e.g.,
3-formyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole, FTCRI, IC50 ) 0.23 µM). Conversely,
analogues with non-hydrogen-bonding substituents at the 3-position (e.g., 3-methyl-2,5,6-
trichloro-1-(â-D-ribofuranosyl)indole) were much less active (IC50 ) 32 µM) than those with
the requisite hydrogen-bonding capacity. The 5′-O-acyl analogue of FTCRI was obtained as an
intermediate and also found to be a potent inhibitor of HCMV (IC50 < 0.1 µM). The synthesis
of some additional 5′-O-acylated analogues did not provide a compound with increased antiviral
activity. None of the indole nucleosides had significant activity against HSV-1, and none were
cytotoxic to uninfected cells in their antiviral dose range. Results obtained from the antiviral
evaluations have validated our hypothesis that hydrogen bonding at the 3-position is required
for antiviral activity in this series of chlorinated indole nucleosides.
Introduction
trichloro-1-(â-D-ribofuranosyl)benzimidazole (1, TCRB).9
Although TCRB demonstrated excellent antiviral activ-
ity and selectivity in vitro, it was degraded (via glyco-
sidic 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. Included among these com-
pounds is 2,5,6-trichloro-1-(â-D-ribofuranosyl)indole11
(TCRI, 3). TCRI should be more stable to glycosidic bond
cleavage because protonation of the heterocyclic base
is a likely mechanism involved in enzymatic degrada-
tion, and the indole heterocycle is much more difficult
to protonate than benzimidazole owing to the basic
nitrogen at the benzimidazole 3-position (see Figure 1).
Although TCRI itself is inactive against HCMV, it was
hypothesized that the installation of a hydrogen-bonding
substituent at the 3-position of the indole ring would
act as a surrogate for the 3-nitrogen in TCRB, thus
restoring antiviral activity. We now describe the syn-
thesis of a series of chlorinated indole nucleosides with
exocyclic groups at the 3-position of the heterocycle and
the effect that these groups exert on their antiviral
activity and cytotoxicity.
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 used for
the treatment of HCMV infections, namely, ganciclovir,4
valganciclovir,5 cidofovir,6 foscarnet,7 and fomivirsen.8
All of these compounds suffer limitations, however,
including poor oral bioavailability and toxicity. Fur-
thermore, 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 likely.
The search for new compounds with fewer or less
severe limitations has led our laboratory to synthesize
a wide range of nucleoside analogues, including 2,5,6-
Results and Discussion
Chemistry. For our initial studies in this area, we
elected to investigate the procedures and methods that
would add certain exocyclic groups directly to TCRI at
the 3-position. Because the 3-position of indole is quite
electrophilic, many possibilities were attractive, among
them the Vilsmeier-Haack formylation, Friedel-Crafts
* 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 Chemistry.
§ Department of Biologic and Materials Sciences.
10.1021/jm0400146 CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/13/2004

5754 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
Scheme 1a
Figure 1. Proposed in vivo degradation of TCRB and resis-
tance of indole nucleosides to glycosidic cleavage.
acylations, and electrophilic cyanation with chlorosul-
fonyl isocyanate.12 The known intermediate 2,5,6-
trichloro-1-(2,3-O-isopropylidene-â-D-ribofuranosyl)in-
dole11 (5), produced by a modification of the previous
procedure, was protected as the 5′-O-acetate ester to
provide 6. Formylation and cyanation of 6 proceeded in
moderate yield to provide the expected compounds 7a
and 7b. A reaction of 6 under Friedel-Crafts conditions
with aluminum chloride and either acetyl or propionyl
chloride provided low yields of 7c and 7d. The attempted
trifluoroacetylation using trifluoroacetic anhydride and
aluminum chloride was unsuccessful, but the procedure
of Kiselyov,13 using a mixture of trifluoroacetic anhy-
dride and BF3/SMe2, did provide the desired trifluoro-
acetyl derivative 7e in yields comparable to the previous
Lewis acid-catalyzed acylations.
a Reagents and conditions: (a) Ac2O, 100 °C, 4 h; (b) POCl3,
DMF, 70 °C, 16 h or CSI, CH2Cl2, 20 °C, 16 h, then DMF, 20 °C,
1 h or RCOCl, AlCl3, 20 °C, 1 h or TFAA, BF3/SMe2, 20 °C, 90
min; (c) 90% TFA, 20 °C, 2 min; (d) NaOMe, MeOH, 20 °C, 15-90
min.
Scheme 2a
All of the analogues thus produced were deprotected
in a two-part procedure. The acetonide was first hydro-
lyzed using 90% aqueous trifluoroacetic acid to provide
the 5′-O-acetylated intermediates 8a-e. The acetate
esters were then hydrolyzed using methanolic sodium
methoxide to provide the fully deprotected nucleosides
9a-e. The order in which the protecting groups are
removed is very important. If the fully protected nucleo-
side analogue 7a is treated first with sodium methoxide,
an anhydro nucleoside is produced as the only product
(see Scheme 1).
Our initial antiviral screening indicated that the 5′-
O-acetylated indole nucleoside analogue 8a was par-
ticularly potent against HCMV. To further explore this
interesting activity, we decided to synthesize a number
of 8a analogues with other acyl protecting groups at the
5′-position. To minimize the number of synthetic trans-
formations necessary for the completion of this series,
the 5′-unprotected nucleoside 10a was required. How-
ever, because this intermediate could not be synthesized
by a partial deprotection of 7a (because of anhydro
nucleoside formation, vide supra), another strategy was
needed to provide 10a (Scheme 2). Therefore, we used
the easily hydrolyzed trifluoroacetate ester as a “tem-
porary protecting group”. The precursor 5 was trifluo-
roacetylated with trifluoroacetic anhydride, and the
crude material was subjected to either formylation or
cyanation reactions under the previous conditions.
Aqueous workup was sufficient in this case to cleave
the trifluoroacetate ester and provide 10a and 10b in
a Reagents and conditions: (a) TFAA, CH2Cl2, 20 °C, 90 min,
then POCl3, DMF, 70 °C, 16 h or CSI, CH2Cl2, 20 °C, 16 h, then
DMF, 20 °C, 1 h, aqueous workup; (b) (RCO)2O, DMAP, pyridine,
120 °C, 12 min; (c) 90% TFA, 20 °C, 2 min.
reasonable yield without the formation of any anhydro
nucleoside.
We then carried out the desired acylations with either
propionic or butyric anhydride and DMAP in pyridine

3-Substituted 2,5,6-Trichloroindole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5755
Scheme 3a
a Reagents and conditions: (a) NaNH2, NH3(l), -78 °C, 30 min, then MeI, -78 °C, 30 min; (b) HNO3, H2SO4, 5 °C, 35 min; (c) H2, PtO2,
Na2SO4, 20 °C, 4 h, then AcOH, 20 °C, 2 h; (d) POCl3, CH3CN, 100 °C, 1 h, then imidazole, 100 °C, 16 h; (e) NaH, THF, 0 °C, 10 min, then
18, THF/toluene, 60 °C, 16 h; (f) NaH, THF, 0 °C, 10 min, then 20, THF/toluene, 20 °C, 3 h; (g) NaOMe, MeOH, 20 °C, 2 h; (h) TBDPSCl,
pyridine, 20°C, 24 h, then MsCl, pyridine, 20 °C, 4 h; (i) t-BuOK, DMSO/H2O 20 °C, 10 min; (j) OsO4, NMO, acetone/H2O, 20 °C, 18 h.
with heating to provide 11a and 11b in low yield (less
harsh conditions did not produce the desired acylated
compounds). Treatment of 10a with methyl chlorofor-
mate and pyridine provided the carbonate 11c in good
yield. The 5′-O-acylated nucleosides 11a-c were then
treated with 90% aqueous trifluoroacetic acid to yield
the target compounds 12a-c.
Our antiviral screening assays indicated that the
addition of a hydrogen-bonding substituent at the
3-position did indeed impart good antiviral activity to
this series of compounds. With these promising initial
biological results, a more extensive exploration of the
structure-activity relationship (SAR) in this series was
undertaken.
These studies were initiated in an effort to determine
what other factors might be involved in the binding of
this class of compounds to their target. Synthesis of the
3-methyl derivative would be useful to determine whether
the hydrogen-bonding effects are actually required for
activity or whether steric and van der Waals contacts
are sufficient. No efficient method for the electrophilic
introduction of simple alkyl groups was available, so it
was necessary to synthesize the requisite chlorinated
3-methylindole heterocycle and couple that heterocycle
with an appropriate glycosyl donor. Ethyl 3,4-dichlo-
rophenylacetate (13) was deprotonated with a mixture
of sodium amide in liquid ammonia and then methy-
lated with methyl iodide.14 By optimization of the
amount of sodium amide and methyl iodide used, little
of the dialkylated product was obtained. The resulting
dichlorophenylpropionate ester 14 was then nitrated
with HNO3/H2SO4. Reduction of the nitro group followed
by an acid-catalyzed ring closure provided the oxindole
16. Compound 16 was then chlorinated with phosphorus
oxychloride and imidazole to yield 3-methyl-2,5,6-
trichloroindole (17).
Unfortunately, the sodium salt method used to syn-
thesize TCRI11 did not work in the case of the 3-methyl
derivative. Because the synthesis of 19 using the
R-chlorosugar 1815 and the indole 17 was unsuccessful,
the desired riboside was synthesized via the manipula-
tion of a 2′-deoxyribofuranoside. Thus, 3-methyl-2,5,6-
trichloroindole (17) was deprotonated with sodium
hydride and reacted with the R-chlorosugar 3,5-di-O-
(p-toluoyl)-2-deoxy-R-D-ribofuranosyl chloride16 (20),
which produced the nucleoside 21 in good yield (Scheme
3). The protected nucleoside 21 was deprotected with
sodium methoxide in methanol and converted to the 3′-
O-mesylate after the 5′-position had been protected with
the bulky TBDPS protecting group. Base-promoted
elimination of the mesylate and concomitant removal
of the silyl protecting group resulted in the formation
of the 2,3-dideoxy-2,3-didehydro nucleoside 24, which
was dihydroxylated with osmium tetroxide. Dihydroxy-
lation occurred exclusively on the R-face of the sugar
because of steric hindrance of the â-face and provided
3-methyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole (25)
in good yield.
The 3-chloro and 3-iodo derivatives of the indole
nucleosides were also desirable targets because they
would provide non-hydrogen bonding, electron-with-
drawing substituents at the 3-position. Furthermore,
the 3-iodo derivative could be used in palladium-
catalyzed coupling reactions to further explore the
structural requirements of the 3-position. Direct elec-
trophilic halogenation of the protected indole nucleoside
6 using several different procedures was unsuccessful
(data not presented), so a strategy similar to that used

5756 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
Scheme 4a
(31b). Compound 31b was first protected with 2,2-
dimethoxypropane and chloromethyl methyl ether to
avoid the side reaction of boronic ester formation during
the Suzuki coupling. The resulting 2′,3′-O-isopropyl-
idene-5′-O-methoxymethyl intermediate 33 was then
subjected to the coupling conditions as described by
Huff19 (Scheme 5). The protected 3-aryl nucleoside
analogues 34a and 34b were synthesized in good yield
and then deprotected in wet methanolic HCl to produce
the desired analogues 35a and 35b.
Another desirable synthetic target was the homoal-
dehyde 38. This extended aldehyde can be compared to
the 3-acetyl derivative 7c. Both of these compounds
have very similar steric bulk and hydrogen-bonding
capacity, but the homoaldehyde has more conforma-
tional flexibility. This compound was synthesized from
the protected 3-formylindole nucleoside 7a (Scheme 6).
Wittig olefination of the aldehyde using the phosphorus
ylide derived from (methoxymethyl)triphenylphospho-
nium chloride provided the vinyl ether 36 as a mixture
of cis and trans isomers. The aldehyde was unmasked
and the sugar was deprotected by treatment with wet
methanolic HCl, but the aldehyde was immediately
converted in situ to the dimethyl acetal 37, which was
not rigorously characterized. The acetal was then
hydrolyzed with 90% aqueous trifluoroacetic acid to
provide the desired nucleoside 38.
It has been established that 2-bromo-5,6-dichloro-1-
(â-D-ribofuranosyl)benzimidazole (BDCRB) is more ac-
tive against HCMV than TCRB.9 The structural simi-
larity of the benzimidazole nucleosides compared to the
indole nucleosides synthesized in the present investiga-
tion prompted us to initiate some studies on the
synthesis of the 2-bromo analogues of selected indole
nucleosides. The carboxaldehyde 9a and its 5′-O-acetyl
analogue 8a were very active and selective inhibitors,
and we therefore chose the 2-bromo analogues of these
compounds (42 and 43, respectively) as synthetic tar-
gets.
In a methodology analogous to the synthesis of 8a and
9a, 2-bromo-5,6-dichloro-1-(2,3-O-isopropylidene-â-D-ri-
bofuranosyl)indole11 (39) was acetylated at the 5′-
position with acetic anhydride in pyridine. The protected
nucleoside was then subjected to Vilsmeier-Haack
formylation conditions using a mixture of phosphorus
oxybromide and DMF (use of phosphorus oxychloride
led to halogen exchange at the 2-position; data not
presented). Deprotection of the 3-formyl intermediate
41 with 90% aqueous trifluoroacetic acid provided the
desired nucleoside 42. Further reaction of 42 with
methanolic sodium methoxide provided the fully depro-
tected nucleoside 43 (Scheme 7).
Biological Evaluation. The compounds synthesized
above were tested for activity against HCMV and HSV-1
and for cytotoxicity. Although none of the compounds
demonstrated potent and selective inhibition of HSV-1
replication, our initial exploration into substituents at
the 3-position provided several active and selective
compounds against HCMV replication. The 3-formyl
analogue 9a was the most potent against HCMV with
an IC50 of 0.23 µM, whereas the 3-acetyl analogue 9c
was somewhat less cytotoxic and therefore more selec-
tive. Further increasing the length of the acyl chain at
the 3-position (i.e., 3-propionyl analogue 9d) resulted
a Reagents and conditions: (a) NCS or NIS, CH3CN, 20 °C, 1
h; (b) NaH, THF, 0 °C, 10 min, then 20, THF/toluene, 20 °C, 3 h;
(c) NaOMe, MeOH, 20 °C, 2 h; (d) TBDPSCl, pyridine, 20°C, 24 h,
then MsCl, pyridine, 20 °C, 4 h; (e) t-BuOK, DMSO/H2O, 20 °C,
10 min; (f) OsO4, NMO, acetone/H2O, 20 °C, 18 h.
in the synthesis of the 3-methyl analogue 25 was
initiated. The 3-halogenated heterocycles 2,3,5,6-tetra-
chloroindole (26a) and 3-iodo-2,5,6-trichloroindole (26b)
were prepared easily by a reaction of 2,5,6-trichloroin-
dole11 (4) with N-chlorosuccinimide and N-iodosuccin-
imide, respectively (Scheme 4). The heterocycles were
deprotonated with sodium hydride and glycosylated
with the R-chlorosugar 20. Deprotection of the 2′-
deoxyribofuranosyl nucleoside intermediates 27a and
27b followed by 5′-O-silylation and 3′-O-mesylation led
to the intermediates 29a and 29b. Base-promoted
elimination followed by dihydroxylation with osmium
tetroxide yielded the expected ribofuranosides 31a and
31b.
Having synthesized the ribofuranosyl derivative of
3-iodo-2,5,6-trichloroindole, our attention turned to the
palladium-catalyzed coupling of the iodinated nucleoside
with other heterocyclic components. Either Suzuki-17 or
Stille-type18 couplings could be used for this purpose,
but the former was selected largely because of the
toxicity of tin derivatives required for Stille couplings.
2-Furylboronic acid and 3-thiopheneboronic acid were
selected as coupling partners because of their avail-
ability, relatively small size, and potential hydrogen-
bonding ability.
The ribofuranosides 35a and 35b were synthesized
from 3-iodo-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole

3-Substituted 2,5,6-Trichloroindole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5757
Scheme 5a
a Reagents and conditions: (a) DMP, p-TsOH, acetone, 20 °C, 15 min; (b) MOMCl, DIPEA, CH2Cl2, 20 °C 16 h; (c) Ar-B(OH)2, Pd(OAc)2,
(o-tol)3P, Na2CO3, DMF/n-PrOH/H2O, 120 °C, 5-15 min; (d) HCl, MeOH/H2O, 60 °C, 45 min.
Scheme 6a
to that of the very potent 9c, we hypothesize that the
additional degrees of rotational freedom afforded by the
methylene bridge resulted in an entropic binding pen-
alty and was therefore less potent.
The 5′-O-acetyl analogues 8b-d, synthesized en route
to the initial series of analogues, had activities compa-
rable to those of their 5′-unprotected congeners (Table
2). One difference was noted for the 3-formyl analogue
8a, which was more potent than the fully deprotected
9a in initial plaque-reduction assays without being
substantially more cytotoxic. This prompted us to
synthesize some other acylated derivatives (12a-c).
However, the other 5′-O-acyl groups and the 5′-carbon-
ate were less active against HCMV than 8a, and no
additional modifications were pursued.
The 2-bromo homologues of 8a and 9a (42 and 43,
respectively) also were potent and selective inhibitors
of HCMV replication. The antiviral activity of the
FTCRI analogue 43 was comparable to FTCRI (9a), and
the activity of 42 was less than its 2-chloro congener
8a. These results are in direct contrast to the relation-
ship of TCRB and its 2-bromo homologue BDCRB,
where the latter is more active and selective than the
2-chloro homologue.9
The results of our HCMV screening assays (Tables 1
and 2) are in good agreement with our initial hypothesis
that hydrogen-bond acceptors are required for the
potent activity of this series of indole nucleosides against
HCMV. Furthermore, this suggests that the difference
in activity between TCRB and TCRI can be explained
in terms of hydrogen bonding and not by changes in the
electronic character of the heterocycle.
a Reagents and conditions: (a) Ph3PdCHOMe, THF, 0 °C, 1 h;
(b) HCl, MeOH/H2O, 60 °C, 1 h; (c) 90% TFA, 20 °C, 2 min.
in a decrease in the activity of the compound. The
3-nitrile 9b was slightly less active and slightly more
toxic than 9a or 9c. The 3-trifluoroacetyl analogue 9e
was also substantially less active than the aforemen-
tioned acyl analogues (Table 1).
The 3-methyl, 3-chloro, and 3-iodo analogues 25, 31a,
and 31b, respectively, were all substantially less active,
but no less cytotoxic, than the analogues with hydrogen-
bonding capacity. This suggests that steric and van der
Waals forces between the nucleosides and their enzy-
matic target are not sufficient for potent antiviral
activity and that electron-withdrawing substituents are
also not sufficient in the absence of hydrogen-bond
acceptors. Interestingly, the nucleosides do not require
hydrogen bonding for cytotoxicity, thereby suggesting
the specific inhibition of a viral target. The presence of
van der Waals interactions is necessary for cytotoxicity,
however, because TCRI (which lacks a substituent at
the 3-position) is not cytotoxic.
Although the 2-furyl and 2-thienyl analogues 35a and
35b could be involved in hydrogen bonding, they were
much less potent than the 3-acyl analogues above.
Perhaps these substituents are too bulky or the ad-
ditional aryl system contributed to unfavorable interac-
tions in the putative viral target. Additionally, the
homoaldehyde 38 was also much less active than either
9a or 9c. Because the homoaldehyde has bulk similar
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.
Chromatography 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 performed 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 the
Supporting Information. 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

5758 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
Scheme 7a
a Reagents and conditions: (a) Ac2O, pyridine, 20 °C, 4 h; (b) POBr3, DMF, 70 °C, 16 h; (c) 90% TFA, 20 °C, 2 min; (d) NaOMe, MeOH,
20 °C, 15-90 min.
Table 1. Antiviral Activity and Cytotoxicity of 3-Substituted Indole Nucleosides
50% inhibitory concentration (µM)
antiviral
cytotoxicity
compd
R2
R3
HCMV plaquea
HSV-1 ELISAb
HFF visualc
KB growthc
9a
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Br
-CHO
0.23
0.55
0.31
2.5
6.2
32
40
15
45
32
45
65
9b
-CN
9c
-COCH3
-COCH2CH3
-COCF3
-CH3
-Cl
-I
-(2-furyl)
-(3-thienyl)
-CH2CHO
-CHO
20
>100d
>100
32
50
9d
20
100
20
9e
15
25
70
32
>100
80
31a
31b
35a
35b
38
38
45
32
12
20
32
65
42
50
32
80
31
45
>100
>100
32
60
4.0
0.32
2.9
0.70
7.4
>100
20
70
43
50
TCRBe
BDCRBe
GCVf
102
130
3.5
238
118
>100
210
>100
>100
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 TCRB and BDCRB (2,5,6-trichloro- and 2-bromo-5,6-dichloro-1-(â-D-ribofuranosyl)benzimidazole, respectively) were
published previously as compounds 9 and 11, respectively, in ref 9. f Averages from 108, 33, and 3 experiments, respectively, using
ganciclovir (GCV).
DPX300 spectrometer. Chemical shift values for 1H 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 spec-
trometry facility and elemental analysis facility, respectively.
2,5,6-Trichloro-1-(2,3-O-isopropylidene-â-D-ribofuran-
osyl)indole (5). (5 was prepared on a large scale by a
modification of the procedure of Chen.11) 2,3-O-Isopropylidene-
5-O-tert-butyldimethylsilyl-â-D-ribofuranose15 (18.27 g, 60.0
mmol) was dissolved in dry toluene (120 mL) to which was
added dry carbon tetrachloride (25 mL, 40 g, 260 mmol). The
solution was cooled to -20 °C in an ice/salt bath, and
hexamethylphosphorus triamide (13.0 mL, 11.5 g, 72 mmol)
was added dropwise over 20 min. Once the addition was
complete, the resulting cloudy suspension was stirred at -20
°C for an additional 30 min, and then the suspension was
poured into cold brine (500 mL). The organic layer was
separated, and the aqueous layer was extracted with an
additional portion of cold toluene (200 mL). The combined
organic extracts were dried over MgSO4 and filtered, and the
filtrate was diluted to 400 mL total volume to yield a solution
of crude R-chlorosugar 18 in toluene, which was used im-
mediately without further purification.
2,5,6-Trichloroindole11 (11.03 g, 50.0 mmol) was dissolved
in dry THF (200 mL) and cooled to 5 °C on an ice bath. To the
cooled solution was added sodium hydride (60% in mineral oil,
2.5 g, 63 mmol) in small portions. The resulting suspension
was stirred at <10 °C for 10 min until gas evolution had
ceased. The resulting suspension was filtered, and the solids
were rinsed with dry THF (50 mL). The filtrate was diluted
to 400 mL total volume with dry THF, then combined with
the crude solution of 18. The combined solution was heated
on a 70 °C oil bath for 16 h, then cooled to room temperature
and evaporated to yield a red oil. The oil was dissolved in
EtOAc (500 mL), washed with water (200 mL) and brine (200
mL), then dried over MgSO4, filtered, and evaporated to yield
a red oil. The oil was dissolved in CHCl3 (15 mL) and subjected
to column chromatography (100 mm × 1000 mm) on silica gel
with 5:1 hexane/EtOAc. Fractions containing product were
pooled and evaporated to yield 14.41 g (57%) of a clear oil,
which solidified upon standing.
The above solid (14.41 g, 28.4 mmol) was dissolved in dry
DMF (125 mL) to which was added cesium fluoride (8.65 g,

3-Substituted 2,5,6-Trichloroindole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5759
Table 2. Antiviral Activity and Cytotoxicity of Sugar-Modified Indole Nucleosides
50% inhibitory concentration (µM)
antiviral cytotoxicity
compd
R2
R3
R5′
HCMV plaquea
HSV-1 ELISAb
HFF visualc
KB growthc
8a
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Br
-CHO
-OCOCH3
<0.1d
2.5
0.30
3.2
0.22
0.25
0.20
0.32
2.9
0.70
7.4
20
50
32
32
35
70
8b
-CN
-OCOCH3
8c
-COCH3
-COCH2CH3
-CHO
-OCOCH3
-OCOCH3
-OCOCH2CH3
-OCOCH2CH2CH3
-OCOOCH3
-OCOCH3
20
32
40
8d
15
10
9.0
15
15
102
130
3.5
>100e
32
100
50
12a
12b
12c
-CHO
32
40
-CHO
32
50
42
-CHO
32
40
TCRBf
BDCRBf
GCVg
238
118
>100
210
>100
>100
a Plaque reduction assays were performed in duplicate wells as described in Experimental Section. 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 Experimental Section. d <0.1 indicates an IC50 less than
the lowest concentration tested. e >100 indicates an IC50 greater than the highest concentration tested. f Data for TCRB and BDCRB
(2,5,6-trichloro- and 2-bromo-5,6-dichloro-1-(â-D-ribofuranosyl)benzimidazole, respectively) were published previously as compounds 9
and 11, respectively, in ref 9. g Averages from 108, 33, and 3 experiments, respectively, using ganciclovir (GCV).
57 mmol). The suspension was stirred vigorously at room
temperature for 2 h, and then the solvent was removed under
vacuum (0.5 mmHg, 40 °C). The residual oil was suspended
in brine (300 mL) and extracted with EtOAc (3 × 150 mL).
The combined organic extracts were dried over MgSO4, filtered,
and evaporated to yield a yellow oil. The oil was dissolved in
CHCl3 (10 mL) and subjected to column chromatography (100
mm × 1000 mm) on silica gel with 2:1 hexane/EtOAc. Frac-
tions containing product were pooled and evaporated to yield
10.27 g (92%) of 511 as a clear syrup. Rf ) 0.5 (2:1 hexane/
EtOAc). 1H NMR and 13C NMR results match literature
precedent.11
2,5,6-Trichloro-1-(2,3-O-isopropylidene-5-O-acetyl-â-D-
ribofuranosyl)indole (6). Compound 511 (544 mg, 1.4 mmol)
was dissolved in acetic anhydride (20 mL) and heated to 100
°C for 4 h. The solution was then cooled to room temperature
and evaporated to dryness. The viscous residue was suspended
in 5% aqueous Na2CO3 (30 mL) and extracted with EtOAc (2
× 40 mL). The combined organic extracts were washed with
brine (15 mL), dried over MgSO4, filtered, and evaporated to
yield a 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 503 mg (84%) of 6 as a
clear oil. Rf ) 0.3 (5:1 hexane/EtOAc).
8.1 mmol) was dissolved in dry CH2Cl2 (100 mL) to which was
added chlorosulfonyl isocyanate (1.05 mL, 1.71 g, 12.1 mmol).
The resulting solution was stirred at room temperature for
16 h, then dry DMF (5.0 mL) was added and the solution
stirred for an additional 1 h. Water (50 mL) was added, and
the biphasic mixture was stirred vigorously for 15 min and
then poured into 10% NaHCO3 (200 mL). The aqueous suspen-
sion was extracted with EtOAc (2 × 100 mL) and the combined
organic extracts were dried over MgSO4, filtered, and evapo-
rated to yield a yellow oil. The oil was dissolved in CHCl3 (4
mL) and subjected to column chromatography (50 mm × 450
mm) on silica gel with 1:1 hexane/EtOAc. Fractions containing
product were pooled and evaporated to yield 2.01 g (54%) of
7b as a pale-yellow solid: mp 154-155 °C; Rf ) 0.5 (2:1
hexane/EtOAc).
3-Acetyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
acetyl-â-D-ribofuranosyl)indole (7c). Compound 6 (341 mg,
0.78 mmol) was dissolved in dry CH2Cl2 (10 mL) to which were
added acetyl chloride (84 µL, 93 mg, 1.2 mmol) and anhydrous
aluminum(III) chloride (160 mg, 1.2 mmol). The resulting
solution was stirred at room temperature for 1 h and then
poured into 10% aqueous NaHCO3 (100 mL). The aqueous
suspension was extracted with EtOAc (2 × 50 mL), and 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 2:1 hexane/EtOAc. Fractions
containing product were pooled and evaporated to yield 129
mg (34%) of 7c as a white solid: mp 129-130 °C; Rf ) 0.4 (2:1
hexane/EtOAc).
3-Propionyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-
O-acetyl-â-D-ribofuranosyl)indole (7d). Compound 6 (435
mg, 1.0 mmol) was treated with propionyl chloride (130 µL,
138 mg, 1.5 mmol) and anhydrous aluminum(III) chloride (200
mg, 1.5 mmol) as per 7c above to yield 200 mg (41%) of 7d as
a pale-yellow solid. A portion was recrystallized from boiling
CHCl3/hexane: mp 141-142 °C; Rf ) 0.5 (2:1 hexane/EtOAc).
3-Trifluoroacetyl-2,5,6-trichloro-1-(2,3-O-isopropyl-
idene-5-O-acetyl-â-D-ribofuranosyl)indole (7e). A solution
of trifluoroacetic anhydride (0.35 mL, 0.52 g, 2.5 mmol) in dry
CH2Cl2 was cooled to -78 °C, and boron trifluoride methyl
sulfide complex (0.21 mL, 0.26 g, 2.0 mmol) was added
dropwise. The resulting mixture was stirred for 20 min, with
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
acetyl-â-D-ribofuranosyl)indole (7a). Compound 6 (1.00 g,
2.3 mmol) was dissolved in dry DMF (40 mL) to which was
added phosphorus oxychloride (1.1 mL, 1.8 g, 11.8 mmol). The
resulting solution was stirred at room temperature for 10 min,
then heated on a 60 °C oil bath for 16 h. The resulting orange
solution was evaporated under high vacuum (0.5 mmHg, 40
°C), and the residual oil was poured into 10% aqueous NaHCO3
(200 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 yellow oil. The
oil was dissolved in CHCl3 (2 mL) and subjected to column
chromatography (50 mm × 450 mm) on silica gel with 1:1
hexane/EtOAc. Fractions containing product were pooled and
evaporated to yield 0.55 g (52%) of 7a as a pale-yellow solid:
mp 148-149 °C; Rf ) 0.3 (2:1 hexane/EtOAc).
3-Cyano-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
acetyl-â-D-ribofuranosyl)indole (7b). Compound 6 (3.50 g,

5760 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
slow warming to room temperature, and then compound 6
(0.43 g, 0.99 mmol) was added in one portion. The mixture
was stirred at room temperature for 90 min and then poured
into 5% aqueous Na2CO3 (25 mL). The aqueous suspension was
extracted with CHCl3 (2 × 25 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 (40 mm × 350 mm)
on silica gel with 1:1 hexane/EtOAc. Fractions containing
product were pooled and evaporated to yield 0.18 g (35%) of
7e as a colorless foam. Rf ) 0.4 (2:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(5-O-acetyl-â-D-ribofurano-
syl)indole (8a). Compound 7a (540 mg, 1.2 mmol) was
dissolved in 90% aqueous TFA (10 mL) and stirred at room
temperature for 5 min. The solvent was then removed under
vacuum, and the residue was dissolved in EtOAc (100 mL).
The organic solution was washed with 10% aqueous NaHCO3
(50 mL), then dried over MgSO4, filtered, and evaporated to
yield a white powder, which was recrystallized from EtOAc/
hexane to yield 420 mg (86%) of 8a as a white solid: mp 154-
155 °C; Rf ) 0.5 (10% MeOH/CHCl3). Anal. (C16H14Cl3NO6‚1/
4EtOAc) C, H, N.
3-Cyano-2,5,6-trichloro-1-(5-O-acetyl-â-D-ribofuranosyl)-
indole (8b). Compound 7b (2.45 g, 5.3 mmol) was treated with
90% aqueous TFA (25 mL) as per 8a above, and the resulting
solid recrystallized from boiling hexane/CHCl3 to yield 1.85 g
(83%) of 8b as a white solid: mp 103-104 °C; Rf ) 0.6 (10%
MeOH/CHCl3). Anal. (C16H13Cl3N2O5) C, H, N.
3-Acetyl-2,5,6-trichloro-1-(5-O-acetyl-â-D-ribofuranosyl)-
indole (8c). Compound 7c (375 mg, 0.79 mmol) was treated
with 90% aqueous TFA (10 mL) as per 8a above, and the
resulting solid recrystallized from CHCl3/hexane to yield 318
mg (93%) of 8c as a white solid: mp 178-179 °C; Rf ) 0.5
(10% MeOH/CHCl3). Anal. (C17H16Cl3NO6) C, H, N.
3-Propionyl-2,5,6-trichloro-1-(5-O-acetyl-â-D-ribofuran-
osyl)indole (8d). Compound 7d (178 mg, 0.36 mmol) was
treated with 90% aqueous TFA (10 mL) as per 8a above, and
the resulting solid recrystallized from warm MeOH to yield
152 mg (93%) of 8d as a white solid: mp 168-169 °C; Rf )
0.6 (10% MeOH/CHCl3). Anal. (C18H18Cl3NO6) C, H, N.
3-Trifluoroacetyl-2,5,6-trichloro-1-(5-O-acetyl-â-D-ribo-
furanosyl)indole (8e). Compound 7e (130 mg, 0.24 mmol)
was treated with 90% aqueous trifluoroacetic acid (10 mL) as
per 8a above and evaporated to yield a pale-yellow oil. The oil
was dissolved in a minimum of CHCl3 and added dropwise to
rapidly stirred hexane (25 mL). The solids were triturated for
15 min, then filtered and rinsed with hexane (5 mL) to yield
98 mg (82%) of 8e as a pale-yellow powder: mp 71-74 °C; Rf
) 0.4 (10% MeOH/CHCl3).
3-Formyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole
(9a). Compound 8a (148 mg, 0.35 mmol) was dissolved in dry
MeOH (20 mL) to which was added sodium methoxide (21 mg,
0.39 mmol). The solution was stirred at room temperature for
30 min, and the solvent was then removed under vacuum. The
residue was suspended in brine (50 mL), and the suspension
was extracted with EtOAc (2 × 50 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to
yield a white solid. The solid was dissolved in MeOH (1 mL)
and subjected to column chromatography (40 mm × 350 mm)
on silica gel with 20% MeOH/CHCl3. Fractions containing
product were pooled and evaporated, then recrystallized from
boiling EtOAc/hexane to yield 55 mg (41%) of 9a as a white
powder: mp 216-218 °C; Rf ) 0.3 (10% MeOH/CHCl3). Anal.
(C14H12Cl3NO5‚1/2H2O) C, H, N.
yield 170 mg (81%) of 9c as a white solid: mp 249-250 °C; Rf
) 0.4 (10% MeOH/CHCl3). Anal. (C15H14Cl3NO5) C, H, N.
3-Propionyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)in-
dole (9d). Compound 8d (90 mg, 0.21 mmol) was treated with
sodium methoxide (14 mg, 0.26 mmol) as per 9a above, and
the resulting solid recrystallized from boiling EtOAc to yield
60 mg (67%) of 9d as a white solid: mp 239-240 °C; Rf ) 0.3
(10% MeOH/CHCl3). Anal. (C16H16Cl3NO5) C, H, N.
3-Trifluoroacetyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)-
indole (9e). Compound 8e (94 mg, 0.19 mmol) was treated
with sodium methoxide (12 mg, 0.22 mmol) as per 9a above,
and the resulting solid recrystallized from MeOH/H2O to yield
48 mg (56%) of 9e as a pale-yellow powder: mp 179-180 °C;
Rf ) 0.3 (10% MeOH/CHCl3). Anal. (C15H11Cl3F3NO5) C, H,
N.
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-â-D-ri-
bofuranosyl)indole (10a). Compound 511 (4.70 g, 12.0 mmol)
was dissolved in dry CH2Cl2 (50 mL) to which was added
trifluoroacetic anhydride (8.0 mL, 12 g, 57 mmol). The solution
was stirred at room temperature for 1 h, the solvent was
removed under vacuum, and the residue was dried under high
vacuum (0.5 mmHg, 30 °C) for 1 h. The residue was then
dissolved in dry DMF (100 mL), and phosphorus oxychloride
(6.0 mL, 9.9 g, 64 mmol) was added in one portion. The
resulting orange solution was heated on a 60 °C oil bath for
16 h and cooled to room temperature, and the solvent was
removed under high vacuum (0.5 mmHg, 40 °C). The residual
oil was poured into 10% aqueous NaHCO3 (300 mL, foaming!),
and the aqueous suspension was extracted with EtOAc (2 ×
100 mL). The combined organic extracts were dried over
MgSO4, filtered, and evaporated to yield a dark-orange oil. The
oil was dissolved in CHCl3 (3 mL) and subjected to column
chromatography (50 mm × 450 mm) on silica gel with 1:1
hexane/EtOAc. Fractions containing product were pooled and
evaporated to yield a clear oil, which was crystallized from
CH2Cl2/hexane to yield 3.18 g (63%) of 10a as a white solid:
mp 183-184 °C; Rf ) 0.4 (1:1 hexane/EtOAc).
3-Cyano-2,5,6-trichloro-1-(2,3-O-isopropylidene-â-D-ri-
bofuranosyl)indole (10b). Compound 511 (744 mg, 1.9 mmol)
was dissolved in dry CH2Cl2 (20 mL) to which was added
trifluoroacetic anhydride (3.0 mL, 4.5 g, 21 mmol). The solution
was stirred at room temperature for 1 h. The solvent was then
removed under vacuum, and the residue was dried under high
vacuum (0.5 mmHg, 30 °C) for 1 h. The residue was dissolved
in dry CH2Cl2 (20 mL), and chlorosulfonyl isocyanate (0.25 mL,
0.41 g, 2.9 mmol) was added in one portion. The resulting
solution was stirred at room temperature for 1 h, dry DMF
(1.0 mL) was added, and the solution was stirred for an
additional 16 h. Water (5 mL) was added, and the biphasic
suspension was stirred vigorously for 15 min, then poured into
10% aqueous NaHCO3 (50 mL), and the aqueous suspension
was extracted with EtOAc (2 × 50 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to
yield a pale-yellow oil. The oil was dissolved in CHCl3 (3 mL)
and subjected to column chromatography (50 mm × 450 mm)
on silica gel with 1:1 hexane/EtOAc. Fractions containing
product were pooled and evaporated to yield a white solid,
which was recrystallized from EtOAc/hexane to yield 296 mg
(37%) of 10b as a white solid: mp 191-192 °C; Rf ) 0.6 (1:1
hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
propionyl-â-D-ribofuranosyl)indole (11a). Compound 10a
(315 mg, 0.75 mmol) was dissolved in propionic anhydride (6
mL) to which was added dry pyridine (1.5 mL) and 4-(di-
methylamino)pyridine (90 mg, 0.75 mmol). The resulting
solution was heated on a 120 °C oil bath for 12 min and cooled
to room temperature, and the solvent was removed under
vacuum (0.5 mmHg, 50 °C). The residual oil was dissolved in
EtOAc (75 mL), washed with 0.25 M aqueous HCl (50 mL),
10% aqueous NaHCO3 (50 mL), and brine (50 mL), then 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 2:1
hexane/EtOAc. Fractions containing product were pooled and
3-Cyano-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole (9b).
Compound 8b (1.95 g, 4.6 mmol) was treated with sodium
methoxide (300 mg, 5.6 mmol) as per 9a above, and the
resulting solid recrystallized from boiling EtOAc/hexane to
yield 1.45 g (83%) of 9b as a white solid: mp 237-238 °C; Rf
) 0.3 (10% MeOH/CHCl3). Anal. (C14H11Cl3N2O4) C, H, N.
3-Acetyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole (9c).
Compound 8c (232 mg, 0.53 mmol) was treated with sodium
methoxide (35 mg, 0.65 mmol) as per 9a above, and the
resulting solid recrystallized from boiling EtOAc/hexane to

3-Substituted 2,5,6-Trichloroindole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5761
evaporated to yield 175 mg (49%) of 11a as a pale-yellow
solid: mp 153-154 °C; Rf ) 0.4 (2:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
butyryl-â-D-ribofuranosyl)indole (11b). Compound 10a
(250 mg, 0.59 mmol) was treated with butyric anhydride (8
mL) and 4-(dimethylamino)pyridine (75 mg, 0.61 mmol) as per
11a above to yield 75 mg (26%) of 11b as a pale-yellow solid:
mp 165-166 °C; Rf ) 0.4 (2:1 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
methoxycarbonyl-â-D-ribofuranosyl)indole (11c). Com-
pound 10a (167 mg, 0.40 mmol) was dissolved in CH2Cl2 (10
mL) and dry pyridine (0.5 mL). A solution of methyl chloro-
formate (0.5 mL) in CH2Cl2 (10 mL) was added dropwise over
30 min. The resulting solution was stirred at room tempera-
ture for 12 h. The solvent was then removed under vacuum,
and the residual oil was dissolved in EtOAc (75 mL). The
organic solution was washed with 0.25 M aqueous HCl (50
mL), 10% aqueous NaHCO3 (50 mL), and brine (50 mL), then
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
2:1 hexane/EtOAc. Fractions containing product were pooled
and evaporated to yield a pale-yellow solid, which was
recrystallized from CHCl3/hexane to yield 171 mg (90%) of 11c
as a pale-yellow solid: mp 120-121 °C; Rf ) 0.4 (2:1 hexane/
EtOAc).
3-Formyl-2,5,6-trichloro-1-(5-O-propionyl-â-D-ribofuran-
osyl)indole (12a). Compound 11a (169 mg, 0.35 mmol) was
treated with 90% aqueous TFA (10 mL) as per 8a above, and
the resulting solid recrystallized from boiling EtOAc/hexane
to yield 112 mg (72%) of 12a as a pale-yellow solid: mp 139-
140 °C; Rf ) 0.6 (10% MeOH/CHCl3). Anal. (C17H16Cl3NO6) C,
H, N.
3-Formyl-2,5,6-trichloro-1-(5-O-butyryl-â-D-ribofurano-
syl)indole (12b). Compound 11b (164 mg, 0.33 mmol) was
treated with 90% aqueous TFA (10 mL) as per 8a above, and
the resulting solid recrystallized from boiling EtOAc/hexane
to yield 111 mg (74%) of 12b as a pale-yellow solid: mp 128-
129 °C; Rf ) 0.6 (10% MeOH/CHCl3). Anal. (C18H18Cl3NO6) C,
H, N.
(1 × 250 mL), and brine (1 × 100 mL), then dried over MgSO4,
filtered, and evaporated to yield 9.63 g (98%) of 15 as a pale-
yellow oil, which crystallized upon standing 4-5 days: mp
102-105 °C; Rf ) 0.4 (9:1 hexane/EtOAc).
3-Methyl-5,6-dichlorooxindole (16). Compound 15 (9.63
g, 34.7 mmol) was dissolved in toluene (100 mL), to which was
added sodium sulfate (50 g) and platinum(IV) oxide (100 mg).
This suspension was shaken under 20-30 psi of hydrogen at
room temperature for 4 h. The resulting gray suspension was
diluted with EtOAc (25 mL) and filtered. The solids were
rinsed with 80% toluene/EtOAc (125 mL) and discarded, and
the filtrate was evaporated to yield a yellow oil. The oil was
dissolved in glacial AcOH (50 mL) and stirred at room
temperature for 2 h. The solvent was then removed under
vacuum to yield a damp, orange solid. This solid was recrystal-
lized from boiling hexane to yield 3.72 g (50%) of 16 as a white
solid: mp 202-203 °C; Rf ) 0.5 (1:1 hexane/EtOAc).
3-Methyl-2,5,6-trichloroindole (17). Compound 16 (3.67
g, 17.0 mmol) was suspended in dry CH3CN (100 mL) and
heated on a 100 °C oil bath until completely dissolved.
Phosphorus oxychloride (3.2 mL, 5.3 g, 34 mmol) was added
in one portion, and the resulting solution was gently refluxed
for 1 h. A solution of imidazole (2.90 g, 42 mmol) in dry CH3-
CN (25 mL) was slowly added, and the resulting cloudy
solution was gently refluxed with vigorous stirring for 16 h.
The reaction mixture was then cooled to room temperature
and diluted with EtOAc (100 mL). The suspension thus
obtained was filtered, and the solids were rinsed with EtOAc
(100 mL) and discarded. The filtrate was evaporated to provide
an orange solid, which was dissolved in a minimum of EtOAc
and adsorbed onto 20 mL of silica gel. The adsorbed material
was placed on a pad of silica gel (50 mm × 75 mm) and eluted
with 4:1 hexane/EtOAc until no more product was obtained.
The eluent was evaporated to yield a white solid, which was
recrystallized from EtOAc/hexane to yield 1.54 g (39%) of 17
as a white solid: mp 155-156 °C; Rf ) 0.7 (3:1 hexane/EtOAc).
3-Methyl-2,5,6-trichloro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-
â-D-ribofuranosyl]indole (21). Compound 17 (1.15 g, 4.9
mmol) was dissolved in dry THF (25 mL) to which was added
60% sodium hydride in mineral oil (0.25 g, 6.3 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 chloride16 (20,
1.91 g, 4.9 mmol) in toluene (25 mL). The resulting solution
was stirred at room temperature for 3 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 syrup. The crude material was
dissolved in CHCl3 (4 mL) and subjected to column chroma-
tography (50 mm × 450 mm) on silica gel with 3:1 hexane/
EtOAc. Fractions containing product were pooled and evapo-
rated to yield 2.42 g (84%) of 21 as a colorless foam. An
analytical sample was prepared by recrystallization from
MeOH/CHCl3: mp 137-139 °C; Rf ) 0.5 (3:1 hexane/EtOAc).
3-Methyl-2,5,6-trichloro-1-(2-deoxy-â-D-ribofuranosyl)-
indole (22). Compound 21 (1.15 g, 1.96 mmol) was suspended
in dry MeOH (75 mL) to which was added sodium methoxide
(0.43 g, 8.0 mmol). The suspension was stirred at room
temperature until the solution cleared (2 h). The solvent was
then 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 clear oil. The residue was dissolved in EtOAc
(2 mL) and subjected to column chromatography (50 mm ×
450 mm) on silica gel with 1:2 hexane/EtOAc. Fractions
containing product were pooled and evaporated to yield a clear
oil, which was coevaporated with CHCl3 (4 × 10 mL) to yield
0.39 g (57%) of 22 as a white powder: mp 66-68 °C; Rf ) 0.4
(1:2 hexane/EtOAc).
3-Formyl-2,5,6-trichloro-1-(5-O-methoxycarbonyl-â-D-
ribofuranosyl)indole (12c). Compound 11c (191 mg, 0.40
mmol) was treated with 90% aqueous TFA (10 mL) as per 8a
above, and the resulting solid recrystallized from MeOH/H2O
to yield 126 mg (72%) of 12c as a pale-yellow solid: mp 148-
149 °C; Rf ) 0.5 (10% MeOH/CHCl3). Anal. (C16H14Cl3NO7) C,
H, N.
Methyl 2-(3,4-Dichlorophenyl)propionate (14). Sodium
amide (3.50 g, 88 mmol) was suspended in liquid ammonia
(250 mL) maintained at -78 °C. To this rapidly stirred
suspension was slowly added a solution of methyl (3,4-
dichlorophenyl)acetate20 (13, 10.96 g, 50.0 mmol) in dry THF
(45 mL). The resulting orange suspension was stirred at -78
°C for 30 min, then methyl iodide (30 g, 210 mmol) was added
in one portion. The reaction mixture was stirred at -78 °C
for 30 min, then quenched with ammonium chloride (20 g),
diluted with EtOAc (200 mL), and allowed to warm to room
temperature over 16 h. The remaining organic suspension was
diluted with H2O (200 mL), and the organic layer was
separated. The aqueous layer was extracted with EtOAc (200
mL), and the combined organic layers were washed with brine,
dried over MgSO4, filtered, and evaporated to yield an orange
oil. The crude oil was distilled on a Kugelrohr (100 °C, 0.5
mmHg) to provide 10.41 g (89%) of 14 as a clear oil: Rf ) 0.3
(9:1 hexane/EtOAc).
Methyl 2-(3,4-Dichloro-6-nitrophenyl)propionate (15).
Compound 14 (8.25 g, 35.4 mmol) was dissolved in concen-
trated sulfuric acid (20 mL) and cooled to >5 °C on an ice/
water bath. Concentrated aqueous nitric acid (2.7 mL, 43
mmol) was added dropwise over 15 min, and the resulting
orange solution was stirred at 0 °C for 20 min. The orange
solution was then poured into ice/water (250 mL) and extracted
with cold Et2O (2 × 250 mL). The combined organic extracts
were washed with water (1 × 250 mL), 10% aqueous NaHCO3
3-Methyl-2,5,6-trichloro-1-(2-deoxy-3-O-methanesulfo-
nyl-5-O-tert-butyldiphenylsilyl-â-D-ribofuranosyl)in-
dole (23). Compound 22 (0.44 g, 1.3 mmol) was dissolved in

5762 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
dry pyridine (20 mL) to which was added tert-butyldiphenyl-
silyl chloride (0.41 g, 1.5 mmol). The solution was stirred at
room temperature for 16 h, then methanesulfonyl chloride (5.0
mL, 7.4 g, 64 mmol) was added, and the reaction mixture was
stirred at room temperature for an additional 4 h. Methanol
(10 mL) was added, and the solution was stirred at room
temperature for 30 min. The solvent was then removed under
vacuum, and the residue was dissolved in EtOAc (100 mL).
The organic suspension was washed with water (100 mL) and
brine (100 mL), then dried over MgSO4, filtered, and evapo-
rated 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 CHCl3. Fractions containing product
were pooled and evaporated to yield 0.53 g (63%) of 23 as a
clear viscous residue. Rf ) 0.6 (CHCl3).
3-Methyl-2,5,6-trichloro-1-(2,3-dideoxy-2,3-didehydro-
â-D-ribofuranosyl)indole (24). Compound 23 (515 mg, 0.77
mmol) was dissolved in dry DMSO (10 mL) to which was added
water (100 µL) and potassium tert-butoxide (0.45 g, 4.0 mmol).
The resulting dark solution was swirled at room temperature
for 10 min and then poured into cold water (150 mL). The
aqueous mixture was extracted with EtOAc (3 × 100 mL), and
the organic extracts were washed successively with brine (150
mL). The combined organic extracts were dried over MgSO4,
filtered, and evaporated to yield an orange oil. The oil was
dissolved in CHCl3 and subjected to column chromatography
(50 mm × 450 mm) on silica gel with 1:1 hexane/EtOAc.
Fractions containing product were pooled and evaporated to
yield 240 mg (93%) of 24 as a clear oil: Rf ) 0.6 (1:1 hexane/
EtOAc).
3-Methyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole (25).
Compound 24 (212 mg, 0.64 mmol) was dissolved in acetone
(8 mL) and water (1 mL) to which were added N-methylmor-
pholine N-oxide (0.20 g, 1.7 mmol) and 2.5% osmium tetroxide
solution in t-BuOH (0.65 mL, 0.065 mmol). The resulting
solution was stirred at room temperature for 2 h, and then
additional N-methylmorpholine N-oxide (0.20 g, 1.7 mmol) was
added. The solution was then stirred at room temperature for
an additional 16 h. The solvent volume was reduced to
approximately 2 mL, and the solution was poured into 5%
aqueous sodium thiosulfate (30 mL). The resulting mixture
was extracted with EtOAc (2 × 30 mL), and the combined
organic extracts were dried over MgSO4, filtered, and evapo-
rated to yield an orange oil. The oil was dissolved in 50%
MeOH/CHCl3 (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 144 mg (62%) of 25 as a white powder: mp 190-191 °C;
Rf ) 0.4 (10% MeOH/CHCl3). Anal. (C14H14Cl3NO4‚1/2H2O) C,
H, N.
2,3,5,6-Tetrachloroindole (26a). 2,5,6-Trichloroindole11 (4,
0.55 g, 2.5 mmol) was dissolved in dry CH3CN (20 mL) to which
was added N-chlorosuccinimide (0.58 g, 4.3 mmol). The result-
ing solution was stirred at room temperature for 1 h, the
solvent was removed under vacuum, and the residue was
suspended in 10% aqueous sodium thiosulfate (100 mL). The
organic suspension was extracted with EtOAc (2 × 50 mL),
and the combined organic extracts were dried over MgSO4,
filtered, and evaporated to yield a pale-yellow solid. The solid
was dissolved in EtOAc (2 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 0.45 g (70%) of 26a as a white solid: mp
162-163 °C; Rf ) 0.5 (hexane/EtOAc 5:1).
tography (50 mm × 450 mm) on silica gel with 5:1 hexane/
EtOAc. Fractions containing product were pooled and evapo-
rated to dryness. The resulting pink solid was recrystallized
from EtOAc/hexane to yield 3.02 g (87%) of 26b as a pink
solid: mp 153-154 °C; Rf ) 0.5 (hexane/EtOAc 5:1).
2,3,5,6-Tetrachloro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-â-D-
ribofuranosyl]indole (27a). Compound 26a (1.14 g, 4.5
mmol) was treated with 60% sodium hydride in mineral oil
(0.40 g, 10 mmol) and 3,5-di-O-(p-toluoyl)-2-deoxy-R-D-ribo-
furanosyl chloride16 (20, 1.82 g, 4.7 mmol) as per 21 above to
yield 2.27 g (83%) of 27a as a pale-yellow solid: mp 123-124
°C; Rf ) 0.6 (3:1 hexane/EtOAc).
3-Iodo-2,5,6-trichloro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-â-
D-ribofuranosyl]indole (27b). Compound 26b (4.87 g, 14.1
mmol) was treated with 60% sodium hydride in mineral oil
(0.80 g, 20 mmol) and compound 2016 (5.49 g, 14.1 mmol) in
toluene (50 mL) as per 21 above, and the resulting solid
recrystallized from CHCl3/MeOH to yield 7.26 g (74%) of 27b
as a pale-yellow powder: mp 115-117 °C; Rf ) 0.6 (3:1 hexane/
EtOAc).
2,3,5,6-Tetrachloro-1-(2-deoxy-â-D-ribofuranosyl)in-
dole (28a). Compound 27a (2.24 g, 3.7 mmol) was suspended
in absolute MeOH (250 mL) to which was added potassium
carbonate (1.53 g, 11 mmol). The suspension was stirred
vigorously at room temperature for 2 h, after which time the
solution clarified. The solvent was then removed under
vacuum, and the residue was suspended in 40 mL of brine
and extracted with EtOAc (2 × 40 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to
yield a pale-yellow oil. The oil was adsorbed onto silica gel (50
mL) and then placed onto a silica gel column (50 × 100 mL).
The column was eluted with 3:1 hexane/EtOAc until no more
methyl p-toluoylate was eluted. Then the column was further
eluted with 1:3 hexane/EtOAc until the product was completely
eluted from column. The solvent was then removed under
vacuum to yield a clear residue, which was recrystallized from
CHCl3/hexane to yield 0.93 g (68%) of 28a as a pale-yellow
powder: mp 132-133 °C; Rf ) 0.4 (10% MeOH/CHCl3).
3-Iodo-2,5,6-trichloro-1-(2-deoxy-â-D-ribofuranosyl)in-
dole (28b). Compound 27b (1.75 g, 2.5 mmol) was treated with
sodium methoxide (0.41 g, 7.6 mmol) as per 22 above, and the
resulting solid recrystallized from CHCl3/hexane to yield 0.91
g (78%) of 28b as a white solid: mp >100 °C (dec); Rf ) 0.3
(10% MeOH/CHCl3).
2,3,5,6-Tetrachloro-1-(2-deoxy-3-O-methanesulfonyl-5-
O-tert-butyldiphenylsilyl-â-D-ribofuranosyl)indole (29a).
Compound 28a (0.90 g, 2.4 mmol) was treated with tert-
butyldiphenylsilyl chloride (0.87 g, 3.2 mmol) and methane-
sulfonyl chloride (1.0 mL, 1.48 g, 13 mmol) as per 23 above to
yield 1.45 g (87%) of 29a as a colorless foam. Rf ) 0.5 (3:1
hexane/EtOAc).
3-Iodo-2,5,6-trichloro-1-(2-deoxy-3-O-methanesulfonyl-
5-O-tert-butyldiphenylsilyl-â-D-ribofuranosyl)indole (29b).
Compound 28b (1.92 g, 4.1 mmol) was treated with tert-
butyldiphenylsilyl chloride (1.50 g, 5.5 mmol) and methane-
sulfonyl chloride (2.0 mL, 3.0 g, 25 mmol) as per 23 above to
yield 2.75 g (85%) of 29b as a colorless foam: mp 64-70 °C;
Rf ) 0.4 (3:1 hexane/EtOAc).
2,3,5,6-Tetrachloro-1-(2,3-dideoxy-2,3-didehydro-â-D-ri-
bofuranosyl)indole (30a). Compound 29a (1.27 g, 1.8 mmol)
was treated with potassium tert-butoxide (1.05 g, 9.4 mmol)
as per 24 above to yield 0.30 g (46%) of 30a as a red oil. Rf )
0.5 (1:1 hexane/EtOAc).
3-Iodo-2,5,6-trichloro-1-(2,3-dideoxy-2,3-didehydro-â-
D-ribofuranosyl)indole (30b). Compound 29b (2.69 g, 3.5
mmol) was treated with potassium tert-butoxide (1.95 g, 17.4
mmol) as per 24 above to yield 0.86 g (56%) of 30b as a slightly
red oil. Rf ) 0.5 (1:1 hexane/EtOAc).
3-Iodo-2,5,6-trichloroindole (26b). 2,5,6-Trichloroindole11
(4, 2.21 g, 10.0 mmol) was dissolved in 30 mL of dry CH3CN
to which was added N-iodosuccinimide (2.70 g, 12.0 mmol).
The orange solution was stirred at room temperature for 30
min, then the solvent was removed under vacuum. To the
residual dark-red solid was added 25 mL of 10% sodium
thiosulfate and 25 mL of 10% Na2CO3. The aqueous suspension
was extracted with EtOAc (2 × 50 mL). The combined organic
extracts were dried over MgSO4, filtered, and evaporated to
yield a red solid. The solid was subjected to column chroma-
2,3,5,6-Tetrachloro-1-(â-D-ribofuranosyl)indole (31a).
Compound 30a (0.30 g, 0.85 mmol) was treated with N-
methylmorpholine N-oxide (0.50 g, 4.2 mmol) and 2.5% os-
mium tetroxide solution in tert-BuOH (1.0 mL, 0.10 mmol) as
per 25 above, and the solid recrystallized from 50% MeOH/
CHCl3 and hexane to yield 0.60 g (48%) of 31a as a white

3-Substituted 2,5,6-Trichloroindole Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5763
powder: mp 197-198 °C; Rf ) 0.3 (10% MeOH/CHCl3). Anal.
(C13H11Cl4NO4) C, H, N.
yield 175 mg (76%) of 34b as an orange oil. Rf ) 0.4 (5:1
hexane/acetone).
3-Iodo-2,5,6-trichloro-1-(â-D-ribofuranosyl)indole (31b).
Compound 30b (0.77 g, 1.7 mmol) was treated with N-
methylmorpholine N-oxide (1.02 g, 8.6 mmol) and 2.5% os-
mium tetroxide solution in tert-BuOH (1.7 mL, 0.17 mmol) as
per 25 above, and the solid recrystallized from 50% MeOH/
CHCl3 and hexane to yield 0.60 g (72%) of 31b as a white
solid: mp 201-202 °C; Rf ) 0.3 (10% MeOH/CHCl3). Anal.
(C13H11Cl3INO4) C, H, N.
3-(2-Furyl)-2,5,6-trichloro-1-(â-D-ribofuranosyl)in-
dole (35a). Compound 34a (105 mg, 0.21 mmol) was dissolved
in absolute MeOH (10 mL) to which was added concentrated
aqueous HCl (2 mL). The resulting suspension was heated on
a 60 °C oil bath for 45 min, cooled to room temperature, and
evaporated until no more MeOH remained. The remaining
aqueous suspension was diluted with brine (25 mL) and
extracted with EtOAc (2 × 40 mL). The combined organic
extracts were washed with 10% NaHCO3 (25 mL), dried over
MgSO4, filtered, and evaporated to yield a dark oil. The oil
was dissolved in 10% MeOH/CHCl3 (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 63 mg (65%) of 35a as a pale-gray
powder: mp 139-140 °C; Rf ) 0.3 (10% MeOH/CHCl3). Anal.
(C17H14Cl3NO5) C, H, N.
3-(3-Thienyl)-2,5,6-trichloro-1-(â-D-ribofuranosyl)in-
dole (35b). Compound 34b (175 mg, 0.34 mmol) was treated
with absolute MeOH (10 mL) and aqueous HCl (2 mL) as per
35a above, and the resulting solid recrystallized from MeOH/
H2O to yield 110 mg (75%) of 35b as a tan solid: mp 152-153
°C; Rf ) 0.4 (10% MeOH/CHCl3). Anal. (C17H14Cl3NO4S) C, H,
N.
3-[1-(2-Methoxy)vinyl]-2,5,6-trichloro-1-(2,3-O-isopro-
pylidene-5-O-acetyl-â-D-ribofuranosyl)indole (36). Meth-
oxymethyltriphenylphosphonium chloride (0.70 g, 2.0 mmol)
was suspended in dry THF (25 mL), and the suspension was
cooled to 0 °C. A solution of n-butyllithium (1.6 M) in hexane
(1.0 mL, 1.6 mmol) was added dropwise over 15 min, resulting
in an orange suspension to which was added a solution of
compound 7a (441 mg, 0.95 mmol) in dry THF (10 mL). The
resulting yellow suspension was stirred at 0 °C for 1 h, then
poured into brine (50 mL) and H2O (10 mL). The aqueous
suspension was extracted with EtOAc (2 × 50 mL), and the
combined extracts were dried over MgSO4, filtered, and
evaporated to yield a yellow-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 clear
oil. The oil was coevaporated twice with MeOH to yield 185
mg (40%) of 36 as a white solid, which was an inseparable 7:3
mixture of trans/cis isomers. A portion was recrystallized from
warm MeOH to provide an analytical sample: mp 135-136
°C; Rf ) 0.4 (3:1 hexane/EtOAc).
3-Formylmethyl-2,5,6-trichloro-1-(â-D-ribofuranosyl)-
indole (38). Compound 36 (185 mg, 0.38 mmol) was dissolved
in absolute MeOH (10 mL) to which was added concentrated
aqueous HCl (2 mL). The resulting suspension was heated on
a 60 °C oil bath for 1 h, then cooled to room temperature and
evaporated until no more MeOH remained. The remaining
aqueous suspension was diluted with brine (25 mL) and
extracted with EtOAc (2 × 40 mL). The combined organic
extracts were washed with 10% NaHCO3 (25 mL), dried over
MgSO4, filtered, and evaporated to yield a yellow oil. The oil
was dissolved in 90% aqueous TFA (10 mL), and the solution
was stirred at room temperature for 2 min. The solvent was
evaporated until approximately 1 mL remained, and the
remainder was poured into 10% aqueous NaHCO3 (50 mL).
The aqueous suspension was extracted with EtOAc (2 × 50
mL), and the combined organic extracts were dried over
MgSO4, filtered, and evaporated to yield a white powder. The
solid was dissolved in MeOH (1 mL) and subjected to column
chromatography (40 mm × 350 mm) on C18 reverse-phase
silica gel with 75% MeOH/H2O. Fractions containing product
were pooled and evaporated to yield 93 mg (62%) of 38 as a
tan powder: mp 119-121 °C; Rf ) 0.2 (10% MeOH/CHCl3).
Anal. (C15H14Cl3NO5) C, H, N.
3-Iodo-2,5,6-trichloro-1-(2,3-O-isopropylidene-â-D-ribo-
furanosyl)indole (32). Compound 31b (2.22 g, 4.6 mmol) was
dissolved in dry acetone (50 mL) to which were added 2,2-
dimethoxypropane (10 mL) and p-toluenesulfonic acid hydrate
(20 mg). The solution was stirred at room temperature for 15
min. The solvent was then removed under vacuum, and the
residue was suspended in 10% NaHCO3 (100 mL). The aqueous
suspension was extracted with EtOAc (2 × 75 mL), and the
combined organic extracts were dried over MgSO4, filtered, and
evaporated to yield a pale-yellow oil. The oil was diluted with
absolute MeOH (50 mL) and glacial acetic acid (2 mL). The
resulting suspension was stirred at room temperature for 1
h, at which time the solids had completely dissolved. The
solvent was then removed under vacuum, and the residue was
suspended in 10% NaHCO3 (100 mL). The aqueous suspension
was extracted with EtOAc (2 × 75 mL), and the combined
organic extracts were dried over MgSO4, filtered, and evapo-
rated to yield a clear 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 white solid,
which was recrystallized from CHCl3/hexane to yield 2.17 g
(90%) of 32 as a white solid: mp 78-80 °C; Rf ) 0.5 (2:1
hexane/EtOAc).
3-Iodo-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-meth-
oxymethyl-â-D-ribofuranosyl)indole (33). Compound 32
(1.00 g, 1.9 mmol) was dissolved in dry CH2Cl2 (50 mL) to
which were added diisopropylethylamine (4 mL, 23 mmol) and
chloromethyl methyl ether (0.75 mL, 0.80 g, 9.9 mmol). The
solution was stirred at room temperature for 16 h. The solvent
was then removed under vacuum, and the residue was
suspended in 10% NaHCO3 (75 mL). The aqueous suspension
was extracted with EtOAc (2 × 50 mL), and the combined
organic extracts were dried over MgSO4, filtered, and evapo-
rated to yield an orange 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 a clear oil, which
was recrystallized from warm hexane to yield 0.76 (70%) of
33 as a white solid: mp 120-121 °C; Rf ) 0.6 (3:1 hexane/
EtOAc).
3-(2-Furyl)-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-O-
methoxymethyl-â-D-ribofuranosyl)indole (34a). Com-
pound 33 (250 mg, 0.44 mmol) and 2-furanboronic acid (60 mg,
0.53 mmol) were suspended in 1-propanol (4 mL) and dry DMF
(1.0 mL). The suspension was stirred at room temperature for
10 min, then palladium(II) acetate (20 mg, 0.09 mmol) and
tri-o-tolylphosphine (84 mg, 0.28 mmol) were added, followed
by 2.0 M aqueous Na2CO3 (400 µL, 0.80 mmol) and H2O (1.0
mL). The resulting suspension was heated on a 120 °C oil bath
for 15 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 185 mg (83%) of
34a as an orange oil. Rf ) 0.3 (5:1 hexane/EtOAc).
3-(3-Thienyl)-2,5,6-trichloro-1-(2,3-O-isopropylidene-5-
O-methoxymethyl-â-D-ribofuranosyl)indole (34b). Com-
pound 33 (250 mg, 0.44 mmol) was treated with 3-thiophenebo-
ronic acid (69 mg, 0.54 mmol), palladium(II) acetate (20 mg,
0.09 mmol), tri-o-tolylphosphine (84 mg, 0.28 mmol), and 2.0
M aqueous Na2CO3 (400 µL, 0.80 mmol) as per 34a above to
2-Bromo-5,6-dichloro-1-(2,3-O-isopropylidene-5-O-
acetyl-â-D-ribofuranosyl)indole (40). 2-Bromo-5,6-dichloro-
1-(2,3-O-isopropylidene-â-D-ribofuranosyl)indole11 (39, 1.35 g,
3.1 mmol) was dissolved in dry pyridine (12 mL) to which was
added acetic anhydride (4 mL). The resulting solution was

5764 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Williams et al.
stirred at room temperature for 4 h, and the solvent was then
removed under high vacuum (0.5 mmHg, 40 °C). The residual
oil was dissolved in EtOAc (100 mL), washed successively with
0.25 M aqueous HCl (100 mL), 10% NaHCO3 (100 mL), brine
(25 mL), then dried over MgSO4, filtered, and evaporated to
afford a clear 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 1.41 g (95%) of 40 as a
colorless foam: mp 49-54 °C; Rf ) 0.6 (2:1 hexane/EtOAc).
3-Formyl-2-bromo-5,6-dichloro-1-(2,3-O-isopropylidene-
5-O-acetyl-â-D-ribofuranosyl)indole (41). Compound 40
(0.47 g, 0.98 mmol) was dissolved in dry DMF (15 mL) to which
was added phosphorus oxybromide (2.2 g, 7.6 mmol). The
resulting suspension was stirred vigorously for 10 min at room
temperature and then heated on a 70 °C oil bath for 16 h. The
resulting solution was cooled to room temperature and evapo-
rated under high vacuum (0.5 mmHg, 40 °C) to yield an orange
oil. The oil was poured into cold 10% NaHCO3 (150 mL), and
the resulting aqueous suspension was extracted with EtOAc
(2 × 100 mL). The combined organic extracts were washed
with brine (50 mL), dried over MgSO4, filtered, and evaporated
to yield an orange oil. The oil was dissolved in CHCl3 (1 mL)
and subjected to column chromatography (40 mm × 350 mm)
on silica gel with 2:1 hexane/EtOAc. Fractions containing
product were pooled and evaporated to yield 278 mg (56%) of
41 as a white solid: mp 178-179 °C; Rf ) 0.3 (2:1 hexane/
EtOAc).
3-Formyl-2-bromo-5,6-dichloro-1-(5-O-acetyl-â-D-ribo-
furanosyl)indole (42). Compound 41 (257 mg, 0.51 mmol)
was treated with 90% aqueous trifluoroacetic acid (10 mL) as
per 8a above, and the resulting solid recrystallized from EtOAc
and hexane to yield 220 mg (93%) of 42 as a white solid: mp
159-160 °C; Rf ) 0.5 (10% MeOH/CHCl3). Anal. (C16H14BrCl2-
NO6‚1/4 hexane) C, H, N.
3-Formyl-2-bromo-5,6-dichloro-1-(â-D-ribofuranosyl)-
indole (43). Compound 42 (101 mg, 0.22 mmol) was treated
with sodium methoxide (25 mg, 0.46 mmol) as per 9a above,
then dissolved in MeOH (1 mL) and subjected to column
chromatography (40 mm × 350 mm) on C18 reverse-phase
silica gel with 75% MeOH/H2O. Fractions containing product
were pooled and evaporated to yield a white solid, which was
recrystallized from MeOH/H2O to yield 43 mg (47%) of 43 as
a pale-tan powder: mp 210-211 °C; Rf ) 0.2 (10% MeOH/
CHCl3). Anal. (C14H12BrCl2NO5) 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.21
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.22 High titer HSV-1 stocks were prepared by infecting
KB cells at a moi of <0.1 also as detailed previously.22 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.23 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.
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 employed24 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 and HSV-1 at 100 pfu/well were added in
quadruplicate wells. 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 2.0 N H2SO4 (50 µL) after 15
min, 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
absorbance 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 uninfected cells.22 (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.25 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 concen-
trations. 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.
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-acetyl-
pyridine thiosemicarbazone for cytotoxicity) were used in all
assays.
Acknowledgment. We thank Julie M. Breitenbach
and Kathy Z. Borysko for expert performance of anti-
viral and cytotoxicity assays. These studies were sup-
ported by Training Grant T32-GM07767 and Research
Grants U19-AI31718 and P01-AI46390 from the Na-
tional Institutes 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.
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