Monocyclic L-nucleosides with type 1 cytokine-inducing activity

Article abstract of DOI:10.1021/jm9905514

A series of 1,2,4-triazole L-nucleosides were synthesized and evaluated for their ability to stimulate type i cytokine production by activated human T cells in direct comparison to the known active agent ribavirin. Among the compounds prepared, 1-β-L-ribofuranosyl-1,2,4-triazole-3-carboxamide (5, ICN 17261) was found to be the most uniformly potent compound. Conversion of the 3-carboxamide group of 5 to a carboxamidine functionality resulted in 1-β-L- ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (10), which induced cytokine levels comparable to 5 for two of the three type 1 cytokines examined. Modification of the carbohydrate moiety of 5 provided compounds of reduced activity. Significantly, ICN 17261 offers interesting immunomodulatory potential for the treatment of diseases where type I cytokines play an important role.

Full text of DOI:10.1021/jm9905514

J . Med. Chem. 2000, 43, 1019-1028
1019
Mon ocyclic L-Nu cleosid es w ith Typ e 1 Cytok in e-In d u cin g Activity
Kanda S. Ramasamy,*,† Robert C. Tam,‡ J osie Bard,‡ and Devron R. Averett§,#
Department of Chemistry, Department of Immunology, and ICN Corporate Research, ICN Pharmaceuticals, Inc.,
3300 Hyland Avenue, Costa Mesa, California 92626
Received November 4, 1999
A series of 1,2,4-triazole L-nucleosides were synthesized and evaluated for their ability to
stimulate type 1 cytokine production by activated human T cells in direct comparison to the
known active agent ribavirin. Among the compounds prepared, 1-â-L-ribofuranosyl-1,2,4-
triazole-3-carboxamide (5, ICN 17261) was found to be the most uniformly potent compound.
Conversion of the 3-carboxamide group of 5 to a carboxamidine functionality resulted in 1-â-
L-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (10), which induced cytokine levels
comparable to 5 for two of the three type 1 cytokines examined. Modification of the carbohydrate
moiety of 5 provided compounds of reduced activity. Significantly, ICN 17261 offers interesting
immunomodulatory potential for the treatment of diseases where type 1 cytokines play an
important role.
Ch a r t 1. Structure of Ribavirin
In tr od u ction
Selective modulation of the immune system offers an
important opportunity to control many diseases. The
balance between immune protection from offending
agents versus the pathologic effects of immune dysfunc-
tion can be altered by changes in the levels of two major
types of cytokines secreted by subsets of T-helper cells.1,2
Type 1 cytokines such as interleukin 2 (IL-2), interferon
gamma (IFN-γ), and tumor necrosis factor alpha (TNF-
R) generally enhance cell-mediated immunity, whereas
the type 2 cytokines such as IL-4, IL-5, IL-6, IL-9, IL-
10, and IL-13 are primarily involved in enhancing
humoral immune responses.3 Appropriately balanced
type 1 and type 2 responses are essential in host
defense, but inappropriate levels of response occur. Such
inappropriate responses reduce host defense capability
and can promote a wide range of immunopathological
reactions.4-13 Thus, the ability to selectively modulate
the cytokine profile could be of therapeutic benefit in a
wide variety of disease states.
Ribavirin (1-â-D-ribofuranosyl-1,2,4-triazole-3-carbox-
amide; Chart 1) is a nucleoside analogue that has
demonstrated efficacy in treating viral diseases both as
monotherapy (respiratory syncytial virus)14 and in
combination therapy with interferon alpha (hepatitis C
virus).15 Recently reported studies indicate that the in
vivo utility of ribavirin can result not only from direct
inhibition of viral replication but also from its ability
to enhance T cell-mediated immunity.16-18 This immu-
nomodulatory effect of ribavirin is demonstrable in vitro
by measuring the levels of type 1 cytokines produced
by activated T cells from both humans and mice19 and
by other measures. The induction of a type 1 cytokine
bias by ribavirin is functionally significant in vivo in
murine systems.20
Our interest in both synthesizing novel L-nucleoside
analogues and ascertaining whether the interesting
immunomodulatory properties of ribavirin are limited
to nucleosides of D-configuration led us to examine the
L-nucleoside derivatives of ribavirin. A number of
investigators have reported that certain â-L-nucleosides
have antiviral activity and may differ from their â-D-
congeners in a variety of other biologic properties
including toxicity.21-23 However, the immunologic prop-
erties of â-L-nucleosides have not been previously
reported. Also, in the majority of the studies reported,
the investigators focused on compounds having biologic
nucleobase moieties with altered sugars. We report here
the synthesis of a series of L-nucleosides structurally
related to ribavirin, a D-nucleoside in which the hetero-
cycle is a triazole rather than a purine or pyrimidine.
Our studies show that the L-enantiomer of ribavirin, 5
(ICN 17261), has type 1 cytokine-enhancing activity that
is similar to that of ribavirin. In addition, other struc-
turally related L-nucleosides such as 23 and 10 also
show immunologic activity. Additional studies are un-
derway to further evaluate the therapeutic potential of
these compounds.
Ch em istr y
Our synthetic approach for the preparation of triazole
L-ribofuranosyl nucleosides is shown in Scheme 1. The
key intermediate sugar, viz. 1,2,3,5-tetra-O-acetyl-L-
ribose (1), was prepared from commercially available
L-ribose according to a similar procedure used for the
synthesis of 1,2,3,5-tetra-O-acetyl-D-ribose.24 Condensa-
* Corresponding author. Tel: (714)545-0100 × 2008. Fax: (714)-
668-3141. E-mail: kramasamy@icnpharm.com.
† Department of Chemistry.
‡ Department of Immunology.
§ ICN Corporate Research.
# Present address: Averett Consulting, 26 Trinity, Irvine, CA 92612.
10.1021/jm9905514 CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/16/2000

1020 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Ramasamy et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (i) Ac2O, Py, DMF; (ii) 20% phosgene/toluene, Py,
CH2Cl2; (iii) 1 N NaOMe, MeOH; (iv) MeOH/NH3, NH4Cl.
Sch em e 3a
a
Reagents: (i) MeOH, HCl; (ii) Ac2O, Py; (iii) AcOH, Ac2O,
H2SO4; (iv) methyl 1,2,4-triazole-3-carboxylate (2), bis(p-nitrophen-
yl)phosphate, 165-175 °C; (v) MeOH/NH3.
tion of 1 with methyl 1,2,4-triazole-3-carboxylate (2)25
in the presence of a catalytic amount of bis(p-nitrophen-
yl)phosphate26 at 165-70 °C for 25 min under vacuum
followed by aqueous workup provided the fully protected
nucleosides 3 and 4 in 78% and 11% yields, respectively.
Stirring a solution of 3 in methanolic ammonia in a steel
bomb at room temperature for 12 h gave 1-â-L-ribofura-
nosyl-1,2,4-triazole-3-carboxamide (5) in 90% yield.
Similarly, compound 4 was treated with methanolic
ammonia to provide 1-â-L-ribofuranosyl-1,2,4-triazole-
5-carboxamide (6). The structures of 3 and 4 are based
a
Reagents: (i) 1,3-dichloro-1,1,3,3-tetraisopropyl-1,3-O-disilox-
ane, TEA, Py, DMF; (ii) phenyl chlorothionoformate, TEA, CH2Cl2;
(iii) diphenylsilane, AIBN, dioxane; (iv) Et3N‚HF, CH2Cl2.
1
on the H NMR spectra of 5 and 6 that were obtained
nolic ammonia containing 1 equiv of ammonium chloride
for 16 h at room temperature gave the target 1-â-L-
ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochlo-
ride (10).
respectively from 3 and 4. The positions of the signals
for H5, H3, and H1′ are in close agreement with those
reported26 for the corresponding D-nucleosides. The
chemical shift of H1′ in 6 is δ 6.78, a downfield shift of
0.98 ppm, comparing with that of H1′ of 5 (δ 5.80). This
can be explained by a stronger inductive effect on the
H1′ induced by the presence of a closer C5-H amide
group. On the other hand, the electronegative anomeric
center has less inductive effect on the further C3-H in
6, which is reflected in the chemical shift of C3-H (δ
8.16), an upfield shift of 0.71 ppm in comparison with
that of C5-H in 5 (δ 8.87).
To investigate the qualitative structure-activity re-
lationships with respect to immunological properties, we
converted the 3-carboxamide functionality of 5 to an
amidine hydrochloride group (Scheme 2). Thus, treat-
ment of 5 with acetic anhydride in pyridine/dimethyl-
formamide mixture for 12 h at room temperature under
argon atmosphere afforded triacetyl derivative 7 as an
amorphous solid. Dehydration of the carboxamide group
in 7 was achieved by exposure of 7 to 20% phosgene in
toluene at 0 °C for a 2-h period under argon atmosphere
and afforded a clean product 8 in 73% yield. Reaction
of 8 with freshly prepared 1 N sodium methoxide
solution furnished the corresponding methyl imidate 9
in good yield. The imidate 9 on treatment with metha-
2′-Deoxy nucleosides are an important class of me-
dicinal agents, chiefly because of their pronounced
antiviral activity. We explored this type of structural
modification in our series as well. The 2′-deoxy deriva-
tive of 5 was prepared from 5 as shown in Scheme 3.
The 3′- and 5′-hydroxyl groups in 5 were selectively
protected with Markiewicz reagent (TIPSiCl2)27 to pro-
vide silyl derivative 11. The silyl derivative 11 on
further reaction with phenyl chlorothionoformate af-
forded 12 in 62% yield. Radical-mediated deoxygen-
ation28 of 12 with diphenylsilane in the presence of
AIBN in dioxane under reflux furnished 13. Deprotec-
tion of the silyl group of 13 with Et3N‚HF, followed by
purification of the crude product on a silica gel column,
yielded the 2′-deoxy nucleoside 14.
In an effort to study the immunomodulatory proper-
ties of other L-nucleosides, the synthesis of L-xylofura-
nosyl nucleosides was designed. For the synthesis of
L-xylofuranosyl nucleosides, we utilized the reported29
1,2-di-O-acetyl-3,5-di-O-benzoyl-L-xylofuranose (15) as
a chiral starting material, which was prepared from
L-xylose. Using the acid-catalyzed fusion procedure
(Scheme 4), condensation of 15 with 2 provided 16,

Type 1 Cytokine-Inducing Monocyclic L-Nucleosides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1021
Sch em e 4a
Sch em e 5a
a
a
Reagents: (i) Me2CO, H2SO4, CuSO4; (ii) NH4OH; (iii) HCl/
Reagents: (i) Me2CO, H2SO4; (ii) 0.2% HCl/H2O; (iii) C6H5COCl,
H2O; (iv) NaHCO3, H2O; (v) C6H5COCl, TEA, CHCl3; (vi) AcOH,
Ac2O, H2SO4; (vii) methyl 1,2,4-triazole-3-carboxylate, bis(p-ni-
trophenyl)phosphate, 165-175 °C; (viii) MeOH/NH3.
Py, CH2Cl2; (iv) PDC, Ac2O, CH2Cl2; (v) p-toluenesulfonylhydra-
zine, EtOH; (vi) NaCNBH3, HCl, MeOH, THF; (vii) NaOAc‚3H2O,
EtOH; (viii) AcOH, Ac2O, H2SO4; (ix) methyl 1,2,4-triazole-3-
carboxylate, bis(p-nitrophenyl)phosphate, 165-175 °C; (x) MeOH/
NH3.
which on treatment with methanolic ammonia afforded
1-â-L-xylofuranosyl-1,2,4-triazole-3-carboxamide (17) in
89% yield.
Sch em e 6a
In addition to the ribose, 2′-deoxy, and xylose nucleo-
sides described above, we also prepared 3′-deoxy ana-
logues. To synthesize 3′-deoxy L-nucleosides, we turned
our attention to a different sugar, viz. 1,2-di-O-acetyl-
5-O-benzoyl-L-ribose (21). Even though the intermediate
21 was reported in a communication,30 no detailed
procedure was given. We elected to synthesize 21 from
L-xylose. L-Xylose was transformed into a known inter-
mediate 18 by following the literature report.31 Com-
pound 18 was heated with p-toluenesulfonylhydrazine
to give the corresponding hydrazide intermediate, which
on reduction with sodium cyanoborohydride in the
presence of HCl afforded 19 as a crystalline product.
Treatment of 19 with sodium acetate in ethanol at
reflux temperature provided the protected 3-deoxy sugar
2030 in good yield.32 Stirring of 20 with AcOH and acetic
anhydride in the presence of concentrated H2SO4 at
room temperature gave the required sugar 21. Acid-
catalyzed26 fusion of 21 and 2 gave 22 in 77% yield
(Scheme 5), which on subsequent treatment with meth-
anolic ammonia yielded the target 3′-deoxy-L-nucleoside
23 as a colorless solid.
a
Reagents: (i) Me2CO, 2,2-dimethoxypropane, HCl; (ii) Ph3P,
Br2, CH2Cl2; (iii) Pd/C, KOH/H2O, MeOH; (iv) AcOH, Ac2O, H2SO4;
(v) methyl 1,2,4-triazole-3-carboxylate, HMDS, SnCl4, CH3CN; (vi)
MeOH/NH3.
The â-L-configuration of the synthesized nucleosides
1
was confirmed by comparison of the H NMR spectra
with those of the corresponding D-isomers.26,34 In addi-
tion, the optical rotation of the L-compounds was found
to have a specific rotation opposite to that of the
D-compounds.
Finally, the synthesis of 5′-deoxy L-nucleoside 29 was
pursued. The required intermediate sugar 27 was
prepared as depicted in Scheme 6. Reaction of L-ribose
with methanolic HCl solution in acetone in the presence
of 2,2′-dimethoxypropane gave oil 24. Treatment of 24
with bromine and Ph3P provided 5-bromo derivative 25.
Hydrogenation of 25 with 10% Pd/C in the presence of
potassium hydroxide afforded the protected 5-deoxy
sugar 26 in excellent yield. Compound 26 was trans-
formed to the key intermediate 27 by stirring 26 with
acetic anhydride and concentrated H2SO4 in glacial
AcOH for 12 h. Glycosylation of 2 with 27 was ac-
complished using Vorbru¨ggen conditions.33 Accordingly,
2 was persilylated with hexamethyldisilazane under
reflux to give the corresponding silylated derivative
which on treatment with 27 in the presence of SnCl4 at
0 °C gave 28. Exposure of 28 to methanolic ammonia
afforded 1-(5-deoxy-â-L-ribofuranosyl)-1,2,4-triazole-3-
carboxmide (29) in 72% yield.
Resu lts a n d Discu ssion
Recently we showed that ribavirin can enhance
antiviral type 1 cytokines and suppress type 2 cytokine
expression in human T cells.19 These results prompted
us to synthesize the L-isomer (5) of ribavirin and
evaluate its impact on cytokine production. This would
allow us to predict the influence of the sugar configu-
ration on cytokine profile as well as to design and
synthesize nucleosides with a specific cytokine profile.
Interestingly, compound 5 had the same cytokine pat-
tern as ribavirin (see Table 1 and Figure 1).
On the basis of the above result, compound 5 was
selected as a lead upon which further structure-activity
relationship studies were conducted. As shown in
Schemes 1-6, L-nucleoside analogues of ribavirin were
synthesized in which the carboxamide group as well as
the sugar portions were modified while retaining the

1022 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Ramasamy et al.
Ta ble 1. Effect of L-Nucleosides of Ribavirin on
SEB-Stimulated T Cell Expression of the Type 1 Cytokines
IL-2, IFN-γ, and TNF-R
indeed, compound 5 was the most potent of the L-
nucleosides tested with respect to type 1 cytokine
induction and was the only L-nucleoside that strongly
enhanced production of all three type 1 cytokines. The
furanose 2′-OH appears to be important for full activity,
since conversion of the 2′-OH of 5 to hydrogen, generat-
ing the 2′-deoxy compound 14, resulted in total loss of
activity. Also, manipulation of the 3′-OH (5 to 17, 23)
resulted in retention of IFN-γ activity, partial loss of
TNF-R activity, and total loss of IL-2-enhancing activity.
This result indicates that there is considerable flexibility
with respect to allowable modifications at the 3′-position
of the sugar portion of the molecule. Transformation of
the 5′-hydroxyl to a hydrogen (29) resulted in the loss
of IFN-γ- and IL-2-inducing activity with retention of
TNF-R-inducing activity. It is clear from the results of
this study that free hydroxyls in the 2′- and 5′-positions
are essential. Substitutions on the heterocycle also are
important. Switching from a 3-carboxamide to a 5-car-
boxamide (5 to 6) exerted a strongly negative influence
on bioactivity for all three type 1 cytokines, whereas
conversion of the amide group in compound 5 to a
amidine functionality (10) did not reduce activity. These
results show that the substitution position in the
triazole ring is critical for activity.
% of activated controlb
treatmenta
SEB
IL-2c
100
IFN-γc
TNF-Rc
100
100
SEB + ribavirin
143 ( 18
131 ( 12
108 ( 9
100 ( 14
115 ( 5
102 ( 12
99 ( 7
131 ( 6
122 ( 3
110 ( 10
119 ( 7
114 ( 15
102 ( 12
111 ( 10
128 ( 7
124 ( 4
144 ( 7
119 ( 11
132 ( 6
109 ( 11
125 ( 16
128 ( 7
120 ( 6
SEB + 5
SEB + 14
SEB + 17
SEB + 23
SEB + 29
SEB + 6
SEB + 10
133 ( 6
a
T cell-derived cytokine levels from five individual human
donors were determined in cell-free supernatants by ELISA.
b
Compound numbers are shown in bold. Data are shown col-
lectively as mean percentage of activated control ((standard
deviation) for all cytokines. Percentage of activated control is
calculated as the ratio of activated T cell cytokine level in the
presence of test nucleoside over the cytokine level of untreated
activated T cells × 100%. Zero effect on cytokine levels by test
nucleosides would give a percentage of activated control value of
100%. c The absolute level (pg/mL ( standard deviation) of SEB-
induced type 1 cytokine secretion was for IL-2, 640 ( 36; for IFN-
γ, 462 ( 37; and for TNF-R, 223 ( 27. Resting levels were <30
pg/mL for all cytokines.
glycosylation site and pentose sugar moiety. All the
compounds were evaluated for the type 1 cytokine-
enhancing activity in activated human T cells in com-
parison with ribavirin (used as positive control). The
influence of the L-nucleosides on the cytokine pattern
induced by the bacterial superantigen staphylococcal
enterotoxin B (SEB) was examined in human T cells
from five normal donors using protocols previously
described elsewhere.19 The secreted levels of the type 1
cytokines IL-2, TNF-R, and IFN-γ were determined in
the cell-free supernatants by ELISA. The effects of these
L-nucleosides on IL-2 levels are shown in Table 1. These
data showed that 5, 10, and ribavirin, at 5 µM, signifi-
cantly increased IL-2 above activated control levels with
a mean percentage increase of 31%, 33%, and 43%,
respectively. Table 1 also shows the effect on IFN-γ
levels. Here, 5, 10, 17, and ribavirin, at 5 µM, signifi-
cantly enhanced IFN-γ above activated control levels
with a mean percentage increase of 25%, 28%, 19%, and
31%, respectively. Finally TNF-R levels were increased
substantially above activated control levels by all L-
nucleosides except 23 (Table 1). However 5 and 17
appeared to be more potent enhancers of TNF-R,
augmenting activated control levels by 44% and 32%,
respectively (ribavirin 24%).
Previously we showed that the induction of activated
type 1 cytokine levels by ribavirin exhibits a bell-shaped
dose-versus-response curve, with a peak at 5 µM.19 We
evaluated the dose-response profiles of this series of
L-nucleosides from 1.25 to 10 µM with respect to type 1
cytokine induction (Figure 1). In general, the dose-
response profiles reveal three categories of type 1
cytokine-inducing activity: active, partially active, and
inactive L-nucleosides. The active and partially active
nucleosides all have a similar bell-shaped profile as
ribavirin.
The results presented here show that the type 1
cytokine-enhancing activity is retained in an analogue
in which the D-ribose moiety of ribavirin is replaced by
L-ribose (5). However, these two compounds differ
markedly with respect to other biologic properties.35
Additional structural changes can have a significant
effect on this biologic activity. To our knowledge, this
report is the first demonstration that selected L-nucleo-
sides possess immunomodulatory activity. The effect on
type 1 cytokines by these L-nucleosides offers significant
potential for the treatment of those diseases in which
type 1 cytokines play a critical role.
Exp er im en ta l Section
Melting points were measured on a Haake Bu¨chler capillary
melting point apparatus and are uncorrected. Nuclear mag-
netic resonance (1H and 13C NMR) spectra were recorded on
Varian mercury 300 MHz spectrometer. The chemical shifts
are expressed in δ values (ppm) relative to tetramethylsilane
as internal standard. Optical rotations were performed on a
J ASCO DIP-370 digital polarimeter. Elemental analyses were
performed by Quantitative Technologies Inc., Whitehouse, NJ .
Thin-layer chromatography (TLC) was performed on plates of
silica gel 60F254 coated on aluminum sheets (5 × 10 cm; EM
Science) using different solvents prepared freshly. ICN silica
gel 18-32 (60 Å) was used for flash column chromatography.
All solvents used were reagent grade. Most of the dry solvents
were purchased from Fluka and used as such without further
purification. Most of the reactions were conducted under argon
atmosphere. Evaporations were carried out under reduced
pressure with the bath temperature below 35 °C.
1,2,3,5-Tetr a -O-a cetyl-â-L-r ibofu r a n ose (1). To a stirred
solution of L-ribose (50.0 g, 333.33 mmol) in anhydrous
methanol (500 mL) at room temperature was added a freshly
prepared dry methanlic HCl (40 mL, prepared by bubbling dry
HCl gas into methanol at 0 °C to a weight increase of 4 g) via
syringe during 15-min period under argon atmosphere. After
the addition of methanolic HCl, the reaction mixture was
allowed to stir at room temperature for 3-4 h. Dry pyridine
(100 mL) was added and evaporated to dryness. The residue
was dissolved in dry pyridine (100 mL) and evaporated to
dryness under high vacuum below 40 °C. The process was
repeated second time with additional dry pyridine (100 mL).
The residue was dissolved in dry pyridine (250 mL) and cooled
Collectively these data demonstrate that several
structural modifications can affect this immunologic
activity. Introduction of L-ribose for the D-ribose of
ribavirin allowed retention of type 1 cytokine activity;

Type 1 Cytokine-Inducing Monocyclic L-Nucleosides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1023
F igu r e 1. Dose-dependent modulation of SEB-induced type 1 cytokine responses following treatment with L-nucleoside analogues
of ribavirin in human T cells. Comparison of the dose-dependent (1.25-10 µM) augmentation by L-nucleoside analogues of ribavirin
of IL-2 (left panel), IFN-γ (middle panel), and TNF-R (right panel) levels in SEB-stimulated T cells from five individual human
donors. Cytokine levels were determined in cell-free supernatants by ELISA. Human donors, A-E, were determined in cell-free
supernatants by ELISA. Data are shown as percentage of activated control for all cytokines. Percentage of activated control is
calculated as the ratio of activated T cell cytokine level in the presence of test nucleoside over the cytokine level of untreated
activated T cells × 100%. Zero effect on cytokine levels by test nucleosides would give a percentage of activated control value of
100%. The absolute level (pg/mL ( standard deviation) of SEB-induced type 1 cytokine secretion was as shown in Table 1.
in an ice bath to 0 °C under argon atmosphere. To this cold
stirred solution was added acetic anhydride (100 mL) via a
dropping funnel during a 15-min period. After the addition of
acetic anhydride, the reaction was allowed to stir at room
temperature under exclusion of moisture for 24 h. The reaction
mixture was evaporated to dryness. The residue was parti-
tioned between ethyl acetate (400 mL) and water (400 mL)
and extracted in ethyl acetate. The aqueous layer was ex-
tracted again with ethyl acetate (100 mL). The combined ethyl
acetate extract was washed with water (400 mL), saturated
NaHCO3 (2 × 300 mL), water (300 mL) and brine (200 mL).
The organic extract was dried over anhydrous sodium sulfate
and filtered and the filtrate evaporated to dryness. The residue
was coevaporated with dry toluene (2 × 150 mL) at high
vacuum. The dried oily residue (92 g, 95%) was used as such
for the following reaction without further characterization.
The syrup (92 g) from the above reaction was dissolved in
glacial acetic acid (300 mL) and treated with acetic anhydride
(75 mL) at room temperature. The solution was cooled to 0 to
5 °C in an ice bath under argon atmosphere. Concentrated H2-
SO4 (21 mL) was added slowly during 15-min period. After
the addition of H2SO4, the reaction mixture was stirred at room
temperature for 14 h and poured on crushed ice (500 g), and
stirred until ice melts. Water (500 mL) was added and
extracted with chloroform (2 × 300 mL). The chloroform
extract was washed with water (3 × 400 mL), saturated
NaHCO3 (2 × 300 mL), water (200 mL) and brine (200 mL).
The washed organic extract was dried over anhydrous sodium
sulfate, filtered and evaporated to dryness to give an oily
residue (99 g). The residue was coevaporated with dry toluene
(200 mL) and dissolved in ethyl ether (200 mL) which on
cooling at -10 °C for a day provided colorless crystals. The
crystalline solid was filtered, washed with hexanes:ether (2:
mmol), 1,2,3,5-tetra-O-acetyl-â-L-ribofuranose (63.66 g, 200
mmol) and bis(p-nitrophenyl)phosphate (1 g) was placed in a
RB flask (500 mL). The flask was placed in a preheated oil
bath at 165-175 °C under water aspirator vacuum with
stirring for 25 min. The acetic acid displaced was collected in
an ice-cold trap that is placed between aspirator and the RB
flask. The flask was removed from the oil bath and allowed to
cool. When the temperature of the flask reached roughly to
60-70 °C, ethyl acetate (300 mL) and saturated NaHCO3 (150
mL) were introduced and extracted in ethyl acetate. The
aqueous layer was extracted again with ethyl acetate (200 mL).
The combined ethyl acetate extract was washed with saturated
NaHCO3 (300 mL), water (200 mL) and brine (150 mL). The
organic extract was dried over anhydrous sodium sulfate and
filtered and the filtrate evaporated to dryness. The residue
was dissolved in ethanol (100 mL) and diluted with methanol
(60 mL) which on cooling at 0 °C for 12 h provided colorless
crystals. The solid was filtered, washed with minimum cold
ethanol (20 mL) and dried at high vacuum over solid sodium
hydroxide to give 60 g (78%). The filtrate was evaporated to
dryness and purified on silica column using CHCl3 f EtOAc
(9:1) as the eluent. Two products were isolated from the
filtrate: fast moving product 8.5 g (11%) and slow moving
product 5 g (6.5%). The slow moving product matched with
crystallized product. The fast moving product was found to be
4 and obtained as a foam. The combined yield of 3 was 65 g
(84%): mp 108-110 °C; 1H NMR (CDCl3) δ 2.11 (s, 3H,
COCH3), 2.12 (s, 3H, COCH3), 2.13 (s, 3H, OCH3), 3.99 (s, 3H,
COCH3), 4.22 (dd, 1H), 4.46 (m, 2H), 5.55 (t, 1H, J ) 6.0 Hz),
5.75 (m, 1H), 6.05 (d, 1H, C1′H J ) 3.6 Hz), 8.41 (s, 1H, C5H).
Anal. (C15H19N3O9) C, H, N. 4: 1H NMR (CDCl3) δ 2.02 (s, 3H,
COCH3), 2.10 (s, 3H, COCH3), 2.12 (s, 3H, OCH3), 4.00 (s, 3H,
COCH3), 4.14 (m, 1H), 4.42 (m, 2H), 5.76 (t, 1H), 5.81 (m, 1H),
6.94 (d, 1H, C1′H J ) 2.1 Hz), 8.03 (s, 1H, C3H). Anal.
(C15H19N3O9) C, H, N.
1, 50 mL) and dried to give 60.5 g (60%) of 1: mp 57-59 °C;
1
[R]20 +12.1 (c 2.47, CHCl3); H NMR (CDCl3) δ 2.07 (s, 3H,
D
COCH3), 2.09 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 2.13 (s,
3H, COCH3), 4.14 (m, 1H), 4.36 (m, 2H), 5.34 (m, 2H, C2′H &
C3′H), 6.16 (s, 1H, C1′H).
Met h yl 1-(2,3,5-Tr i-O-a cet yl-â-L-r ib ofu r a n osyl)-1,2,4-
t r ia zole-3-ca r b oxyla t e (3) a n d Met h yl 1-(2,3,5-Tr i-O-
a cetyl-â-L-r ibofu r a n osyl)-1,2,4-tr ia zole-5-ca r boxyla te (4).
A mixture of methyl 1,2,4-triazole-3-carboxylate (25.4 g, 200
1-â-L-R ibofu r a n osyl-1,2,4-t r ia zole-3-ca r boxa m id e (5).
Methyl 1-(2,3,5-tri-O-acetyl-â-L-ribofuranosyl)-1,2,4-triazole-
3-carboxylate (62 g, 161 mmol) was placed in a steel bomb and
treated with freshly prepared methanolic ammonia (350 mL,
prepared by passing dry ammonia gas into dry methanol at 0
°C until saturation) at 0 °C. The steel bomb was closed and
stirred at room temperature for 18 h. The steel bomb was

1024 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Ramasamy et al.
cooled to 0 °C and opened and the content evaporated to
dryness. The residue was treated with dry ethanol (100 mL)
and evaporated to dryness. The residue obtained was tritu-
rated with acetone to give a solid, which was filtered and
washed with acetone. The solid was dried overnight at room
temperature and dissolved in hot ethanol (600 mL) and water
(10 mL) mixture. The volume of the ethanol solution was
reduced to 150 mL by heating and stirring on a hot plate. The
hot ethanol solution on cooling provided colorless crystals,
which was filtered, washed with acetone and dried under
vacuum. Further concentration of the filtrate gave additional
material: total yield 35 g (89%); mp 177-179 °C; [R]20D +35.3
(c 10, H2O); 1H NMR (Me2SO-d6) δ 3.46 (m, 1H, C5′H), 3.60
(m, 1H, C5′H), 3.94 (m, 1H, C4′H), 4.12 (m, 1H), 4.34 (m, 1H),
4.95 (t, 1H, C5′OH), 5.22 (d, 1H), 5.60 (d, 1H), 5.80 (d, 1H, J )
3.9 Hz, C1′H), 7.64 (bs, 1H, NH2), 7.84 (bs, 1H, NH2), 8.87 (s,
1H, C5H); 13C NMR (Me2SO-d6) δ 61.8, 70.2, 74.4, 86.0, 91.6,
144.9, 157.4, 160.6. Anal. (C8H12N4O5) C, H, N.
Meth yl 1-(â-L-Ribofu r a n osyl)-1,2,4-tr ia zole-3-ca r box-
im id a te (9). To a stirred suspension of 8 (5.6 g, 15.91 mmol)
in dry methanol (60 mL) at room temperature was added 1 N
sodium methoxide (10 mL) and the mixture was stirred at
room temperature overnight. The clear solution was treated
with Dowex H+ resin (washed with dry methanol before use)
till the pH of the solution was 4. The resin was filtered and
the filtrate evaporated to dryness. The residue was purified
on silica column using CH2Cl2 f MeOH as the eluent. The
pure product was pooled and evaporated to dryness to afford
2.5 g (61%) of 9: 1H NMR (Me2SO-d6) δ 3.46 (m, 1H, C5′H),
3.62 (m, 1H, C5′H), 3.85 (s, 3H, OCH3), 3.94 (m, 1H), 4.12 (m,
1H), 4.34 (m, 1H), 4.98 (t, 1H, C5′OH), 5.24 (d, 1H), 5.62 (d,
1H), 5.84 (d, 1H, J ) 4.2 Hz, C1′H), 8.96 (s, 1H, C5H). Anal.
(C9H14N4O5) C, H, N.
1-(â-L-Ribofu r an osyl)-1,2,4-tr iazole-3-car boxam idin e Hy-
d r och lor id e (10). To a mixture of 9 (0.6 g, 2.33 mmol) and
ammonium chloride (0.13 g, 2.35 mmol) in a steel bomb was
added a solution of methanol saturated at 0 °C with dry
ammonia gas. The bomb was closed and the reaction mixture
was allowed to stir at room temperature overnight. The steel
bomb was cooled to 0 °C and opened carefully and the content
was evaporated to dryness. The residue was crystallized from
acetonitrile-ethanol to yield 0.5 g (77%) of 10: mp >180 °C
dec; [R]20D +31.4 (c 1.91, H2O); 1H NMR (Me2SO-d6) δ 3.50 (m,
1H, C5′H), 3.60 (m, 1H, C5′H), 3.94 (m, 1H), 4.13 (m, 1H), 4.35
(m, 1H), 5.00 (bs, 1H), 5.26 (bs, 1H), 5.64 (bs, 1H), 5.81 (d,
1H, J ) 3.9 Hz, C1′H), 7.34 (bs, 2H), 8.92 (s, 1H, C5H); 13C
NMR (Me2SO-d6) δ 61.6, 70.4, 74.3, 86.0, 91.6, 144.8, 157.2,
160.6. Anal. (C8H14N5O4Cl) C, H, N.
1-â-L-R ibofu r a n osyl-1,2,4-t r ia zole-5-ca r b oxa m id e (6).
Methyl 1-(2,3,5-tri-O-acetyl-â-L-ribofuranosyl)-1,2,4-triazole-
5-carboxylate (9.63 g, 25.0 mmol) was treated with freshly
prepared methanolic ammonia at 0 °C as described for
compound 5. The residue was treated with dry ethanol (100
mL) and evaporated to dryness. The residue obtained was
triturated with acetone and filtered. The residue was dissolved
in ethanol (100 mL). The volume of the ethanol solution was
reduced to 50 mL by heating and stirring on a hot plate. The
hot ethanol solution on cooling provided colorless crystals,
which was filtered, washed with acetone and dried under
vacuum over P2O5. The solid was found to be moisture sensitive
and on exposure to atmosphere became a viscous material:
1-[3,5-O-(1,1,3,3-Tetr a isop r op yl-1,3-O-d isiloxa n yl)-â-L-
r ibofu r a n osyl]-1,2,4-tr ia zole-3-ca r boxa m id e (11). To a
stirred solution of compound 5 (4.88 g, 20 mmol) in dry
pyridine (40 mL) and dry dimethylformamide (40 mL) at 0 °C
under argon atmosphere was added triethylamine (5.05 g, 50
mmol) followed by 1,3-dichloro-1,1,3,3-tetraisopropyl-1,3-O-
disiloxane (TIPSiCl2; 7.88 g, 25 mmol) during a 30-min period.
After the addition of TIPSiCl2, the reaction was allowed to stir
at room temperature for 12 h and evaporated to dryness. The
residue was partitioned between saturated NaHCO3 (100 mL)
and dichloromethane (150 mL) and extracted in dichlo-
romethane. The organic extract was washed with water (100
mL) and brine (50 mL), dried over anhydrous sodium sulfate
and evaporated to dryness. The residue was purified by flash
chromatography over silica gel using CHCl3 f acetone as the
eluent. The pure fractions were pooled and concentrated to
give 7.0 g (72%) of 11 as a foam: 1H NMR (CDCl3) δ 1.02-
1.08 (m, 24H, 4 × isopropyl-H), 3.98-4.20 (m, 3H, C4′ & C5′H),
4.48 (d, 1H), 4.62 (m, 1H), 5.95 (s, 1H, C1′H), 6.16 (bs, 1H, NH2),
6.98 (bs, 1H, NH2), 8.41 (s, 1H, C5H). Anal. (C20H38N4O6Si2)
C, H, N.
1-[2-O-P h en ylth iofor m ate-3,5-O-(1,1,3,3-tetr aisopr opyl-
1,3-O-d isiloxa n yl)-â-L-r ibofu r a n osyl]-1,2,4-tr ia zole-3-ca r -
boxa m id e (12). To a stirred solution of compound 11 (4.0 g,
8.23 mmol) in dry pyridine (20 mL) and dry dichloromethane
(50 mL) at 0 °C under argon atmosphere was added triethyl-
amine (2.53 g, 25 mmol) followed by phenyl chlorothionofor-
mate (4.25 g, 24.7 mmol) during a 15-min period. The reaction
was stirred at 0 °C for 1 h and at room temperature for 12 h
and evaporated to dryness. The residue was partitioned
between saturated NaHCO3 (100 mL) and ethyl acetate (150
mL) and extracted in ethyl acetate. The organic extract was
washed with water (100 mL) and brine (50 mL), dried over
anhydrous sodium sulfate and evaporated to dryness. The
residue was purified by flash chromatography over silica gel
using CH2Cl2 f EtOAc as the eluent. The pure fractions were
pooled and concentrated to give 3.17 g (62%) of the pure
compound as a foam: 1H NMR (CDCl3) δ 1.08-1.19 (m, 24H,
4 × isopropyl-H), 4.00-4.22 (m, 3H, C4′ & C5′H), 4.82 (m, 1H),
6.0 (bs, 1H, NH2), 6.14 (d, 1H), 6.17 (s, 1H, C1′H), 6.96 (bs,
1H, NH2), 7.11-7.45 (m, 5H, PhH), 8.47 (s, 1H, C5H). Anal.
(C27H42N4O7SSi2) C, H, N.
yield 4.7 g (77%); mp becomes viscous > 75 °C and melts at
1
122-125 °C; [R]20 +40.6 (c 1.33, H2O); H NMR (Me2SO-d6)
D
δ 3.40 (m, 1H, C5′H), 3.54 (m, 1H, C5′H), 3.86 (m, 1H, C4′H),
4.20 (m, 1H), 4.38 (m, 1H), 4.78 (t, 1H, C5′OH), 5.18 (d, 1H),
5.44 (d, 1H), 6.78 (d, 1H, J ) 3.9 Hz, C1′H), 8.14 (bs, 1H, NH2),
8.16 (s, 1H, C3H), 8.28 (bs, 1H, NH2); 13C NMR (Me2SO-d6) δ
62.2, 70.3, 74.2, 85.7, 90.8, 147.8, 149.4, 158.7. Anal. (C8H12N4O5)
C, H, N.
1-(2,3,5-Tr i-O-a cet yl-â-L-r ib ofu r a n osyl)-1,2,4-t r ia zole-
3-ca r boxa m id e (7). A mixture of 5 (14.0 g, 57.38 mmol), acetic
anhydride (23.46 g, 230.0 mmol) and triethylamine (25.25 g,
250.0 mmol) was allowed to stir at room temperature in dry
pyridine:dimethylformamide mixture (1:1, 100 mL) for 12 h.
The reaction mixture was evaporated too dryness to give an
oily residue. The residue was partitioned between ethyl acetate
(500 mL) and saturated NaHCO3 (150 mL) and extracted in
ethyl acetate. The organic extract was washed with water (100
mL) and brine (100 mL), dried over anhydrous sodium sulfate
and evaporated to dryness. The residue was purified on silica
column using CH2Cl2 f EtOAc as the eluent. The pure product
was pooled and evaporated to dryness to provide 17 g (80%)
of 7: 1H NMR (CDCl3) δ 2.11 (s, 3H, COCH3), 2.12 (s, 3H,
COCH3), 2.14 (s, 3H, OCH3), 4.22 (m, 1H), 4.49 (m, 2H), 5.56
(m, 1H), 5.60 (m, 1H), 6.06 (d, 1H, J ) 3.3 Hz), 6.24 (bs, 1H,
NH2), 7.04 (bs, 1H, NH2), 8.38 (s, 1H, C5H). Anal. (C14H18N4O8)
C, H, N.
1-(2,3,5-Tr i-O-a cet yl-â-L-r ib ofu r a n osyl)-1,2,4-t r ia zole-
3-ca r bon itr ile (8). To an ice-cold solution of 7 (12.95 g, 35.0
mmol) in dry dichloromethane (250 mL) and dry pyridine (50
mL) under argon atmosphere was added 20% phosgene in
toluene (50 mL) during 30 min period. After the addition, the
reaction mixture was allowed to stir at 0- 10 °C during a 2-h
period. The reaction mixture was poured onto crushed ice (500
g) and extracted with dichloromethane (2 × 250 mL). The
combined organic extract was washed with water (3 × 100 mL)
and brine (100 mL), dried over anhydrous sodium sulfate and
evaporated to dryness. The residue was purified on silica
column using CH2Cl2 f EtOAc as the eluent. The pure product
was pooled and evaporated to dryness to provide 9.0 g (73%)
of 8: 1H NMR (CDCl3) δ 2.11 (s, 3H, COCH3), 2.13 (s, 3H,
COCH3), 2.15 (s, 3H, OCH3), 4.22 (dd, 1H), 4.40-4.50 (m, 2H),
5.56 (m, 1H), 5.66 (m, 1H), 6.02 (d, 1H, J ) 3.3 Hz), 8.39 (s,
1H, C5H). Anal. (C14H16N4O7) C, H, N.
1-[2-Deoxy-3,5-O-(1,1,3,3-tetr a isop r op yl-1,3-O-d isiloxa -
n yl)-â-L-r ibofu r a n osyl]-1,2,4-tr ia zole-3-ca r boxa m id e (13).

Type 1 Cytokine-Inducing Monocyclic L-Nucleosides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1025
Compound 12 (3.17 g, 5.09 mmol) was dissolved in dry dioxane
(60 mL) and bubbled with dry argon for 15 min. To this
solution were added 2,2′-azobis(isobutyronitrile) (0.82 g, 5
mmol) and diphenylsilane (1.84 g, 10 mmol) and heated at
reflux for 12 h. The reaction was cooled and evaporated to
dryness and the residue was purified by flash chromatography
over silica gel using CH2Cl2 f EtOAc as the eluent. The pure
fractions were pooled and concentrated to give 1.2 g (50%) of
the pure compound as a foam: 1H NMR (CDCl3) δ 1.02-1.08
(m, 24H, 4 × isopropyl-H), 2.60 & 2.78 (2m, 2H, C2′H), 3.92
(m, 1H, C4′H), 4.02 (m, 2H, C5′H), 4.62 (m, 1H, C3′H), 5.74 (bs,
1H, NH2), 6.12 (d, 1H, C1′H), 7.0 (bs, 1H, NH2), 8.43 (s, 1H,
C5H). Anal. (C20H38N4O5Si2) C, H, N.
1-(2-Deoxy-â-L-r ibofu r a n osyl)-1,2,4-tr ia zole-3-ca r boxa -
m id e (14). Compound 13 (1.1 g, 2.34 mmol) was dissolved in
dry dichloromethane (10 mL) and treated with triethylamine
trihydrofluoride (10 mL). The reaction was stirred at room
temperature for 3 h and evaporated to dryness. The residue
was purified by flash chromatography over silica gel using CH2-
Cl2 f MeOH as the eluent. The pure fractions were pooled
and concentrated to give 0.25 g (47%) of the pure compound
as a foam: [R]20D -12 (c 1.33, H2O); 1H NMR (CDCl3) δ 2.32 &
2.58 (2m, 2H, C2′H), 3.40-3.60 (m, 2H, C5′H), 3.82 (m, 1H,
C4′H), 4.38 (m, 1H, C3′H), 4.86 (t, 1H, C5′OH), 5.28 (d, 1H,
C3′OH), 6.22 (t, 1H, C1′H), 7.62 (bs, 1H, NH2), 7.84 (bs, 1H,
NH2), 8.81 (s, 1H, C5H); 13C NMR (Me2SO-d6) δ 62.5, 70.9, 71.9,
85.4, 88.3, 147.3, 151.0, 158.7. Anal. (C8H12N4O4) C, H, N.
C5′OH), 5.32 (d, 1H), 5.75 (s, 1H), 5.97 (d, 1H, J ) 4.2 Hz, C1′H),
7.61 (bs, 1H, NH2), 7.84 (bs, 1H, NH2), 8.62 (s, 1H, C5H); 13C
NMR (Me2SO-d6) δ 60.0, 74.7, 80.4, 85.2, 94.4, 144.3, 156.3,
160.6. Anal. (C8H12N4O5) C, H, N.
1,2-O-Isop r op ylid en e-5-O-b en zoyl-r-L-er yth r o-p en t o-
fu r a n os-3-u lose (18). Compound 18 was prepared from
L-xylose in 65% yield (overall) by the method reported:31 1H
NMR (CDCl3) δ 1.44 & 1.52 (2s, 2 CH3), 4.42 (m, 1H, C5′H),
4.50 (m, 1H, C5′H), 4.60-4.72 (m, 2H, C2′ & C4′H), 6.14 (d, 1H,
J ) 4.4 Hz, C1′H), 7.0.42-7.97 (m, 5H, PhH).
1,2-O-Isop r op ylid en e-3-d eoxy-3-(p-tolu en esu lfon ylh y-
d r a zin o)-5-O-ben zoyl-r-L-r ibofu r a n ose (19). A mixture of
18 (60 g, 205.5 mmol) and p-toluenesulfonyl hydrazide (40.92
g, 220 mmol) in dry ethanol (500 mL) was heated at reflux for
4 h. The reaction mixture was cooled and the precipitate was
filtered, washed with ether and dried to give 45 g (48%) of
white solid: mp 178-181 °C.
To a stirred solution of the above solid (10 g, 21.74 mmol)
in tetrahydrofuran (40 mL) and methanol (40 mL) was added
a trace of methyl orange (indicator) at room temperature. To
this stirred solution was added sodium cyanoborohydride (0.63
g, 10 mmol) followed by methanolic HCl (saturated) drop by
drop keeping the color of the solution at red-yellow transition
point. The mixture was stirred at room temperature for 1 h.
A second portion of sodium cyanoborohydride (0.63 g, 10 mmol)
was added followed by dropwise addition of methanolic HCl
during a 1-h period to maintain the pH of the reaction mixture
between 2.5 and 4.0. The reaction was stirred for an additional
1 h, neutralized with saturated NaHCO3 solution and evapo-
rated to dryness. The residue was partitioned between water
(100 mL) and dichloromethane (200 mL) and extracted in
dichloromethane. The organic extract was washed with brine
(100 mL), dried and evaporated to dryness to give 10 g (99%)
of a white solid: mp 152-154 °C; 1H NMR (CDCl3) δ 1.30 and
1.45 (2s, 6H, isopropyl-CH3), 2.32 (s, 3H, Ph-CH3), 3.28 (m,
1H), 3.75 (m, 2H), 3.84 (m, 1H), 4.20 (m, 1H), 4.67 (m, 1H),
5.82 (d, 1H), 7.24-8.02 (m, 9H, PhH). Anal. (C22H26N2O7S) C,
H, N.
1,2-Isop r op ylid en e-3-d eoxy-5-O-b en zoyl-r-L-er yth r o-
p en tofu r a n ose (20). A mixture of 19 (10 g, 21.65 mmol) and
sodium acetate trihydrate (12.0 g, 88.1 mmol) in ethanol (200
mL) was heated at reflux for 2 h and cooled. The reaction was
filtered and the solid was washed with ethanol and dichlo-
romethane. The combined filtrate was evaporated and ex-
tracted in ethyl acetate (200 mL) upon partition with water
(100 mL). The organic extract was washed with brine (100 mL),
dried and evaporated to dryness to give 5.25 g (87%) of 20 as
an oil: 1H NMR (CDCl3) δ 1.32 and 1.53 (2s, 6H, isopropyl-
CH3), 1.74 (m, 1H, C2′H), 2.15 (dd, 1H, C2′H), 4.34 (m, 1H,
C5′H), 4.52 (m, 2H, C4′ & C5′H), 4.76 (m, 1H, C2′H), 5.86 (d,
1H, C1′H), 7.40 (m, 2H, PhH), 7.53 (m, 1H, PhH), 8.04 (m, 2H,
PhH). Anal. (C15H18O5) C, H.
1,2-Di-O-acetyl-5-O-ben zoyl-3-deoxy-L-r ibofu r an ose (21).
A solution of 20 (21 g, 75.54 mmol) was coevaporated with dry
toluene (100 mL), dissolved in glacial acetic acid (200 mL) and
acetic anhydride (40 mL), and cooled to 0 °C in an ice bath
under argon atmosphere. To this cold stirred solution was
added concentrated H2SO4 (15 mL) during 30 min. After the
addition of H2SO4, the reaction mixture was stirred at room
temperature for 24 h, poured into crushed ice (1000 g) and
stirred for 30 min. The aqueous solution was extracted with
chloroform (3 × 300 mL). The organic extract was washed with
water (3 × 300 mL), saturated NaHCO3 (2 × 400 mL) and
brine (100 mL), dried and evaporated to dryness to afford 24.33
g (100%) of 21 as an oil: [R]20D -1.9 (c 0.98, acetone); 1H NMR
(CDCl3) δ 2.08 (s, 6H, 2 × COCH3), 2.04-2.30 (m, 2H, C2′H),
4.32-4.76 (m, 3H, C4′ & C5′H), 5.20-5.32 (m, 1H, C2′H), 6.20
(s, 1/3H, C1′H), 6.85 (d, 2/3H, C1′H), 7.40-7.60 (m, 3H, PhH),
8.14 (m, 2H, PhH).
1,2-Di-O-a cetyl-3,5-d i-O-ben zoyl-L-xylofu r a n ose (15).
Compound 15 was prepared from L-xylose without purification
of the intermediates in 50% (overall) yield by the method
1
reported:29 [R]20 -16.9 (c 0.96, acetone); H NMR (CDCl3) δ
D
2.06 & 2.08 (2s, 6H, 2 × COCH3), 4.40-4.60 (m, 2H), 5.50-
5.62 (m, 2H), 5.90 (t, 1H, 6.96 (d, 1H), 7.00-7.60 (m, 6H, PhH),
8.03 (m, 4H, PhH).
Meth yl 1-(2-O-Acetyl-3,5-d i-O-ben zoyl-â-L-xylofu r a n o-
syl)-1,2,4-tr ia zole-3-ca r boxyla te (16). A mixture of methyl
1,2,4-triazole-3-carboxylate (1.27 g, 10.0 mmol), compound 15
(4.42 g, 10.0 mmol) and bis(p-nitrophenyl)phosphate (20.0 mg)
was placed in a pear-shaped flask (50 mL). The flask was
placed in a preheated oil bath at 165-175 °C under water
aspirator vacuum with stirring for 25 min. The acetic acid
displaced was collected in an ice-cold trap that is placed
between aspirator and the pear-shaped flask. The flask was
removed from the oil bath and allowed to cool. When the
temperature of the flask reached roughly to 60-70 °C, ethyl
acetate (100 mL) and saturated NaHCO3 (50 mL) were
introduced and extracted in ethyl acetate. The aqueous layer
was extracted again with ethyl acetate (50 mL). The combined
ethyl acetate extract was washed with saturated NaHCO3 (50
mL), water (50 mL) and brine (50 mL). The organic extract
was dried over anhydrous sodium sulfate and filtered and the
filtrate evaporated to dryness. The residue was purified by
flash chromatography over silica gel using hexane f EtOAc
as the eluent. The pure product was pooled together and
evaporated to give 4.0 g (79%) of 16: 1H NMR (CDCl3) δ 2.22
(s, 3H, COCH3), 3.96 (s, 3H, OCH3), 4.72 (m, 2H), 4.98 (m, 1H),
5.79 (t, 2H), 6.15 (s, 1H), 7.43 (m, 4H, PhH), 7.59 (m, 2H, PhH),
7.81 (d, 2H, PhH), 8.00 (d, 2H, PhH), 8.61 (s, 1H, C5H). Anal.
(C25H23N3O9) C, H, N.
1-(â-L-Xylofu r a n osyl)-1,2,4-tr ia zole-3-ca r boxa m ide (17).
Compound 16 (3.5 g, 6.88 mmol) was placed in a steel bomb
and treated with freshly prepared methanolic ammonia (100
mL, prepared by passing dry ammonia gas into dry methanol
at 0 °C until saturation) at 0 °C. The steel bomb was closed
and stirred at room temperature for 18 h. The steel bomb was
cooled to 0 °C and opened and the content evaporated to
dryness. The residue was treated with dry ethanol (100 mL)
and evaporated to dryness. The residue obtained was tritu-
rated with acetone and the acetone was decanted. This was
repeated for two more times to remove acetamide. The residue
was dissolved in minimum amount of ethanol, which on cooling
Meth yl 1-(2-O-Acetyl-5-O-ben zoyl-3-deoxy-â-L-r ibofu r a -
n osyl)-1,2,4-tr ia zole-3-ca r boxyla te (22). The procedure was
the same as that described for compound 16. Materials used:
methyl 1,2,4-triazole-3-carboxylate (1.27 g, 10.0 mmol), com-
pound 21 (3.54 g, 11.0 mmol) and bis(p-nitrophenyl)phosphate
inside refrigerator gave crystals: yield 1.5 g (89%); mp 172-
1
175 °C; [R]20 +22 (c 0.49, H2O); H NMR (Me2SO-d6) δ 3.72
D
(m, 2H, C5′H), 4.02 (m, 1H, C4′H), 4.24 (m, 2H), 4.76 (t, 1H,

1026 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Ramasamy et al.
(20.0 mg). The crude product was purified by flash chroma-
tography over silica gel using CHCl3 f EtOAc as the eluent.
The pure product was pooled together and evaporated to give
3.0 g (77%) of 22: 1H NMR (CDCl3) δ 2.03 (s, 6H, 2 × COCH3),
2.32 (m, 1H, C2′H), 2.62 (m, 1H, C2′H), 3.99 (s, 3H, OCH3),
4.42-4.68 (m, 2H, C5′H), 4.82 (m, 1H, C4′H), 5.62 (d, 1H, C2′H),
6.04 (s, 1H, C1′H), 7.40-7.60 (m, 3H, PhH), 8.00 (m, 2H, PhH),
8.40 (s, 1H, C5H). Anal. (C18H19N3O7) C, H, N.
1,2,3-Tr i-O-a cetyl-5-d eoxy-L-r ibofu r a n ose (27). Com-
pound 26 (13.3 g, 71.0 mmol) was dissolved in glacial acetic
acid (100 mL) and acetic anhydride (40 g, 392 mmol) and cooled
to 5 °C. To this cold stirred solution was added concentrated
H2SO4 (7 mL) during 30 min. After the addition, the reaction
mixture was stirred at room temperature for 12 h, poured on
ice (200 g) and stirred for 1 h. The aqueous layer was extracted
with ethyl acetate (2 × 150 mL). The organic layer was washed
with saturated NaHCO3 (200 mL), water (200 mL) and brine
(100 mL), dried and evaporated to dryness to give 9.0 g (49%)
of 27 as an oil: 1H NMR (CDCl3) δ 1.24-1.40 (m, 3H, C5′CH3),
2.04-2.20 (m, 9H, 3 × COCH3), 4.18-4.40 (m, 2H), 5.00-5.40
(m, 3H), 6.99 (d, 1H).
Met h yl (2,3-Di-O-a cet yl-5-d eoxy-â-L-r ib ofu r a n osyl)-
1,2,4-tr ia zole-3-ca r boxyla te (28). 1,2,4-Triazole-3-carboxyl-
ate (2.54 g, 20.0 mmol) in hexamethyldisilazane (100 mL) was
heated at reflux for 4 h and evaporated to dryness. The residue
was dissolved in dry acetonitrile (100 mL) and cooled to 0 °C
under argon atmosphere. To this cold stirred solution was
added 1,2,3-tri-O-acetyl-5-deoxy-L-ribofuranose (5.20 g, 20
mmol) followed by SnCl4 (5.2 g, 20 mmol) during a 15-min
period. The reaction mixture was allowed to stir at room
temperature for 12 h, cooled to 0 °C, quenched with saturated
NaHCO3 solution and filtered, and the precipitate was washed
with acetonitrile (100 mL). The combined filtrate was evapo-
rated to dryness. The residue was partitioned between ethyl
acetate (200 mL) and saturated NaHCO3 (50 mL) and ex-
tracted in ethyl acetate. The aqueous layer was extracted again
with ethyl acetate (100 mL). The combined ethyl acetate
extract was washed with saturated NaHCO3 (150 mL), water
(100 mL) and brine (50 mL). The organic extract was dried
over anhydrous sodium sulfate and filtered and the filtrate
evaporated to dryness. The residue was purified by flash
chromatography over silica gel using hexane f EtOAc as the
eluent. The pure product was pooled together and evaporated
to give 3.5 g (53%) of 28: 1H NMR (CDCl3) δ 1.44 (d, 3H,
C5′CH3), 2.10 (s, 6H, 2 × COCH3), 3.99 (s, 3H, OCH3), 4.40
(m, 1H), 5.24 (m, 1H), 5.72 (m, 1H), 5.99 (d, 1H, C1′H), 8.38 (s,
1H, C5H). Anal. (C13H17N3O7) C, H, N.
1-(3-Deoxy-â-L-r ibofu r a n osyl)-1,2,4-tr ia zole-3-ca r boxa -
m id e (23). Compound 23 was prepared by following the
procedure used for the preparation of 17. Materials used: 23
(0.9 g, 2.31 mmol) and methanolic ammonia (70 mL, prepared
by passing dry ammonia gas into dry methanol at 0 °C until
saturation) at 0 °C. The crude product was dissolved in
minimum amount of ethanol, which on cooling inside refrig-
erator gave 0.4 g (76%) of crystals: mp 128-131 °C; [R]20
D
+11.4 (c 0.56, H2O); 1H NMR (Me2SO-d6) δ 1.84 (m, 1H, C3′H),
2.10 (m, 1H, C3′′H), 3.52-3.64 (m, 2H, C5′H), 4.38-4.58 (m,
2H, C2′ & C4′H), 4.97 (t, 1H, C5′OH), 5.72 (d, 1H), 5.84 (s, 1H),
7.62 (bs, 1H, NH2), 7.82 (bs, 1H, NH2), 8.84 (s, 1H, C5H); 13C
NMR (Me2SO-d6) δ 24.2, 63.4, 75.8, 82.3, 94.9, 144.5, 157.8,
161.1. Anal. (C8H12N4O4) C, H, N.
1-Met h oxy-2,3-O-isop r op ylid en e-L-r ib ofu r a n ose (24).
L-Ribose was suspended in dry methanol (200 mL) and 2,2-
dimethoxypropane (100 mL) and mixed with dry acetone (700
mL). To this stirred suspension was added methanolic HCl
(20 mL, saturated at 0 °C) during a 15-min period. After the
addition, the reaction mixture was allowed to stir at room
temperature overnight. Pyridine (100 mL) was added and
evaporated to dryness. The residue was partitioned water (500
mL) and ether (400 mL) and extracted in ether. The aqueous
layer was extracted with ether (2 × 200 mL). The combined
ether layer was dried and concentrated to give 62 g (92%) of
crude product. The crude product was used as such for the
next reaction: 1H NMR (CDCl3) δ 1.33 (s, 3H, isopropylidene-
CH3), 1.50 (s, 3H, isopropylidene-CH3), 3.30 (dd, 1H), 3.45 (s,
3H, OCH3), 3.58-3.74 (m, 2H, C5′H), 4.42 (m, 1H, C5′OH), 4.61
(d, 1H), 4.84 (d, 1H), 4.99 (s, 1H, C1′H). Anal. (C9H16O5) C, H.
1-Met h oxy-2,3-O-isop r op ylid en e-5-b r om o-L-r ib ofu r a -
n ose (25). Compound 24 (55.0 g, 269 mmol) was dissolved in
dry dichloromethane (900 mL) and cooled to 0 °C under argon
atmosphere. To this cold solution was added Ph3P (141.5 g,
540 mmol) followed by bromine (85.86 g, 540 mmol). The
reaction mixture was refluxed for 4 h, cooled, and added to
methanol (50 mL). The dichloromethane layer was washed
with water (2 × 500 mL), saturated NaHCO3 (2 × 500 mL),
water (2 × 500 mL) and brine (200 mL), dried and evaporated
to dryness. The residue was triturated with hexane:ethyl
acetate (8:2, 1000 mL) and filtered. The precipitate was
washed with hexane:ethyl acetate (8:2, 500 mL). The combined
filtrate was evaporated to give 79 g of crude product. The crude
product was placed on top of silica column packed in hexane
and eluted with hexane:ethyl acetate (9:1, 1000 mL). The pure
fractions were collected and evaporated to give 62.8 g (87%)
of 25 as an oil: 1H NMR (CDCl3) δ 1.33 (s, 3H, isopropylidene-
CH3), 1.49 (s, 3H, isopropylidene-CH3), 3.32-3.46 (m, 5H, C5′H
& OCH3), 4.42 (m, 1H), 4.62 (d, 1H), 4.77 (d, 1H), 5.02 (s, 1H).
Anal. (C9H15O4Br) C, H, N, Br.
1-(5-Deoxy-â-L-r ibofu r a n osyl)-1,2,4-tr ia zole-3-ca r boxa -
m id e (29). Compound 28 (0.9 g, 2.75 mmol) was treated with
freshly prepared methanolic ammonia (50 mL) as described
for the preparation of compound 23. The residue was dissolved
in minimum amount of ethanol, which on cooling inside
refrigerator gave crystals: yield 0.45 g (72%); mp 178-180 °C;
[R]20 -70 (c 1.1, H2O); 1H NMR (Me2SO-d6) δ 1.22 (d, 3H,
D
C5′CH3), 3.62 (m, 1H, C4′H), 4.16 (m, 1H), 4.44 (m, 1H), 5.54
(d, 1H), 5.77 (d, 1H), 5.86 (d, 1H, J ) 5.4 Hz, C1′H), 7.64 (bs,
1H, NH2), 7.84 (bs, 1H, NH2), 8.80 (s, 1H, C5H); 13C NMR (Me2-
SO-d6) δ 18.4, 79.9, 80.9, 81.7, 91.8, 145.1, 157.8, 160.6. Anal.
(C8H12N4O4) C, H, N.
Biologica l Tests. 1. P r ep a r a tion of Hu m a n T Cells a n d
Activa tion in Vitr o. Peripheral blood mononuclear cells were
isolated from healthy donors by density gradient centrifugation
followed by T cell enrichment using Lymphokwik (One Lambda,
Canoga Park, CA). Contaminating monocytes were removed
by adherence to plastic. Purified T cells were >99% CD2+, <1%
HLA-DR+, and <5% CD25+ and were maintained in RPMI-
AP5 (RPMI-1640 medium containing 5% autologous plasma,
1% L-glutamine, 1% penicillin/streptomycin, and 0.05% 2-mer-
captoethanol). For determination of cytokine protein levels, T
cells (0.2 × 106 cells in a volume of 0.2 mL) were activated by
the addition of 80 ng of staphyloccocal enterotoxin B (SEB;
Sigma, St. Louis, MO) and incubated in 96-well plates in the
presence of 0-10 µM of various L-nucleosides or ribavirin for
48 h at 37 °C. Following activation, supernatants were
analyzed for cell-derived cytokine production.
2. Extr a cellu la r Cytok in e An a lyses. Human cytokine
levels were determined in cell supernatants, following ap-
propriate dilution, using ELISA kits specific for IL-2, IFN-γ,
and TNF-R (Biosource, Camarillo, CA). All ELISA results were
expressed as pg/mL. Data are shown as percentage of activated
control calculated as the ratio of activated T cell cytokine level
in the presence of test nucleoside over the cytokine level of
1-Met h oxy-2,3-O-isop r op ylid en e-5-d eoxy-L-r ib ofu r a -
n ose (26). Compound 25 (41.0 g, 153.6 mmol) was dissolved
in dry methanol (200 mL) and treated with potassium hydrox-
ide (8.96 g, 160 mmol) dissolved in water (10 mL). To this
solution was added Pd/C (10%, 3.0 g) and hydrogenated at 45
psi for 12 h. The reaction mixture was filtered and washed
with methanol (100 mL) and the filtrate evaporated to dryness.
The residue was partitioned between water (100 mL) and
dichloromethane (200 mL) and extracted in dichloromethane.
The organic layer was washed with brine (100 mL), dried and
evaporated to dryness to give 27.0 g (94%) of pure product as
an oil: 1H NMR (CDCl3) δ 1.22-1.30 (m, 6H, isopropylidene-
CH3 & C5′CH3), 1.45 (s, 3H, isopropylidene-CH3), 3.30 (s, 3H,
OCH3), 4.32 (m, 1H), 4.49 (d, 1H), 4.60 (d, 1H), 4.91 (1, 1H).
Anal. (C9H16O4) C, H.

Type 1 Cytokine-Inducing Monocyclic L-Nucleosides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1027
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levels by test nucleosides would give a percentage of activated
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