Design, synthesis, and antiviral evaluation of some polyhalogenated indole C-nucleosides

Article abstract of DOI:10.1080/15257770500265646

□ 2,5,6-Trichloro-1-(β-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and se

Full text of DOI:10.1080/15257770500265646

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Design, Synthesis, and Antiviral Evaluation of Some
Polyhalogenated Indole C-nucleosides
Jiong J. Chen a , Yuan Wei a , John D. Williams b , John C. Drach b c & Leroy B. Townsend a c
a Department of Chemistry, College of Literature, Sciences and the Arts, University of
Michigan, Ann Arbor, Michigan, USA
b Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann
Arbor, Michigan, USA
c Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan, USA
Version of record first published: 16 Aug 2006.
To cite this article: Jiong J. Chen , Yuan Wei , John D. Williams , John C. Drach & Leroy B. Townsend (2005): Design,
Synthesis, and Antiviral Evaluation of Some Polyhalogenated Indole C-nucleosides, Nucleosides, Nucleotides and Nucleic Acids,
24:10-12, 1417-1437
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1417–1437, 2005
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500265646
DESIGN, SYNTHESIS, AND ANTIVIRAL EVALUATION OF SOME
POLYHALOGENATED INDOLE C-NUCLEOSIDES
ᮀ
Jiong J. Chen and Yuan Wei
Department of Chemistry, College of Literature,
Sciences and the Arts, University of Michigan, Ann Arbor, Michigan, USA
ᮀ
John D. Williams
Department of Medicinal Chemistry, College of Pharmacy,
University of Michigan, Ann Arbor, Michigan, USA
ᮀ
John C. Drach
Department of Medicinal Chemistry, College of Pharmacy, and
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan, USA
ᮀ
Leroy B. Townsend
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, USA
ᮀ
2,5,6-Trichloro-1-( -D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5,6-dichloro-1-( -D-ribo-
furanosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-( -D-ribofuranosyl)benzimid-
azole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV ac-
tivity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole
nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione
and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5,6-dichloro-3-( -D-
ribofuranosyl)indole (12). 3-( -D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12
in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted
2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole
aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.
Keywords
Indole nucleoside; C-Nucleoside; Nucleoside analog; Antiviral; TCRB;
Human cytomegalovirus (HCMV)
This manuscript is dedicated to the memory of Dr. John A. Montgomery.
Received 12 October 2004; accepted 18 April 2005.
We thank Julie M. Breitenbach and Kathy Z. Borysko 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 Institutes of Health.
Address correspondence to Dr. Leroy B. Townsend, Department of Medicinal Chemistry, College
of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065. Fax: 734/764-7547; E-mail: ltownsen@
umich.edu
1417

1418
INTRODUCTION
J. J. Chen et al.
A series of 2-substituted benzimidazole nucleosides have been synthe-
sized in our group and displayed strong antiviral activities.[1,2] The lead com-
pounds, including 2,5,6-trichloro-1-(β-D-ribofuranosyl)benzimidazole (1,
TCRB, Figure 1), 2-bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole
(2, BDCRB, Figure 1) and 2-benzylthio-5,6-dichloro-1-(β-D-ribofuranosyl)
benzimidazole (3, BTDCRB, Figure 1), have shown potent activity against
HCMV with low cellular toxicity at concentrations inhibiting viral growth
(TCRB: IC50 = 2.8 µM, CC50 = 238 µM, BDCRB: IC50 = 0.7 µM, CC50
=
118 µM, BTDCRB: IC50 = 22 µM, CC50 = 100 µM in a plaque assay in hu-
man foreskin fibroblasts). However, the instability of the glycosyl bond of
TCRB has been one of the major problems in its clinical development. It was
found that TCRB disappears rapidly from the bloodstream of animals (rats
or monkeys) following intravenous or oral dosage.[3] The disappearance of
TCRB from the blood correlated with increased blood concentration of the
aglycon, 2,5,6-trichlorobenzimidazole. This indicated that the lability of the
glycosidic bond is at least partially responsible for the rapid disappearance
of TCRB from the bloodstream.
Indole, a heterocycle that is structurally similar to benzimidazole, has the
potential of bearing the same substitution patterns as that of TCRB, BDCRB
and BTDCRB. Therefore, we proposed to synthesize some polyhalogenated
indole nucleosides (Figure 2) as 1-deaza analogs of the antiviral benzimi-
dazole nucleosides (Figure 1). Because the proposed indole C-nucleosides
should resist enzymatic cleavage at the glycosyl bond, they may potentially
solve the unstable glycosyl bond problem of TCRB. Several polyhalogenated
indole N-nucleosides have been synthesized in our group as 3-deaza analogs
of TCRB,[4–8] but the synthesis of indole C-nucleosides poses unique syn-
thetic challenges and would help us further elucidate the structural and
chemical requirements for activity in this series. In light of substantial struc-
ture activity relationship (SAR) studies of TCRB,[1,2,9–13] a limited number
of 5,6-dichloroindole C-ribosides should be sufficient to evaluate the poten-
tial antiviral activity of this type of nucleosides as analogs of TCRB.
FIGURE 1 Some polyhalogenated benzimidazole nucleosides with activity against HCMV.

Antiviral Evaluation of Some C-Nucleosides
1419
FIGURE 2 Proposed polyhalogenated indole C-nucleosides.
RESULTS AND DISCUSSION
Chemistry
The synthesis of indole C-nucleosides has been of interest for some
time.[14,15] The previous synthesis generally involved a coupling of indole
aglycons and sugar donors to directly provide indole C-nucleosides. In order
to use this synthetic approach, some 2-substituted (benzylthio and chloro)
5,6-dichloroindoles were required as aglycons for our targeted nucleosides.
Although 2-benzylthio-5,6-dichloroindole was readily available,[4] the 2-
chloro analog had not been reported in the literature at the beginning of
our study. Thus, we initially decided to prepare a C-nucleoside of 2-benzyl-
thio-5,6-dichloroindole, and then transform this nucleoside to the C-nucleo-
sides of the 2-chloro analog.
In previous research from our laboratory,[4] we had established that
2-benzylthio-5,6-dichloroindole did not undergo a coupling with certain
ribofuranosyl donors to yield an indole N-nucleoside due to the bulky 2-ben-
zylthio group. Therefore, we assumed that the direct coupling procedures
of Sokolova and Cornia could encounter a similar problem. However,
an indole-2-thione has been known to react with some ketones at the 3-
position in a Knoevenagel type fashion.[16] If the ketones were replaced
with a ribofuranose derivative, a condensation of 5,6-dichloroindole-2-
thione (1) and the ribofuranose derivative, followed by a benzylation, could
lead directly to a 2-benzylthio-5,6-dichloroindole C-nucleoside. Indeed,
when a mixture of compound 1 and 2,3-O-isopropylidene-D-ribofuranose in
MeOH was treated with sodium carbonate, a precipitate was formed. This
precipitate was likely the salt intermediate 5 (Scheme 1). Without isolation,
this salt was treated with benzyl bromide to afford 2-benzylthio-5,6-dichloro-
3-(2,3-O-isopropylidene-α-D-ribofuranosyl)indole (6) and 2-benzylthio-5,6-
dichloro-3-(2,3-O-isopropylidene-β-D-ribofuranosyl)indole (7) in a 2:1 ratio.
It should be noted that the ring closure to form possible pyranose isomers
was not observed, since the presence of a five-membered isopropylidene ring

1420
J. J. Chen et al.
SCHEME 1 Reagents and conditions: (a) Na2CO3, MeOH, 20 C, 20 h; (b) BnBr, acetone, 20 C,
30 min; (c) Raney Ni, EtOH, 20 C, 1 h; (d) Ac2O, Et3N, CH3CN, 20 C, 24 h; (e) 90% TFA, 20 C,
10 min; (f) Na2CO3, MeOH, 20 C, 2 h.

Antiviral Evaluation of Some C-Nucleosides
1421
strongly favors the formation of a second fused, five-membered ring.[17] The
Knoevenagel reaction has been applied in the C-nucleoside synthesis area
with limited successes.[18,19] Ketones and carboxamides were generally used
to couple with sugars, and harsh reaction conditions were usually required,
which resulted in low yields and some epimerizations.[20] To the best of our
knowledge, this is the first example of a thioamide Knoevenagel reaction
being used in a C-nucleoside synthesis. The mild reaction conditions may
find some use in the synthesis of other C-nucleosides.
Some difficulties were encountered in our efforts to determine the ano-
meric configurations of compounds 6 and 7. The chemical shift difference
for the H-1 protons suggested that compound 7 was the α-anomer, since
the peak for an α-anomer’s H-1ꢁ is generally at a lower field than that of a
β-anomer (Table 1).[21] However, the ∆δ value observed for the isopropy-
lidene group of 7 was larger than the ∆δ value for 6, which suggested that
7 was the β-anomer.[21] This β-anomeric assignment was further supported
by the 13C NMR data, since δ values of a β-anomer’s two methyls on the
isopropylidene group are generally larger than those of the α-anomer’s
methyls. The bulky benzylthio group may have exhibited some substituent
effects on the H-1s of 6 and 7. Thus, 5,6-dichloro-3-(2,3-O-isopropylidene-
α-D-ribofuranosyl)indole and 5,6-dichloro-3-(2,3-O-isopropylidene-β-D-ribo-
furanosyl)indole (8 and 9) were prepared from 6 and 7, respectively, to
eliminate this substituent effect. This time, all three NMR spectrum criteria
of 8 and 9 agreed (Table 1) with the general trends and we were confident in
assigning compound 7 as the β-anomer and compound 6 as the α-anomer.
In order to reduce the likelihood of a possible opening of the ribofura-
nose ring and a subsequent cyclization to form the thermodynamically more
stable pyranose isomers under acidic conditions, the 1- and 5ꢁ-positions of
compound 7 were protected with acetyl groups to give 1-acetyl-3-(5-O-ace-
tyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-2-benzylthio-5,6-dichloroindole
(10). The treatment of 10 with aqueous TFA provided 1-acetyl-3-(5-O-acetyl-
β-D-ribofuranosyl)-2-benzylthio-5,6-dichloroindole (7) with a small amount
of the α-anomer (β:α = 3.5:1). Due to the π-electron rich property of
an indole, the protonation of the ribofuranose ring oxygen may be fol-
lowed with an anomerization. This type of anomerization has been reported
TABLE 1 NMR Spectral Data Used to Determine Anomeric Configuration
H-1
(ppm)
δ of CMe2 (ppm)
CMe2 (ppm) in 13C
NMR
Anomeric
configuration
Compound #
in 1H NMR
6
7
8
9
5.04
5.24
5.26
5.08
0.29
0.31
0.23
0.25
24.0, 26.1
25.6, 27.6
24.5, 26.2
25.4, 27.4
α
β
α
β

1422
J. J. Chen et al.
previously.[14] The attempted separation of 11 and the α-anomer by flash
column chromatography proved fruitless. However, pure 11 was obtained by
crystallization from a mixture of EtOAc and hexane. Removal of the acetyl
group of 11 was performed with Na2CO3 in MeOH to afford the target com-
pound, 2-benzylthio-5,6-dichloro-3-(β-D-ribofuranosyl)indole (12).
Compound 12 was treated with Raney Ni (Scheme 2) to afford 5,6-
dichloro-3-(β-D-ribofuranosyl)indole (13), which was then silylated to give
5,6-dichloro-3-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]indole
(14). Treatment of 14 with NCSin CCl at room temperature yielded
4
SCHEME 2 Reagents and conditions: (a) Raney Ni, EtOH, 20 C, 30 min; (b) TBSCl, imidazole, DMF,
20 C, 26 h; (c) NCS, CCl4, 20 C, 24 h; (d) TBAF, THF, 20 C, 30 min; (e) Ac2O, Et3N, CH3CN, 20 C,
30 min; (f) KF, 80% TFA, 20 C, 1 h.

Antiviral Evaluation of Some C-Nucleosides
1423
3-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]-2,5,6-trichloroindole
1
(15) and the α-anomer in a 6.5:1 ratio based on H NMR spectral data.
Pure 15 was obtained in a 51% yield by crystallization from acetonitrile.
The cleavage of TBDMSgroups was effected by treating compound 15 with
TBAF and resulted in the targeted nucleoside, 3-(β-D-ribofuranosyl)-2,5,6-
trichloroindole (16). The N-acetyl derivative 18a was synthesized through
an acylation of 15 with Ac2O and followed by deprotection of the silyl
intermediate 17 under KF-TFA-H2O conditions. Deprotection of 17 with
TBAF caused a substantial cleavage of the labile acetyl group.
Although the above strategy yielded the target compounds 16 and 18a,
it had two major drawbacks: 1) the β-anomer 7 was the minor product of
the coupling reaction; and 2) the synthetic route for the 2-chloroindole
nucleoside 16 was tedious. At the same time, our studies in the indole N-
nucleoside area had resulted in an optimized procedure for the synthesis
of 2,5,6-trichloroindole (19, Scheme 3).[4] Thus, we elected to investigate a
direct coupling method for the synthesis of the 2-haloindole C-nucleosides.
Recently, Yokoyama and coworkers reported a successful preparation of
some indole C-nucleosides under Lewis acid–mediated glycosylation condi-
tions.[22] Thus, we elected to apply this method to our 2,5,6-trichloroindole
substrates. We found that the acyl-protected indoles were stable under
Lewis acid conditions and were reactive toward the desired glycosylation.
The use of an acetyl group is preferred to Yokoyama’s use of a phenylsul-
fonyl group, because the removal of an acetyl group is usually more facile
than that of a phenylsulfonyl group. The coupling between 1-acetyl-2,5,6-
trichloroindole (20a) and 2,3,5-tri-O-benzyl-β-D-ribofuranosyl fluoride[23]
proceeded smoothly at 0◦C. The glycosylation reaction yielded 1-acetyl-
2,5,6-trichloro-3-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)indole (21b) and its α
anomer in a ratio of approximately 4:1 (as determined by integration of the
appropriate 1ꢁ protons in the crude mixture). Similar results were obtained
from the coupling of 1-propionyl-2,5,6-trichloroindole (20b) and the fluoro-
sugar (Scheme 3). The assignment of anomeric configurations were based
on comparisons with Yokoyama’s results,[22] in which the coupling constant
of the β-anomer (J > 6.3 Hz) was always larger than that of the α-anomer
(J < 3.3 Hz). The coupling constants of the β-anomers (J > 8 Hz) in the
current series were much higher than the corresponding α-anomers (J <
3.5 Hz).
Initially, a removal of the benzyl groups from the carbohydrate moiety
under catalytic hydrogenation conditions, i.e., Pearlman’s catalyst (20%
palladium hydroxide on carbon, moisturized) in ethanol under 1 atm
hydrogen, resulted in multiple products. The 1H NMR spectra of all
the products revealed that all of the benzyl groups had been removed
but none of the spectra displayed the characteristic peaks observed for
the previously prepared compound 18a, probably due to overreduction

1424
J. J. Chen et al.
SCHEME 3 Reagents and conditions: (a) Ac2O, pyridine, CH2Cl2, 20 C, 24 h or CH3CH2COCl,
pyridine, 20 C, 16 h; (b) BF3OEt2, CH2Cl2, 0 C, 10 min, (c) H2 (1 atm), 5% Pd/BaSO4, AcOH,
20 C, 45 min; (d) MeNH2, MeOH, 20 C, 30 min; (e) NaH, THF, 0 C, 10 min then MeI or 1-PrI,
20 C, 60 min.

Antiviral Evaluation of Some C-Nucleosides
1425
(i.e., dechlorination of the heterocycle). To avoid the over-reduction, we
elected to use the debenzylation reagent boron trichloride. This reagent has
been used previously for the debenzylation of C-nucleosides,[22,24] although
it generally offers moderate to low yields. Our use of this reagent resulted
in the production of some of the desired product, but in a low yield.
The low yield was partially due to the loss of the labile acetyl group.
In addition, epimerization was also observed. These disappointing results
forced us to reexamine the catalytic hydrogenation conditions. In an effort
to stop or reduce the overreduction, ethyl acetate was tested as the solvent.
Encouragingly, some of the desired product 18a was isolated in a low yield
along with some overreduced products and some starting material. This
prompted us to initiate a thorough study on the effect of solvents on the
reaction. We found that acetic acid worked best as the solvent in this reaction
and yielded 18a as the major product. To further optimize the reaction
conditions, we tested a variety of different catalysts: e.g., 5% Pd/C, 10%
Pd/C, 20% Pd/C, 5% Pd/CaSO4, 5% Rh/C, PtO2, and 5% Pd/BaSO4. The
highest ratio of the desired product in comparison to the side products was
obtained with 5% Pd/BaSO4. Deprotection of the labile acetyl group of 18a
was then effected with methylamine in EtOH at room temperature to afford
the target compound 16 in a quantitative yield.
In order to determine whether different substituents would be toler-
ated at the 1-position of the indole, we decided to alkylate this position with
some simple electrophiles. The protected intermediate 21a was deacetylated
using the methylamine procedure, and the indole nitrogen was then de-
protonated using sodium hydride. The resulting sodium anion was treated
with methyl iodide or 1-iodopropane in situ to provide the desired 1-methyl
analog 23a or the 1-propyl analog 23b. These compounds (23a and 23b)
were debenzylated under the conditions optimized above using 1 atm H2
and 5% Pd/BaSO4 in acetic acid to provide 1-methyl-2,5,6-trichloro-3-(β-
D-ribofuranosyl)indole (24a) and 1-(1-propyl)-2,5,6-trichloro-3-(β-D-ribofur-
anosyl)indole (24b), respectively, in good yield.
Biological Evaluation
The compounds synthesized as described above were tested for antiviral
activity against HCMV and HSV-1 and for cytotoxicity. Although the com-
pounds are structurally similar to the benzimidazole nucleosides, only 12
appeared to have antiviral activity, but this was most likely due to cytotoxi-
city (Table 2). Thus, none of these indole C-nucleosides demonstrated the
potent antiviral activity or lack of cytotoxicity possessed by the benzimidazole
nucleosides, or by a number of the analogous indole N-nucleosides.[5–8] Be-
cause these representative chlorinated indole C-nucleosides were not active,
further analogs were not pursued.

1426
J. J. Chen et al.
TABLE 2 Antiviral Activity and Cytotoxicity of 3-Substituted Indole Nucleosides
50% Inhibitory concentration (µM)
Antiviral Cytotoxicity
HCMV
HSV-1
HFF
KB
No.
R1
R2
plaquea
ELISAb
visualc
growthc
12
16
18a
-H
-H
-COCH3
-Me
-SBn
-Cl
-Cl
32
>100
>100
>100
2.9
>100
>100
>100
>100
102
32
>100
>100
>100
238
50
65
50
24a
-Cl
>100
210
>100
>100
TCRBe
BDCRBe
GCVf
0.70
7.4
130
3.5
118
>100
aPlaque reduction assays were performed in duplicate wells as described in the text.
bCompounds were assayed by ELISA in quadruplicate wells.
cVisual 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.
eData for TCRB and BDCRB published previously as compounds 9 and 11, respectively, in
reference 1.
fAverages from 108, 33, and 3 experiments, respectively, using ganciclovir (GCV).
EXPERIMENTAL
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 a slight
positive pressure of argon. Room temperature is assumed 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 %v.
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, Delaware). Chromatograms were visualized under UV
1
light at 254 nm. H-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. 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). Mass spectrometry and elemental

Antiviral Evaluation of Some C-Nucleosides
1427
analysis for selected compounds (listed in supplemental information) were
performed at the University of Michigan Department of Chemistry Mass
Spectrometry facility and Elemental Analysis facility, respectively.
2-Benzylthio-5,6-dichloro-3-(2,3-O -isopropylidene-α-D-ribofuranosyl)
indole (6) and 2-Benzylthio-5,6-dichloro-3-(2,3-O-isopropylidene-β-D-ribo-
furanosyl)indole (7). A mixture of 5,6-dichloroindol-2-thione (4) (3.00 g,
13.7 mmol), 2,3-O-isopropylidene-D-ribofuranose (3.84 g, 20.5 mmol) and
Na2CO3 (5 g, 47 mmol) in 45 mL of MeOH was stirred at room temperature
for 20 h, and then concentrated to dryness. The residue was dried in vacuo,
treated with 60 mL of acetone and then with benzyl bromide (1.79 mL, 15.1
mmol). The mixture was stirred at room temperature for 30 min. The solids
were removed by filtration. The filtrate was concentrated to dryness. The
residue was purified by flash column chromatography (25% EtOAc/Hex, 4
cm × 10 cm) to give 3.46 g (52%) of 6 as a light yellow foam and 1.70 g
1
(26%) of 7 as a light yellow foam. 6: Rf 0.30 (30% EtOAc/Hex). H NMR
(CDCl3): δ 1.25 (s, 3H), 1.54 (s, 3H), 1.74 (t, 1H, J = 5.9), 3.61 (t, 2H, J =
5.9), 3.86 (s, 2H), 4.34 (m, 2H), 4.63 (m, 1H), 5.04 (d, 1H, J = 3.7), 7.04
(m, 2H), 7.21 (s, 1H), 7.25 (m, 3H), 8.04 (broad s, 1H), 8.06 (s, 1H). 13C
NMR (CDCl3): δ 24.0, 26.1, 41.5, 61.7, 77.9, 82.2, 83.2, 84.2, 111.8, 112.6,
117.2, 123.9, 124.1, 127.0, 127.1, 127.2, 127.4, 128.7, 128.8, 135.5, 138.1. 7: Rf
0.60 (30% EtOAc/Hex). 1H NMR (CDCl3): δ 1.35 (s, 3H), 1.66 (s, 3H), 2.10
(broad s, 1H), 3.92–4.03 (m, 4H), 4.15 (m, 1H), 4.77 (m, 1H), 4.89 (m, 1H),
5.24 (d, 1H, J = 6.1), 7.10 (m, 2H), 7.23 (m, 4H), 7.73 (s, 1H), 7.78 (broad
s, 1H). 13C NMR (CDCl3): δ 25.6, 27.6, 42.4, 62.8, 80.4, 81.4, 84.1, 85.0,
112.3, 115.3, 118.1, 120.7, 124.4, 125.6, 127.4, 127.6, 128.69, 128.72, 129.2,
135.1, 137.9. HRMSfor C 23H23Cl2NO4S: Calcd, 479.0725; Found, 479.0713.
5,6-Dichloro-3-(2,3-O-isopropylidene-α-D-ribofuranosyl)indole (8). A
mixture of compound 6 (500 mg, 1.04 mmol) and Raney Ni (4 g, wet, W-4)
in 15 mL of EtOH was stirred at room temperature for 1 h. The Raney Ni
was removed by filtration. The filtrate was concentrated to dryness, and the
residue was purified by flash column chromatography (90% EtOAc/Hex,
2 cm × 10 cm) to give 250 mg (67%) of 8 as a light white foam. Rf 0.30
1
(90% EtOAc/Hex). H NMR (CDCl3): δ 1.33 (s, 3H), 1.55 (s, 3H), 1.96
(broad s, 1H), 3.75 (m, 2H), 4.35 (m, 1H), 4.86–5.08 (m, 2H), 5.26 (d,
1H, J = 3.6), 7.30 (d, 1H, J = 2.4), 7.41 (s, 1H), 7.86 (s, 1H), 8.25 (broad
s, 1H). 13C NMR (CDCl3): δ 24.5, 26.2, 62.0, 77.4, 82.5, 82.7, 84.0, 110.7,
112.6, 112.8, 121.5, 123.7, 125.6, 125.9, 126.4, 134.9.
5,6-Dichloro-3-(2,3-O-isopropylidene-β-D-ribofuranosyl)indole (9). The
procedure is the same as that described for 8, except that 7 (500 mg, 1.04
mmol) was used instead of 6. A 72% yield (268 mg, 0.75 mmol) of 9 was

1428
J. J. Chen et al.
1
obtained as a white foam. Rf 0.50 (90% EtOAc/Hex). H NMR (CDCl3): δ
1.39 (s, 3H), 1.64 (s, 3H), 3.80 (m, 1H), 3.98 (m, 1H), 4.19 (m, 1H), 4.80
(m, 2H), 5.08 (dd, 1H, J = 5.4, 1.4), 7.24 (d, 1H, J = 2.5), 7.46 (s, 1H),
7.80 (s, 1H), 8.23 (broad s, 1H). 13C NMR (CDCl3): δ 25.4, 27.4, 62.7, 80.7,
81.5, 84.3, 85.0, 112.8, 114.2, 115.2, 120.8, 123.7, 124.1, 125.4, 126.5, 135.4.
HRMSfor C 16H17Cl2NO4: Calcd, 357.0535; Found, 357.0519.
1-Acetyl-3-(5-O-acetyl-2,3-O-isopropylidene-β-D-ribofuranosyl)-2-ben-
zylthio-5,6-dichloroindole (10). To a solution of compound 7 (1.00 g, 2.08
mmol) in 12 mL of CH3CN, Ac2O (0.82 mL, 8.7 mmol) and Et3N (1.21 mL,
8.7 mmol) were added at room temperature. The mixture was stirred for
24 h, and then filtered to give 0.92 g (79%) of 10 as a white solid. The filtrate
was concentrated to dryness. The residue was purified by flash column
chromatography (30% EtOAc/Hex, 1 cm × 10 cm) to give 102 mg (8.7%,
total 88%) of additional 10: mp. 178–179◦C. Rf 0.70 (30% EtOAc/Hex).
1H NMR (CDCl3): δ 1.37 (s, 3H), 1.68 (s, 3H), 2.29 (s, 3H), 2.66 (s, 3H),
4.01 (m, 2H), 4.28 (m, 1H), 4.40–4.52 (m, 2H), 4.81–4.91 (m, 2H), 5.39
(d, 1H, J = 6.1), 7.02 (m, 2H), 7.22 (m, 3H), 7.83 (s, 1H), 8.42 (s, 1H).
13C NMR (CDCl3): δ 21.1, 25.6, 27.5, 28.3, 44.9, 63.5, 80.7, 81.2, 81.7, 84.4,
116.0, 118.2, 120.9, 125.8, 126.9, 127.3, 127.9, 128.5, 128.8, 130.3, 130.5,
136.4, 136.5, 170.8, 170.9. Anal calcd for C27H27Cl2NO6S: C, 57.45; H, 4.82;
N, 2.48. Found: C, 57.58; H, 5.00; N, 2.50.
1-Acetyl-3-(5-O -acetyl-β-D-ribofuranosyl)-2-benzylthio-5,6-dichloroin-
dole (11). Compound 10 (240 mg, 0.43 mmol) was dissolved in 7 mL
of TFA-water (9:1) at room temperature. The mixture was stirred for 10
min, and then concentrated to dryness. The residue was recrystallized from
EtOAc/Hex to give 114 mg (51%) of 11: mp. 135–136◦C. Rf 0.20 (30%
1
EtOAc/Hex). H NMR (CDCl3): δ 2.25 (s, 3H), 2.50 (broad s, 1H), 2.60
(broad s, 1H), 2.72 (s, 3H), 4.00 (s, 2H), 4.04 (m, 1H), 4.20–4.28 (m, 2H),
4.37–4.55 (m, 2H), 5.18 (d, 1H, J = 7.2), 7.01 (m, 2H), 7.23 (m, 3H), 7.85
(s, 1H), 8.41 (s, 1H). 13C NMR (CDCl3): δ 21.1, 28.1, 44.3, 63.6, 70.9, 74.5,
80.0, 82.6, 118.0, 121.0, 126.0, 127.1, 127.3, 127.9, 128.6, 128.9, 130.1, 130.5,
136.3, 136.5, 170.8, 171.1. Anal calcd for C24H23Cl2NO6S: C, 54.97; H, 4.42;
N, 2.67. Found: C, 55.09; H, 4.56; N, 2.65.
2-Benzylthio-5,6-dichloro-3-(β-D-ribofuranosyl)indole (12). Compound
11 (2.80 g, 5.34 mmol) and Na2CO3 (6.0 g, 57 mmol) in 80 mL of MeOH
were stirred at room temperature for 2 h, and then treated with 200 mL
of water. The resulting mixture was concentrated first to remove most of
the organic solvents, and then extracted with EtOAc (3 × 100 mL). The
combined EtOAc extracts were washed with 200 mL of brine and dried over
MgSO4. The organic layer was concentrated to dryness. The residue was

Antiviral Evaluation of Some C-Nucleosides
1429
purified by flash column chromatography (60% EtOAc/Hex, 4 cm × 10 cm)
to give 2.4 g of the crude product, which was crystallized from EtOAc/Hex
to give 1.85 g (79%) of 12 as a white solid. mp 164–165◦C. Rf 0.40 (60%
EtOAc/Hex). 1H NMR (DMSO-d6): δ 3.63 (m, 2H), 3.78 (m, 1H), 4.03 (m,
2H), 4.19 (m, 2H), 4.83 (d, 1H, J = 6.3), 4.89 (m, 2H), 4.96 (t, 1H, J =
5.0), 7.23 (m, 5H), 7.46 (s, 1H), 8.09 (s, 1H), 11.7 (broad s, 1H). 13C NMR
(DMSO-d6): δ 40.7, 61.8, 71.3, 74.7, 77.6, 85.6, 112.2, 117.3, 121.6, 124.3,
125.6, 127.2, 128.4, 128.7, 130.6, 135.7, 137.6. HRMSfor C 20H19Cl2NO4S:
Calcd, 439.0412; Found, 439.0399. Anal calcd for C20H19Cl2NO4S: C, 54.55;
H, 4.35; N, 3.18. Found: C, 54.57; H, 4.51; N, 3.27.
5,6-Dichloro-3-(β-D-ribofuranosyl)indole (13). A mixture of compound
12 (5.0 g, 11 mmol) and Raney Ni (30 g, wet, W-4) in 100 mL of EtOH
was stirred at room temperature for 0.5 h. The Raney Ni was removed by
filtration. The filtrate was concentrated to dryness to give compound 13 as
a white solid. Compound 13 was used in the next reaction without further
1
purification: Rf 0.15 (EtOAc). H NMR (DMSO-d6): δ 3.56 (m, 2H), 3.79
(m, 1H), 3.94 (m, 2H), 4.77 (d, 1H, J = 6.1), 4.85 (m, 2H), 4.93 (d, 1H, J =
5.7), 7.43 (s, 1H), 7.60 (s, 1H), 7.94 (s, 1H), 11.2 (broad s, 1H). 13C NMR
(DMSO-d6): δ 62.0, 71.2, 75.4, 78.0, 84.9, 112.9, 114.5, 120.9, 121.0, 123.3,
125.8, 126.1, 135.5.
5,6-Dichloro-3-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]in-
dole (14). To a solution of compound 13 (used directly from the previous
reaction without purification) in 60 mL of DMF, imidazole (5.0 g, 73
mmol) and TBDMSCl (6.5 g, 43 mmol) were added at room temperature.
The resulting mixture was stirred at room temperature for 26 h, and
then concentrated to dryness. The residue was treated with 100 mL of
EtOAc/Hex (1:1). The solid was removed by filtration and the filtrate
was concentrated to dryness. The residue was purified by flash column
chromatography (10% EtOAc/Hex, 4 cm × 10 cm) to give 5.32 g (73% over
2 steps) of 14 as a white solid: mp. 103–107◦C. Rf 0.60 (15% EtOAc/Hex).
1H NMR (CDCl3): δ −0.45 (s, 3H), −0.17 (s, 3H), 0.10 (s, 3H), 0.11 (s, 3H),
0.16 (s, 6H), 0.74 (s, 9H), 0.95 (s, 9H), 0.96 (s, 9H), 3.86 (m, 2H), 4.04 (m,
1H), 4.10 (m, 1H), 4.20 (m, 1H), 4.97 (d, 1H, J = 8.4), 7.22 (d, 1H, J = 2.4),
7.43 (s, 1H), 7.94 (s, 1H), 8.10 (broad s, 1H). 13C NMR (CDCl3): δ −5.4,
−5.3, −4.5, −4.4, −4.3, 18.0, 18.1, 18.6, 25.8, 25.9, 26.2, 63.9, 73.7, 77.2, 86.3,
112.5, 115.0, 121.8, 123.7, 125.0, 126.0, 135.4. HRMSfor C 31H55Cl2NO4Si3
[M + H]: Calcd, 660.2894; Found, 660.2886.
3-[2,3,5-Tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]-2,5,6-trichloro-
indole (15). A mixture of compound 14 (1.80 g, 2.72 mmol) and NCS
(378 mg, 2.83 mmol) in 20 mL of CCl4 was stirred at room temperature

1430
J. J. Chen et al.
for 24 h, and then concentrated to dryness. The residue was purified by
flash column chromatography (5% EtOAc/Hex, 3 cm × 10 cm), followed
by crystallization from MeCN to give 0.97 g (51%) of 15 as a white solid. mp.
158–164◦C. Rf 0.30 (50% EtOAc/Hex). 1H NMR (CDCl3): δ −0.61 (s, 3H),
−0.19 (s, 3H), 0.11 (s, 3H), 0.16 (s, 3H), 0.19 (s, 6H), 0.74 (s, 9H), 0.96 (s,
9H), 0.99 (s, 9H), 3.89 (m, 2H), 4.05 (m, 1H), 4.22 (m, 2H), 5.05 (d, 1H,
J = 8.4), 7.29 (s, 1H), 7.94 (s, 1H), 8.20 (broad s, 1H). 13C NMR (CDCl3):
δ −5.5, −5.3, −5.2, −4.7, −4.5, −4.3, 17.8, 18.1, 18.7, 25.8, 25.9, 26.3, 63.9,
73.5, 75.4, 75.8, 87.1, 109.5, 111.9, 121.3, 124.6, 125.1, 126.0, 126.4, 133.2.
HRMSfor C 31H54Cl3NO4Si3 [M + NH4]: Calcd, 711.2770; Found, 711.2753.
3-(β-D-Ribofuranosyl)-2,5,6-trichloroindole (16). Method 1: Compound
15 (600 mg, 0.86 mmol) was dissolved in 15 mL of a THF solution of TBAF
(1.0 M). The mixture was stirred at room temperature for 0.5 h. Water (80
mL) was added, and the mixture was extracted with EtOAc (3 × 50 mL).
The combined extracts were washed with brine (70 mL), dried over Na2SO4
and concentrated to dryness. The residue was purified by flash column
chromatography (90% EtOAc/Hex, 2 cm × 10 cm) to give 262 mg (86%)
of 16 as a white solid.
Method 2: Compound 18a (38 mg) was dissolved in 33% methylamine
in EtOH (1 mL) at room temperature. The mixture was stirred at room
temperature for 30 min, and then concentrated to dryness. The residue was
subjected to flash column chromatography (from 5% MeOH/CH2Cl2 to
8% MeOH/CH2Cl2, 1 cm × 10 cm) to give 18 mg, (100%) of 16 as a white
solid, which has the same 1H NMR spectrum as that of the solid prepared in
1
method 1: mp dec >179◦C. Rf 0.35 (EtOAc). H NMR (DMSO-d6): δ 3.61
(m, 2H), 3.79 (m, 1H), 4.03 (m, 2H), 4.72 (d, 1H, J = 8.2), 4.88 (m, 2H,
D2O exchangeable), 4.98 (t, 1H, D2O exchangeable, J = 5.1), 7.51 (s, 1H),
8.07 (s, 1H), 12.2 (broad s, 1H, D2O exchangeable). 13C NMR (DMSO-d6):
δ 61.8, 71.2, 74.3, 76.4, 85.7, 109.3, 112.4, 121.1, 122.2, 124.1, 124.8, 125.4,
133.7. Anal calcd for C13H12Cl3NO4: C, 44.28; H, 3.43; N, 3.97. Found: C,
43.95; H, 3.28; N, 3.82.
1-Acetyl-3-[2,3,5-tri-O-(t-butyldimethylsilyl)-β-D-ribofuranosyl]-2,5,6-tri-
chloroindole (17). To a solution of compound 15 (440 mg, 0.63 mmol) in
15 mL of MeCN, Ac2O (0.8 mL) and Et3N (0.5 mL) were added at room
temperature. The mixture was stirred for 0.5 h, and then filtered to give 360
mg (77%) of 17 as a white solid. The filtrate was concentrated to dryness.
The residue was purified by flash column chromatography (5% EtOAc/Hex,
1 cm × 10 cm) to give an additional 64 mg (14%, total 91%) of 17. mp. 120–
121◦C. Rf 0.40 (50% EtOAc/Hex). 1H NMR (CDCl3): δ −0.56 (s, 3H), −0.15
(s, 3H), 0.11 (s, 3H), 0.12 (s, 3H), 0.19 (s, 3H), 0.20 (s, 3H), 0.76 (s, 9H),
0.96 (s, 9H), 0.98 (s, 9H), 2.79 (s, 3H), 3.91 (m, 2H), 4.06 (m, 1H), 4.20 (m,

Antiviral Evaluation of Some C-Nucleosides
1431
2H), 5.13 (m, 1H), 8.00 (s, 1H), 8.54 (s, 1H). 13C NMR (CDCl3): δ −5.5,
−5.3, −5.2, −4.6, −4.5, −4.2, 17.8, 18.1, 18.7, 25.7, 25.9, 26.3, 27.7, 63.8,
73.3, 75.3, 76.0, 87.7, 117.1, 118.0, 121.1, 123.7, 126.2, 127.9, 129.4, 134.5,
169.3. Anal calcd for C33H56Cl3NO5Si3: C, 44.28; H, 3.43; N, 3.97. Found:
C, 43.95; H, 3.28; N, 3.82.
1-Acetyl-3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (18a). Method 1: A
mixture of compound 17 (300 mg, 0.41 mmol) and KF (700 mg, 12 mmol)
in 7 mL of 80% aqueous TFA was stirred at room temperature for 1 h.
The resulting mixture was concentrated to dryness, and then treated with
50 mL of brine and extracted with EtOAc (2 × 50 mL). The combined
extracts were dried over Na2SO4 and concentrated to dryness. The residue
was purified by flash column chromatography (95% EtOAc/Hex, 2 cm ×
10 cm) to give 108 mg (67%) of 18a as a white solid. Method 2: Compound
21a (324 mg, 0.456 mmol) was suspended in 12 mL of AcOH, and 5%
Pd/BaSO4 (300 mg) was added. The mixture was stirred under 1 atm of H2
for 45 min. The black suspension was filtered through Celite. The filtrate
was concentrated to dryness and the residue was subjected to flash column
chromatography (from 5% MeOH/CH2Cl2 to 8% MeOH/CH2Cl2, 2 cm ×
10 cm) to give 180 mg (90%) of compound 18a as a white solid: mp. 165–
166◦C (EtOAc/Hex). Rf 0.40 (10% MeOH/CH2Cl2). 1H NMR (DMSO-d6):
δ 2.80 (s, 3H), 3.66 (m, 2H), 3.87 (m, 1H), 4.08 (m, 2H), 4.85 (d, 1H, J =
7.7), 5.00 (m, 2H, D2O exchangeable), 5.11 (t, 1H, D2O exchangeable, J =
4.3), 8.37 (s, 1H), 8.43 (s, 1H). 13C NMR (DMSO-d6): δ 27.5, 61.5, 71.1, 74.7,
76.5, 86.3, 117.2, 117.3, 121.8, 123.8, 126.10, 126.14, 127.3, 134.1, 169.9.
HRMSfor C 15H14Cl3NO5: Calcd, 392.9938; Found, 392.9919. Anal calcd
for C15H14Cl3NO5: C, 53.75; H, 7.65; N, 1.90. Found: C, 53.51; H, 7.60;
N, 1.89.
1-Propionyl-3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (18b). The pro-
cedure is the same as that described for 18a (Method 2), except that 21b
(380 mg, 0.56 mmol) was used instead of 21a. A 70% yield (160 mg, 0.39
mmol) of 18b was obtained as a white foam: mp 182–184◦C. Rf 0.28 (10%
MeOH/CHCl3). 1H NMR (DMSO-d6): δ 1.20 (t, 3H), 3.20 (m, 2H) 3.67 (m,
2H), 3.87 (d, 1H), 4.06–4.10 (m, 2H), 4.85 (d, 1H), 4.99–5.01 (m, 2H, D2O
exchangeable), 5.11 (t, 1H, D2O exchangeable), 8.36 (s, 1H), 8.44 (s, 1H).
13C NMR (DMSO-d6): δ 8.9, 32.2, 61.5, 71.1, 74.7, 76.5, 86.3, 117.1, 117.2,
121.8, 123.6, 126.0, 126.1, 127.2, 134.1, 173.7. HRMSfor: C 16H16Cl3NO5 [M
+ Na]: Calcd, 429.9992; Found, 430.0006. Anal calcd for C16H16Cl3NO5: C,
47.02; H, 3.95; N 3.43. Found: C, 47.17; H, 4.15; N, 3.25.
1-Acetyl-2,5,6-trichloroindole (20a). To a solution of compound 194
(4.40 g, 20.0 mmol) in CH2Cl2 (50 mL) and dry pyridine (20 mL) was

1432
J. J. Chen et al.
added Ac2O (10.0 g, 100 mmol) at room temperature. The mixture was
stirred at room temperature for 24 h, and then concentrated to dryness.
The residue was dissolved in EtOAc (100 mL), washed with 1.0 M HCl (50
mL), 10% NaHCO3 (50 mL), brine (20 mL), dried over MgSO4, filtered,
and the filtrate evaporated to dryness. This furnished an orange solid which
was recrystallized from EtOAc/Hex to give 2.77 g (53%) of 20a as a pale-pink
1
solid: mp. 135–137◦C. Rf 0.30 (10% EtOAc/Hex). H NMR (DMSO-d6): δ
2.76 (s, 3H), 6.89 (s, 1H), 7.80 (s, 1H), 8.36 (s, 1H). 13C NMR (DMSO-
d6): δ 27.2, 108.4, 117.4, 120.9, 125.4, 126.2, 127.0, 127.7, 134.3, 169.8. Anal
calcd for C10H6Cl3NO: C, 45.75; H, 2.30; N, 5.34. Found: C, 45.43; H, 2.63;
N, 5.39.
1-Propionyl-2,5,6-trichloroindole (20b). To a solution of compound 19
(1.00 g, 4.5 mmol) in dry pyridine (20 mL) was added propionyl chloride
(2.0 mL, 2.1 g, 23 mmol) at room temperature. The mixture was stirred at
room temperature for 24 h, and then concentrated to dryness. The residue
was dissolved in EtOAc (75 mL), washed with 1.0 M HCl (50 mL), 10%
NaHCO3 (50 mL), brine (20 mL), then dried over MgSO4, filtered, and the
filtrate was evaporated to yield an orange solid. The solid was subjected to
flash column chromatography (3:1 hex:EtOAc, 5 cm × 45 cm) to give 0.64
g (51%) of compound 20b as a white solid: mp. 124–125◦C. Rf 0.65 (25%
EtOAc/Hex). 1H NMR (CDCl3): δ 1.32 (t, 3H), 3.13 (q, 2H), 6.52 (s, 1H),
7.48 (s, 1H), 8.50 (s, 1H). 13C NMR (CDCl3): δ 9.2, 32.7, 108.8, 118.5, 120.5,
124.8, 127.5, 128.1, 129.2, 135.1, 173.6.
1-Acetyl-3-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)-2,5,6-trichloroindole
(21a). To a solution of compound 20a (802 mg, 3.06 mmol) and 2,3,5-
tri-O-benzyl-α,β-D-ribofuranosyl fluoride[23] (800 mg, 1.89 mmol) in 15 mL
of CH2Cl2, boron trifluoride etherate (0.23 mL, 1.89 mmol) was added
dropwise at 0◦C. The resulting mixture was stirred at that temperature for
10 minutes. The reaction was quenched with sat. NaHCO3 (10 mL). The
mixture was extracted with CH2Cl2 (3 × 5 mL). The organic layers were
washed with brine (10 mL), dried over MgSO4 and concentrated to dryness.
The resulting yellow oil was subjected to flash column chromatography
(from 10% EtOAc/Hex to 20% EtOAc/Hex) to give 0.94 g of a crude
product. Recrystallization from EtOAc/Hex provided 590 mg (45%) of
compound 21a as a white solid: mp. 115–116◦C (EtOAc/Hex). Rf 0.70 (30%
1
EtOAc/Hex). H NMR (DMSO-d6): δ 2.78 (s, 3H), 3.59 (m, 1H), 3.71 (m,
1H), 4.19 (m, 2H), 4.23 (m, 1H), 4.33 (d, 1H, J = 12.1), 4.49 (d, 1H, J =
12.1), 4.57 (d, 1H, J = 12.3), 4.64 (q, 2H, J = 9.6), 4.72 (d, 1H, J = 12.3),
5.04 (d, 1H, J = 8.3), 7.07 (m, 5H), 7.32 (m, 10H), 8.02 (s, 1H), 8.37 (s, 1H).
13C NMR (DMSO-d6): δ 27.6, 69.9, 71.3, 71.4, 72.4, 75.1, 76.8, 80.6, 82.9,
115.9, 117.2, 121.2, 124.2, 125.6, 126.0, 127.3, 127.5, 127.6, 127.7, 127.9,

Antiviral Evaluation of Some C-Nucleosides
1433
128.2, 128.3, 134.0, 137.7, 138.0, 138.3, 169.8. Anal calcd for C36H32Cl3NO5:
C, 65.02; H, 4.85; N, 2.11. Found: C, 65.08; H, 5.05; N, 2.10.
1-Propionyl-3-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)-2,5,6-trichloroin-
dole (21b). The procedure is the same as that described for 21a (Method
2), except that 20b (540 mg, 2.0 mmol) was used instead of 20a. A 31%
yield (400 mg, 0.75 mmol) of 21b was obtained as a white crystalline solid:
mp. 94–95◦C. Rf 0.45 (25% EtOAc/Hex). 1H NMR (CDCl3): δ 1.32 (t, 3H),
3.13 (m, 2H), 3.59 (dd, 1H), 3.68 (dd, 1H), 4.11 (dd, 1H), 4.19 (dd, 1H),
4.24 (d, 1H), 4.33 (d, 1H), 4.51 (d, 2H), 4.60 (d, 1H), 4.73 (d, 1H), 4.81
(d, 1H), 5.20 (d, 1H), 7.03–7.13 (m, 5H), 7.28–7.37 (m, 10H), 7.99 (s, 1H),
8.50 (s, 1H). 13C NMR (CDCl3): δ 9.3, 33.0, 34.9, 70.0, 72.6, 73.0, 73.6, 76.1,
81.1, 83.6, 116.9, 118.1, 121.4, 123.5, 125.9, 127.8, 127.9, 128.0, 128.1, 128.2,
128.4, 128.7, 129.4, 134.9, 137.6, 138.0, 138.1, 173.6.
3-(2,3,5-Tri-O-benzyl-β-D-ribofuranosyl)-2,5,6-trichloroindole (22). The
procedure is the same as that described for 16 (Method 2), except that 21a
(2.46 g, 3.7 mmol) was used instead of 18a. A 99% yield (2.27 g, 3.6 mmol) of
22 was obtained as a clear foam: mp. 94–95◦C. Rf 0.44 (1% MeOH/CHCl3).
1H NMR (CDCl3): δ 3.53 (dd, 1H), 3.68 (dd, 1H), 4.11 (dd, 1H), 4.23–4.27
(m, 2H), 4.41 (s, 2H), 4.51 (d, 1H), 4.59 (d, 1H), 4.71 (d, 1H), 4.76 (d,
1H), 5.20 (d, 1H), 7.06–7.14 (m, 5H), 7.25–7.33 (m, 11H), 7.86 (s, 1H),
9.45 (s, 1H). 13C NMR (CDCl3): δ 70.1, 72.5, 73.6, 76.4, 77.4, 82.7, 109.1,
112.4, 121.2, 124.4, 125.2, 125.6, 126.2, 127.8, 127.9, 128.0, 128.3, 128.4,
128.6, 133.8, 137.7, 138.0, 138.1.
1-Methyl-3-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)-2,5,6-trichloroindole
(23a). A solution of compound 22 (0.47 g, 0.75 mmol) in dry THF (10 mL)
was cooled to 0◦C in an ice bath and sodium hydride (60% in mineral oil,
100 mg, 2.5 mmol) was added in one portion. The suspension was stirred
at 0◦C for 15 min, then methyl iodide (1.0 mL, 2.3 g, 16 mmol) was added
and the resulting suspension was allowed to warm to room temperature over
20 min. The solvent was removed under vacuum, and the residue dissolved
in EtOAc (20 mL). The organic suspension was washed with 5% aqueous
sodium thiosulfate (25 mL), brine (20 mL), dried over MgSO4, filtered,
and the filtrate was evaporated to yield a yellow residue. The residue was
subjected to flash column chromatography (2:1 hex:EtOAc, 5 cm × 45
cm) to give 0.45 g (94%) of compound 23a as a clear oil: Rf 0.60 (33%
1
EtOAc/Hex). H NMR (CDCl3): δ 3.59 (dd, 1H), 3.66 (s, 3H), 3.74 (dd,
1H), 4.18 (dd, 1H), 4.29–4.33 (m, 2H), 4.45 (d, 1H), 4.50 (d, 1H), 4.58 (d,
1H), 4.67 (d, 1H), 4.78 (d, 1H), 4.83 (d, 1H), 5.30 (d, 1H), 7.12–7.21 (m,
5H), 7.35–7.41 (m, 11H), 8.00 (s, 1H). 13C NMR (CDCl3): δ 30.2, 70.1, 72.3,
72.6, 73.5, 76.6, 77.3, 82.7, 108.7, 110.8, 121.4, 124.3, 124.7, 126.1, 127.5,
127.6, 127.8, 127.9, 128.1, 128.3, 128.5, 128.6, 135.0, 137.8, 138.1 138.2.

1434
J. J. Chen et al.
1-(1-Propyl)-3-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)-2,5,6-trichloroin-
dole (23b). The procedure is the same as that described for 23a, except that
22 (0.49 g, 0.79 mmol) was treated with 1-iodopropane instead of methyl
iodide. A 100% yield (0.52 g, 0.79 mmol) of 23b was obtained as a clear
1
oil: Rf 0.71 (33% EtOAc/Hex). H NMR (CDCl3): δ 0.90 (t, 3H), 1.75 (q,
2H), 3.53 (dd, 1H), 3.67 (dd, 1H), 4.04 (dt, 2H), 4.11 (q, 2H), 4.21–4.25
(m, 2H), 4.40 (dd, 2H), 4.51 (d, 1H), 4.60 (d, 1H), 4.72 (d, 1H), 4.76 (d,
1H), 5.24 (d, 1H), 7.04–71.4 (m, 5H), 7.26–7.37 (m, 11H), 7.93 (s, 1H). 13
C
NMR (CDCl3): δ 11.4, 23.1, 45.5, 70.1, 72.4, 72.5, 73.5, 76.7, 77.3, 80.9, 82.7,
108.7, 111.0, 124.3, 124.8, 126.0, 127.2, 127.6, 127.8, 127.9, 128.2, 128.3,
128.5, 128.6, 134.5, 137.8, 138.1, 138.2.
1-Methyl-3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (24a). The proce-
dure is the same as that described for 18a (Method 2), except that 23a
(442 mg, 0.69 mmol) was used instead of 21a. A 70% yield (175 mg, 0.48
mmol) of 24a was obtained as a white powder: mp dec >112◦C. Rf 0.37
1
(10% MeOH/CHCl3). H NMR (DMSO-d6): δ 3.49–3.55 (m, 2H), 3.72 (s,
3H), 3.77–3.82 (m, 2H), 3.98 (m, 1H), 4.51 (b, 1H, D2O exchangeable),
4.55 (d, 1H), 4.71 (b, 1H, D2O exchangeable), 4.90 (b, 1H, D2O exchange-
able), 7.79 (s, 1H), 7.88 (s, 1H). 13C NMR (DMSO-d6): δ 30.3, 65.7, 69.6,
70.6, 71.1, 109.8, 112.0, 120.6, 122.2, 124.1, 124.6, 127.3, 134.6. HRMSfor
C14H14Cl3NO4 [M + Na]: Calcd, 387.9886; Found, 387.9877. Anal calcd for
C14H14Cl3NO4·1/4 EtOAc: C, 46.36; H, 4.15; N, 3.60. Found: C, 46.58; H,
4.20; N, 3.65.
1-(1-Propyl)-3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (24b). The pro-
cedure is the same as that described for 18a (Method 2), except that 23b
(442 mg, 0.69 mmol) was used instead of 21a. A 61% yield (167 mg, 0.42
mmol) of 24b was obtained as a white powder: mp dec >135◦C. Rf 0.34 (10%
1
MeOH/CHCl3). H NMR (DMSO-d6): δ 0.86 (t, 3H), 1.68 (m, 2H), 3.50–
3.55 (m, 2H), 3.78–3.18 (m, 2H), 3.97 (s,1H), 4.17 (m, 2H), 4.50 (b, 1H,
D2O exchangeable), 4.55 (d, 1H), 4.69 (b, 1H, D2O exchangeable), 4.78 (b,
1H, D2O exchangeable), 7.79 (s, 1H), 7.93 (s, 1H). 13C NMR (DMSO-d6): δ
10.9, 22.7, 44.8, 65.7, 67.1, 69.5, 70.6, 71.1, 109.9, 112.0, 120.7, 122.2, 124.2,
124.7, 126.8, 134.1. HRMSfor C 16H18Cl3NO4 [M + Na]: Calcd, 416.0199;
Found, 416.0200. Anal calcd for C16H18Cl3NO4·1/4 EtOAc: C, 49.00; H,
4.84; N, 3.36. Found: C, 49.21; H, 4.90; N, 3.31.
Biological Evaluation
Cell Culture Procedures. The routine growth and passage of KB, BSC-1,
and HFF cells was performed in monolayer cultures using minimal essential
medium (MEM) with either Hanks salts [MEM(H)] or Earle salts [MEM(E)]
supplemented with 10% calf serum or 10% fetal bovine serum (HFF cells).

Antiviral Evaluation of Some C-Nucleosides
1435
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
HEPESbuffered salt solution. [25]
Virological Procedures. The Towne strain, plaque-purified isolate Po,
of HCMV was kindly provided by Dr. Mark Stinski, University of Iowa. The
KOSstrain 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 (m.o.i.) of <0.01 plaque-
forming units (p.f.u.) per cell as detailed previously.[26] High titer HSV-1
stocks were prepared by infecting KB cells at an m.o.i. of <0.1 also as detailed
previously.[26] 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.[27] Briefly, HFF or BSC-1 cells were planted as described
above in 96-well cluster dishes and incubated overnight at 37◦C. The next
day cultures were inoculated with HCMV or HSV-1 and serially diluted
1:3 across the remaining eleven columns of the 96-well plate. After virus
adsorption the inoculum was replaced with fresh medium and cultures were
incubated for seven days for HCMV, two or three days for HSV-1. Plaques
were enumerated under 20-fold magnification in wells having the dilution
which gave 5 to 20 plaques per well. Virus titers were calculated according
to the following formula: Titer (p.f.u./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, stained with crystal violet and microscopic plaques enumerated as
described above. Drug effects were calculated as a percentage of reduction
in number of plaques in the presence of each drug concentration compared
to the number observed in the absence of drug.
HSV-1 ELISA. An ELISA was employed[28] to detect HSV-1. Ninety-six-
well cluster dishes were planted with 10,000 BSC-1 cells per well in 200
µL per well of MEM(E) plus 10% calf serum. After overnight incubation
at 37◦C, selected drug concentrations in quadruplicate and HSV-1 at a
concentration of 100 pfu/well were added. Following a 3-day incubation at
37◦C, medium was removed, plates were blocked, rinsed, and horseradish
peroxidase conjugated rabbit anti-HSV-1 antibody was added. Following

1436
J. J. Chen et al.
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 absorbance obtained with virus in the absence of drug.
Cytotoxicity Assays. Two different assays were used for routine cyto-
toxicity testing. (a) Cytotoxicity produced in stationary HFF cells was deter-
mined by microscopic inspection of cells not affected by the virus used in
plaque assays.[26] (b) The effect of compounds during two population dou-
blings of KB cells was determined by crystal violet staining and spectrophoto-
metric quantitation of dye eluted from stained cells as described earlier.[29]
Briefly, 96-well cluster dishes were planted with KB cells at 3000–5000 cells
per well. After overnight incubation at 37◦C, test compound was added in
quadruplicate at six to eight concentrations. Plates were incubated at 37◦C
for 48 h in a CO2 incubator, rinsed, fixed with 95% ethanol, and stained
with 0.1% crystal violet. Acidified ethanol was added and plates read at 570
nm in a spectrophotometer designed to read 96-well ELISA assay plates.
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 in-
hibitory concentrations (IC50s) were calculated from the linear portions of
the regression lines. Samples containing positive controls (acyclovir for HSV-
1, GCV for HCMV, and 2-acetylpyridine thiosemicarbazone for cytotoxicity)
were used in all assays.
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