Synthesis, conformational analysis, and biological activity of C- thioribonucleosides related to tiazofurin

Article abstract of DOI:10.1021/jm990257b

The syntheses of furanthiofurin [5β-D-(4'-thioribofuranosyl)furan-3- carboxamide, 1] and thiophenthiofurin [5β-D-(4'-thioribofuranosyl)thiophene- 3-carboxamide, 2], two C-thioribonucleoside analogues of tiazofurin, are described. Direct trifluoroacetic acid-catalyzed C-glycosylation of ethyl furan-3-carboxylate with 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of α and β anomers. Ethyl 5-(2,3,5-tri-O-benzyl)-β-D-(4'-thioribofuranosyl)furan-3-carboxylate (6β) was debenzylated and then converted into the corresponding amide (furanthiofurin) by reaction with ammonium hydroxide. A similar C- glycosylation of ethyl thiophene-3-carboxylate with 1,2,3,5-tetra-O-acetyl-4- thio-D-ribofuranose catalyzed by stannic chloride afforded an anomeric mixture of 2- and 5-glycosylated regioisomers. Deacetylation of ethyl 5- (2,3,5-tri-O-acetyl)-β-D-(4'-thioribofuranosyl)thiophene-3-carboxylate (13β) with methanolic ammonia and treatment of the ethyl ester with ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration were established by ¹H NMR spectroscopy. Thiophenthiofurin was found to be cytotoxic in vitro toward human myelogenous leukemia K562, albeit 39-fold less than thiophenfurin, while furanthiofurin proved to be inactive. K562 cells incubated with thiophenthiofurin resulted in inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) and an increase in IMP pools with a concurrent decrease in GTP levels. From computational studies it was deduced that, among the C-nucleoside analogues of tiazofurin, activity requires an electrophilic sulfur adjacent to the C-glycosidic bond and an energetically favorable conformer around X = 0°. Among these, the more constrained (least flexible) compounds (tiazofurin and thiophenfurin) are more active than the less constrained thiophenthiofurin. Those compounds which contain a nucleophilic oxygen in place of the thiazole or thiophene (oxazofurin, furanfurin, and furanthiofurin) show the least activity.

Full text of DOI:10.1021/jm990257b

1264
J . Med. Chem. 2000, 43, 1264-1270
Articles
Syn th esis, Con for m a tion a l An a lysis, a n d Biologica l Activity of
C-Th ior ibon u cleosid es Rela ted to Tia zofu r in
Palmarisa Franchetti,* Stefano Marchetti, Loredana Cappellacci, Hiremagalur N. J ayaram,‡
J oel Aaron Yalowitz,‡ Barry M. Goldstein,# J ean-Louis Barascut,† David Dukhan,† J ean-Louis Imbach,† and
Mario Grifantini
Dipartimento di Scienze Chimiche, Universita` di Camerino, Via S. Agostino, 1, 62032 Camerino, Italy, Laboratory for
Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, Department of Biochemistry and
Biophysics, University of Rochester Medical Center, Rochester, New York 14642, and Laboratoire de Chimie Bio-organique,
URA CNRS 488, Universite´ de Montpellier II, 34095 Montpellier Ce´dex 5, France
Received May 28, 1999
The syntheses of furanthiofurin [5â-D-(4′-thioribofuranosyl)furan-3-carboxamide, 1] and
thiophenthiofurin [5â-D-(4′-thioribofuranosyl)thiophene-3-carboxamide, 2], two C-thioribo-
nucleoside analogues of tiazofurin, are described. Direct trifluoroacetic acid-catalyzed C-
glycosylation of ethyl furan-3-carboxylate with 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofura-
nose gave 2- and 5-glycosylated regioisomers, as a mixture of R and â anomers. Ethyl 5-(2,3,5-
tri-O-benzyl)-â-D-(4′-thioribofuranosyl)furan-3-carboxylate (6â) was debenzylated and then
converted into the corresponding amide (furanthiofurin) by reaction with ammonium hydroxide.
A similar C-glycosylation of ethyl thiophene-3-carboxylate with 1,2,3,5-tetra-O-acetyl-4-thio-
D-ribofuranose catalyzed by stannic chloride afforded an anomeric mixture of 2- and 5-glyco-
sylated regioisomers. Deacetylation of ethyl 5-(2,3,5-tri-O-acetyl)-â-D-(4′-thioribofuranosyl)-
thiophene-3-carboxylate (13â) with methanolic ammonia and treatment of the ethyl ester with
ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration
were established by 1H NMR spectroscopy. Thiophenthiofurin was found to be cytotoxic in vitro
toward human myelogenous leukemia K562, albeit 39-fold less than thiophenfurin, while
furanthiofurin proved to be inactive. K562 cells incubated with thiophenthiofurin resulted in
inhibition of inosine 5′-monophosphate dehydrogenase (IMPDH) and an increase in IMP pools
with a concurrent decrease in GTP levels. From computational studies it was deduced that,
among the C-nucleoside analogues of tiazofurin, activity requires an electrophilic sulfur adjacent
to the C-glycosidic bond and an energetically favorable conformer around ø ) 0°. Among these,
the more constrained (least flexible) compounds (tiazofurin and thiophenfurin) are more active
than the less constrained thiophenthiofurin. Those compounds which contain a nucleophilic
oxygen in place of the thiazole or thiophene (oxazofurin, furanfurin, and furanthiofurin) show
the least activity.
In tr od u ction
specific signal transduction pathways. The drug pro-
duces IMPDH inhibition after conversion in sensitive
cells to the active metabolite thiazole-4-carboxamide
adenine dinucleotide (TAD), which binds tightly at the
NADH site and inhibits IMPDH activity. This enzyme
catalyzes the NAD-dependent conversion of inosine 5′-
monophosphate (IMP) to xanthosine monophosphate
(XMP), which is the rate-limiting step in guanine
nucleotide biosynthesis. Thus, IMPDH inhibitors reduce
GTP and deoxyGTP pool levels, and they have been
shown to possess antineoplastic, antiparasitic, antiviral,
and immunosuppressive activity.1
Tiazofurin is a synthetic thiazole C-nucleoside which
has demonstrated clinical efficacy as an antitumor
agent.1 The antitumor activity of tiazofurin results from
a combination of cytotoxic and maturation-inducing
activities. It was found that tiazofurin inhibits inosine
5′-monophosphate dehydrogenase (IMPDH), decreases
cellular GTP concentration, induces differentiation,
down-regulates ras and myc oncogene expression, and
causes apoptosis of K562 human erythroleukemia cells
in a time- and dose-dependent fashion. The tiazofurin-
mediated apoptosis may therefore be linked with the
decrease of GTP and the consequent impairment of
In 1995, we reported the synthesis of thiophenfurin
(5â-D-ribofuranosylthiophene-3-carboxamide) and furan-
furin (5â-D-ribofuranosylfuran-3-carboxamide), two C-
nucleoside isosters of tiazofurin, in which the thiazole
ring was replaced by a thiophene and furan heterocycle,
respectively. While thiophenfurin was found active as
* To whom correspondence should be addressed. Tel: (+39)-0737-
402233. Fax: (+39)-0737-637345. E-mail: frapa@camserv.unicam.it.
‡ Indiana University School of Medicine.
#
University of Rochester Medical Center.
† Universite´ de Montpellier II.
10.1021/jm990257b CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/15/2000

C-Thioribonucleosides Related to Tiazofurin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1265
Sch em e 1
an antitumor agent both in vitro and in vivo, furanfurin
proved to be inactive.2 In sensitive cells, thiophenfurin,
like tiazofurin, is metabolized to a nicotinamide adenine
dinucleotide (NAD) analogue, TFAD, which is a potent
inhibitor of IMPDH and is more active than TAD.3 We
found that the inactivity of furanfurin was due both to
its poor ability to be converted to furan-3-carboxamide
adenine dinucleotide (FFAD) in target cells and to the
poor affinity of this dinucleotide for IMPDH.2,3
To gain further information about the structure-
activity relationships of this type of antitumor C-
nucleosides, we investigated furanfurin and thiophen-
furin analogues in which the furanose ring oxygen O-4′
was replaced by a sulfur atom (furanthiofurin, 1, and
thiophenthiofurin, 2).
Ch em istr y
Furanthiofurin (1) was synthesized by direct C-
glycosylation of ethyl furan-3-carboxylate (3) with
1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose (4)4
(Scheme 1). The glycosylation reaction performed in the
presence of trifluoroacetic acid in methylene chloride
gave 2- and 5-glycosylated regioisomers as a mixture
of R and â anomers (5R,â and 6R,â, 53%). Analytical
samples of 5R, 5â, 6R, and 6â were obtained by semi-
preparative HPLC. The mixture of compounds 5R, 5â,
and 6â was separated from 6R by chromatography on
silica gel. We found it more convenient to separate the
mixture of the 2-glycosylated anomers 5R, 5â and the
isomer 6â after removal of benzyl groups using boron
tribromide in methylene chloride at -78 °C. The de-
blocked ethyl esters 7R, 7â, and 8â were separated by
chromatography on silica gel. The glycosylation position
of these nucleosides was determined by proton spectro-
Crystallographic and ab initio computational studies
showed that thiophenfurin, like tiazofurin, possesses a
conformationally restricted glycosidic bond due to an
energetically favorable intramolecular sulfur-oxygen
interaction. This can be interpreted as an electrostatic
interaction between the positively charged thiophene
sulfur and negatively charged furanose oxygen. This
interaction stabilizes the conformation in which these
heteroatoms are adjacent. In contrast, in furanfurin the
repulsive interaction between the negatively charged
furan and furanose oxygens destabilizes the conformers
in which such atoms are cis (Figure 1).2
1
scopic analysis. The H NMR spectra of compounds 8R
and 8â showed that the signal of the H-5 proton of furan
had disappeared, indicating that the glycosylation posi-
tion was at C-5. On the other hand, in the 1H NMR
spectra of 7R and 7â, the signal of the H-2 proton was
lacking, supporting C-2 glycosylation. The stereostruc-
tures of these compounds were determined by compari-

1266 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Franchetti et al.
Sch em e 2
Ta ble 1. Cytotoxicity and Effect of Compounds on IMPDH
Activity and on Purine Nucleotide Concentration in Human
Myelogenous Leukemia K562 Cells in Culture
son of the chemical shift of the anomeric proton. As
reported in the case of other thioribonucleosides,4 it was
found that the chemical shift of H-1′ of the â anomer is
upfield from the signal of the corresponding H-1′ proton
of the R anomer. Treatment of 8â with ammonium
hydroxide (30%) gave furanthiofurin (1). In a similar
way, ethyl ester 8R was converted to the amide 9, R
anomer of 1.
We found that thiophenthiofurin could not be syn-
thesized by glycosylation of ethyl thiophene-3-carboxy-
late (10) with thioriboside 4, probably owing to the
reduced reactivity of the thiophene ring toward this
electrophilic reagent. We succeeded in the synthesis of
thiophenthiofurin using 1,2,3,5-tetra-O-acetyl-4-thio-D-
ribofuranose (11) as a glycosylation reagent (Scheme 2).
Compound 11 was synthesized starting from D-gulono-
1,4-lactone as reported by Dukhan et al.5 Reaction of
10 with 11 in 1,2-dichloroethane in the presence of
SnCl4 gave a mixture of 2- and 5-glycosylated regioiso-
mers as R and â anomers (12R,â, 13R,â, 56%). Isomer
13R was separated from the mixture of 12R, 12â, and
13â by column chromatography and converted into the
amide 14 by reaction with methanolic ammonia and
then with 30% ammonium hydroxide. Owing to the
impossibility of separating by chromatography the
mixture of 12R, 12â, and 13â isomers, this mixture was
deprotected with methanolic ammonia and then treated
with ammonium hydroxide. Thiophenthiofurin (2) was
separated from the mixture of C-2 isomers by chroma-
tography on silica gel column.
a
Concentration required to inhibit 50% of cell proliferation at
b
48 h. For studies related to IMPDH inhibition and nucleotide
pools, cells were incubated with 2 µM thiophenfurin or 100 µM
furanthiofurin or thiophenthiofurin.
tested concentration of 100 µM. In contrast, thiophenthio-
furin showed cytotoxic activity against K562 cells, albeit
39-fold lower than that of thiophenfurin. The R anomer
of thiophenthiofurin was found inactive as an antitumor
agent. This finding once more confirms that the â
anomeric configuration of these C-nucleosides is impor-
tant for cytotoxic activity.
Furanthiofurin and thiophenthiofurin were also tested
for inhibitory properties against IMPDH from human
myelogenous leukemia K562 cells in culture, as previ-
ously reported.7 For these studies, K562 cells were
exposed to 100 µM each of the inhibitors (2 µM in the
case of thiophenfurin) or saline for 2 h at 37 °C. The
results (Table 1) indicate that IMPDH activity was
inhibited (25%) by the action of furanthiofurin with a
potency comparable to that of thiophenthiofurin (17%
inhibition) and that this inhibition was ∼50-fold lower
than that exhibited by thiophenfurin.
The glycosylation position and the anomeric config-
1
uration of nucleosides 2 and 14 were determined by H
NMR data in DMSO-d6 as reported above.
Biologica l Eva lu a tion
Furanthiofurin, thiophenthiofurin, and their R ano-
mers (9 and 14) were evaluated for their ability to
inhibit the growth of human myelogenous leukemia
K562 cells. Tumor cell proliferation was evaluated by
incubating the cells continuously with either the com-
pounds or saline for 48 h.6 Thiophenfurin was used as
a reference compound. The IC50 (µM) values for these
C-nucleosides are summarized in Table 1. Furanthio-
furin proved to be almost inactive until a maximum
Since inhibition of IMPDH results in perturbation of
nucleotide pools, we examined the influence of furanthio-
furin and thiophenthiofurin on the ribonucleotide con-
centration in K562 cells. The results provided in Table
1 show an increase in IMP levels in the case of
thiophenthiofurin (10.9%) and thiophenfurin (11.8%)
with a concurrent decrease in guanylate concentration.

C-Thioribonucleosides Related to Tiazofurin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1267
F igu r e 1. (Top) Comparison of heteroatom charges for tiazofurin, thiophenfurin, thiophenthiofurin, oxazofurin, furanfurin, and
furanthiofurin. The heterocycle and portion of the ribofuranose or 4-thioribofuranose moiety corresponding to each agent is
illustrated at the top. Charges were calculated at the DFT/6-31G**//RHF/3-21G* level of theory using the model fragment shown
at the top left and partitioned using the NBO method. Where Y ) C-H, the combined charge for the carbon and its attached
proton is listed. (Bottom) Energy vs C-glycosidic torsion angle ø for model fragments of tiazofurin, thiophenfurin, thiophenthiofurin,
oxazofurin, furanfurin, and furanthiofurin. The color of each curve matches that of the symbol above the corresponding agent’s
name. Each point on each curve was obtained at the RHF/3-21G*//3-21G* level. The fragment at the top left is shown in the ø )
0° conformation.
Com p u ta tion a l Stu d ies a n d Discu ssion
anabolite, has been recently determined.13 The structure
indicates an interaction between the SAD electrophilic
heteroatom and the side chain of Gln 334 on the active
site loop. Although the position of the loop is distorted
in this complex by the presence of the 6-Cl analogue of
the IMP substrate, comparison with other structures
suggests that a similar interaction may be maintained
between the electrophilic heteroatom and Gln 441 when
complexed with native substrate.13 The presence of a
nucleophilic oxygen atom in place of the sulfur or
selenium would destabilize this interaction. The pres-
ence of an electrophilic heteroatom would also be
expected to influence binding of the parent compounds
to anabolic enzymes. This may account for the variations
in levels of NAD analogues observed between agents.
The structure of the IMPH-SAD complex also sug-
gests that the conformation about the C-glycosidic bond
in the bound ligand is such that the Se atom and
furanose oxygen remain cis to each other. Thus, con-
formational studies were carried out by ab initio com-
putations as previously reported,2 and the results are
compared in Figure 1 for tiazofurin, thiophenfurin,
thiophenthiofurin, oxazofurin, furanfurin, and furanthio-
furin.
Heteroatom charges for tiazofurin, thiophenfurin,
thiophenthiofurin, oxazofurin, furanfurin, and furanthio-
furin are compared in Figure 1. Charges were obtained
at the DFT/631G**//HF/321G* level of theory using
natural bond order partitioning as implemented in
Gaussian 98.8-10 For those compounds having a C-H
group adjacent to the C-glycosidic carbon, the combined
charge of the carbon and its attached proton is indicated.
While these charges depend to some degree on the
particular computational method employed, relative
values remain consistent over a variety of basis sets and
partitioning methods. Thus, general observations may
be made based on the values shown in Figure 1.
Tiazofurin, thiophenfurin, and thiophenthiofurin all
contain a positively charged sulfur in the unsaturated
five-membered heterocycle. Each of these compounds
demonstrates activity, albeit to varying degrees. In
oxazofurin, furanfurin, and furanthiofurin, the posi-
tively charged sulfur is replaced by a negatively charged
oxygen. These compounds show minimal or no activity.
These observations suggest that an electrophilic atom
adjacent to the C-glycosidic carbon is required for
activity. This requirement may result from one or a
combination of several factors.11,12
Tiazofurin shows a well-defined minimum at ø )
0-20°, close to the conformation maintained in the
enzyme-bound ligand. The barrier to rotation in tiazo-
The crystal structure of human IMPDH complexed
with SAD, the selenium analogue of the active tiazofurin

1268 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Franchetti et al.
column chromatography was used. High-performance liquid
chromatography (HPLC) analysis was carried out on a Waters
apparatus equipped with a model 600 system (detector 486).
Beckman Ultrasphere Si 5U (10 mm × 25 cm) column was
used.
furin is significantly higher than that found in the other
agents, suggesting that the tiazofurin anabolite may
gain an entropic advantage in binding the target.
Among the active agents, tiazofurin remains the most
potent and the most highly constrained.
Eth yl 2-(2,3,5-Tr i-O-ben zyl-4-th io-r-D-r ibofu r a n osyl)-
fu r a n -3-ca r boxyla te (5r), Eth yl 2-(2,3,5-Tr i-O-ben zyl-4-
th io-â-D-r ibofu r a n osyl)fu r a n -3-ca r boxyla te (5â), Eth yl
5-(2,3,5-Tr i-O-b en zyl-4-t h io-r-D-r ib ofu r a n osyl)fu r a n -3-
ca r boxyla te (6r), a n d Eth yl 5-(2,3,5-Tr i-O-ben zyl-4-th io-
â-D-r ibofu r a n osyl)fu r a n -3-ca r boxyla te (6â). To a stirred
solution of 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose
(4) (5 g, 10.44 mmol) in dry CH2Cl2 (60 mL) cooled at 0 °C
was added trifluoroacetic acid (59 mmol, 4.5 mL). After 15 min
under nitrogen atmosphere, the mixture was allowed to warm
to room temperature, and ethyl furan-3-carboxylate (3) (2.13
g, 15.2 mmol) was added. The resulting solution was stirred
for 8 h, neutralized with saturated NaHCO3 (300 ml) and then
extracted with CH2Cl2 (3 × 100 mL). The combined organic
layers were dried (Na2SO4), filtered, and evaporated to dryness
to give an oily residue which was chromatographed on flash
silica gel column eluting with cyclohexanes-EtOAc (97:3). A
mixture of 5R, 5â and 6â (1.16 g, 20%) was separated as first
eluate. TLC (cyclohexanes-EtOAc, 90:10): Rf 0.75.
Evaporation of the following fraction gave 1.56 g (27%) of
6R. TLC (cyclohexanes-EtOAc, 90:10): Rf 0.72. 1H NMR (Me2-
SO-d6): δ 1.30 (t, J ) 7.1 Hz, 3H, OCH2CH3); 3.60 (dd, J )
5.9, 9.9 Hz, 1H, H5′b); 3.75 (dd, J ) 3.7, 10.0 Hz, 2H, H4′,
H5′a); 3.95 (m, 1H, H3′); 4.22 (m, 1H, H2′); 4.25 (q, J ) 7.0
Hz, 2H, OCH2CH3); 4.40-4.55 (m, 7H, CH2C6H5, H1′); 5.80
(s, 1H, H4); 7.20-7.35 (m, 15H, arom.); 7.90 (s, 1H, H2). Anal.
C, H.
Thiophenfurin also shows an energetic minimum close
to the favorable cis conformer (ø ) 20°). However,
replacement of the negatively charged thiazole nitrogen
with the almost neutral C-H group also produces a local
minimum at the unfavorable trans conformation (ø )
180°). Replacement of the oxolane oxygen with a sulfur
in thiophenthiofurin results in a broader, shallower
minimum in the favored region. Thus, any entropic
advantage becomes successively less significant in
thiophenfurin and thiophenthiofurin, respectively, as
conformational constraints diminish. In the inactive
compounds, replacement of the positively charged thia-
zole or thiophene sulfur with a negatively charged
oxygen results in generally lower barriers to rotation.
What constraints do exist are associated with global or
local minima shifted to higher, more unfavorable values
of ø.
It is worth noting that the thiolane sulfur is electroni-
cally unique, mimicking neither the oxolane oxygen nor
the thiazole or thiophene sulfur. Unlike the oxolane
oxygen, the thiolane sulfur has a net positive charge.
However, the magnitude of this charge is reduced to
about one-half that of the more delocalized thiophene
or thiazole sulfur. Thus, the conformational energy
profiles for furanthiofurin and thiophenthiofurin are
more heavily modulated by intramolecular steric and
charge-transfer interactions. Nevertheless, the substitu-
tion of an electrophilic thiolane sulfur in place of the
oxolane oxygen may be also be expected to influence
binding of either the parent compounds or the NAD
analogues to their targets. The net effect of this sub-
stitution is that neither furanthiofurin nor thiophenthio-
furin appears to result in particularly effective IMPDH
inhibition, despite wide variation in activity.
Compounds 5R, 5â, 6R and 6â were separated by HPLC
using cyclohexanes-EtOAc (95:5) as eluents (F 4.4 mL/min,
P1600 psi). HPLC retention times (tR min): 5â, 12.70 min; 5R,
13.48 min; 6â, 14.09 min; 6R, 15.43 min.
Eth yl 2-(4-Th io-r-D-r ibofu r a n osyl)fu r a n -3-ca r boxyla te
(7r), Eth yl 2-(4-Th io-â-D-r ibofu r a n osyl)fu r a n -3-ca r boxy-
la te (7â), a n d Eth yl 5-(4-Th io-â-D-r ibofu r a n osyl)fu r a n -3-
ca r boxyla te (8â). To a solution of 1 M BBr3 in dry CH2Cl2
(1.5 mL) cooled at -78 °C was added dropwise a solution of
the mixture of 5R, 5â and 6â (1.1 g, 1.96 mmol) in dry CH2Cl2
(50 mL). The reaction mixture was stirred for 1 h under
nitrogen atmosphere at the same temperature and then
evaporated to dryness. The residue was coevaporated with
cooled dry CH2Cl2/MeOH (2:1, v/v). After neutralization with
saturated NaHCO3 the mixture was evaporated to obtain a
residue which was washed with cooled anhydrous EtOH and
then filtered off. The filtrate was evaporated to dryness to give
an oily residue which was chromatographed on silica gel
column eluting with CHCl3-MeOH (97:3). Evaporation of the
first fraction gave a mixture of 7R and 7â, which was separated
by preparative TLC using CHCl3-MeOH (90:10) as eluent in
very low yield.
In summary, computational findings remain qualita-
tively consistent with biological results. Active com-
pounds contain an electrophilic sulfur in a delocalized
environment adjacent to the C-glycosidic bond and have
an energetically favorable conformer around ø ) 0°.
Among these, the more constrained (least flexible)
compounds (tiazofurin and thiophenfurin) are more
active than the less constrained thiophenthiofurin.
Those compounds which contain a nucleophilic oxygen
in place of the thiazole or thiophene sulfur appear less
likely to adopt small angles of ø. These compounds
(oxazofurin, furanfurin, and furanthiofurin) show the
least activity.
1
Compound 7R: Rf 0.28. H NMR (Me2SO-d6): δ 1.30 (t, J )
7.1 Hz, 3H, OCH2CH3); 3.50-3.65 (m, 2H, H4′, H5′b); 3.90 (m,
2H, H5′a, H3′); 4.25 (q, J ) 7.0 Hz, 2H, OCH2CH3); 4.28 (m,
1H, H2′); 4.85 (t, J ) 5.3 Hz, 1H, OH); 5.07 (d, J ) 5.1 Hz,
1H, OH); 5.10 (d, J ) 4.7 Hz, 1H, OH); 5.22 (d, J ) 3.9 Hz,
1H, H1′); 6.68 (d, J ) 2.0 Hz, 1H, H4); 7.70 (d, J ) 2.0 Hz,
1H, H5). Anal. C, H.
Exp er im en ta l Section
1
Compound 7â: Rf 0.27. H NMR (Me2SO-d6): δ 1.32 (t, J )
7.1 Hz, 3H, OCH2CH3); 3.45 (m, 1H, H4′); 3.60 (m, 1H, H5′b);
3.75 (m, 1H, H5′a); 4.18 (m, 1H, H3′); 4.41 (q, J ) 7.0 Hz, 2H,
OCH2CH3); 4.45 (m, 1H, H2′); 4.70 (t, J ) 5.2 Hz, 1H, OH);
5.10 (d, J ) 5.2 Hz, 1H, H1′); 5.13 (d, J ) 4.3 Hz, 1H, OH);
5.20 (d, J ) 7.1 Hz, 1H, OH); 6.66 (d, J ) 2.0 Hz, 1H, H4);
7.70 (d, J ) 2.0 Hz, 1H, H5). Anal. C, H.
Melting points were determined on a Bu¨chi apparatus and
are uncorrected. Elemental analyses were determined on a EA
1108 CHNS-O (Fisons Instruments) analyzer. Ultraviolet
spectra were recorded on an HP 8452 A diode array spectro-
photometer driven by an Olivetti M 24 computer. Nuclear
1
magnetic resonance H spectra were determined at 300 MHz
on a Varian VXR-300 spectrometer; chemical shift values are
reported in δ values (ppm) relative to the internal standard
tetramethylsilane. All exchangeable protons were confirmed
by addition of D2O. Thin-layer chromatography (TLC) was
performed on silica gel 60 F254 plates and RP-18 F254 S plates
(Merck); silica gel 60 (Merck) (70-230 and 230-400 mesh) for
Evaporation of the following fraction of column chromatog-
raphy gave 8â as a white solid (255 mg, 45%). Mp: 113-118
1
°C dec. TLC (CHCl3-MeOH, 90:10): Rf 0.25. H NMR (Me2-
SO-d6): δ 1.27 (t, J ) 7.1 Hz, 3H, OCH2CH3); 3.22 (m, 2H,
H4′, H5′b); 3.40 (m, 1H, H5′a); 3.58 (m, 1H, H3′); 4.05 (m, 1H,
H2′); 4.25 (q, J ) 7.0 Hz, 2H, OCH2CH3); 4.32 (d, J ) 7.4 Hz,

C-Thioribonucleosides Related to Tiazofurin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1269
1
1H, H1′); 5.05 (t, J ) 5.3 Hz, 1H, OH); 5.35 (d, J ) 4.3 Hz,
1H, OH); 5.42 (d, J ) 7.0 Hz, 1H, OH); 6.62 (s, 1H, H4); 8.30
(s, 1H, H2). Anal. C, H.
11500), 244 nm (sh, ꢀ 3600). H NMR (Me2SO-d6): δ 3.20 (m,
1H, H4′); 3.45, 3.55 (2m, 2H, H5′b, H5′a); 3.85 (m, 1H, H3′);
4.08 (m, 1H, H2′); 4.46 (d, J ) 7.8 Hz, 1H, H1′); 5.06 (t, J )
5.4 Hz, 1H, OH); 5.18 (d, J ) 4.5 Hz, 1H, OH); 5.26 (d, J ) 6.6
Hz, 1H, OH); 7.15, 7.75 (2 br s, 2H, NH2); 7.35 (s, 1H, H4);
8.02 (s, 1H, H2). Anal. C, H, N.
As a second fraction a very low amount (<0.5%) of C-2
amides as an R,â mixture was separated.
Eth yl 5-(4-Th io-r-D-r ibofu r a n osyl)fu r a n -3-ca r boxyla te
(8r). The title compound was obtained from 6R (1.5 g, 2.68
mmol) as reported for 8â as a white solid (435 mg, 56%). Mp:
125-127 °C. TLC (CHCl3-MeOH, 90:10): Rf 0.31. 1H NMR
(Me2SO-d6): δ 1.30 (t, J ) 7.1 Hz, 3H, OCH2CH3); 3.45 (m,
2H, H4′, H5′b); 3.82 (m, 2H, H5′a, H3′); 4.15 (m, 1H, H2′); 4.22
(q, J ) 7.0 Hz, 2H, OCH2CH3); 4.58 (d, J ) 3.6 Hz, 1H, H1′);
4.85 (t, J ) 5.3 Hz, 1H, OH); 5.10 (d, J ) 6.9 Hz, 1H, OH);
5.16 (d, J ) 5.0 Hz, 1H, OH); 6.62 (s, 1H, H4); 8.28 (s, 1H,
H2). Anal. C, H.
5-(4-Th io-r-D-r ib ofu r a n osyl)t h iop h en e-3-ca r b oxa m -
id e (14). The title compound was obtained from 13R (600 mg,
1.5 mmol) as reported for 2 as a white solid (230 mg, 57%).
Mp: 137-141 °C. TLC (CHCl3-MeOH, 80:20): Rf 0.11. 1H
NMR (Me2SO-d6): δ 3.50 (m, 2H, H4′, H5′b); 3.80-4.05 (m,
3H, H5′a, H3′, H2′); 4.78 (t, J ) 4.3 Hz, 1H, OH); 4.86 (d, J )
3.3 Hz, 1H, H1′); 5.06 (d, J ) 7.2 Hz, 1H, OH); 5.34 (d, J ) 4.9
Hz, 1H, OH); 7.12, 7.70 (2 br s, 2H, NH2); 7.33 (s, 1H, H4); 8.0
(s, 1H, H2). Anal. C, H, N.
Com p u ta tion a l Stu d y. Point charges and energy profiles
for tiazofurin, thiophenfurin, thiophenthiofurin, oxazofurin,
furanfurin and furanthiofurin were obtained using Gaussian
98.8 Charges using the model fragment shown in Figure 1 were
obtained at the DFT/6-31G**//RHF/3-21G* level of theory and
partitioned using the natural bond orbital (NBO) method
incorporated in Gaussian 98.8,9 The density functional single-
point component of the calculation employed the B3LYP three-
parameter hybrid functional.8,10 Charges shown in Figure 1
were also calculated using a variety of basis sets and partition-
ing methods. While magnitudes of individual charges varied
depending on the basis set and partitioning function used,
signs and relative magnitudes of charges remained consistent
with those shown.
Energy profiles were obtained in a manner similar to that
described previously.2 The starting geometry for each model
placed the 3′-deoxy ribose or thioribofuranose ring in a 3′-endo
conformation, the C-glycosidic torsion angle ø at 0°, and the
2′-hydroxyl oxygen proton trans to H-1′. Energies for each
conformer were obtained for values of ø between ( 180°. In
each case, the value of ø was incremented in 20° steps and
fixed. All remaining geometry variables describing the frag-
ment were then fully optimized. The starting geometry at each
value of ø was the optimized geometry obtained at the previous
value. All geometry optimizations used the analytical gradient
method and reduntant internal coordinates.8 Optimized ge-
ometries and associated SCF energies were obtained using the
3-21G* basis set.8 Thus, each point in Figure 1 represents a
calculation at the RHF/3-21G*//3-21G* level.8
An tip r olifer a tive Assa y. Cytotoxicity of tiazofurin and its
analogues against human myelogenous leukemia K562 cells
growing in logarithmic phase in RPMI 1640 medium contain-
ing 10% fetal bovine serum in an atmosphere of air with 5%
CO2 at 37 °C was examined as cited.6 In short, cell suspensions
(0.2 mL; 1 × 105 cells/mL) were transferred into 96-well plates,
appropriate concentration (0.5-100 µM) of compounds pre-
pared in sterile saline or saline was added, mixed and
incubated under conditions described above for 48 h, and then
an aliquot was counted using a Coulter counter. The percent
inhibition of cell proliferation at 48 h in the presence of
compounds compared to saline was expressed as the inhibition
of cell growth. IC50 concentration is expressed as the drug
concentration required to inhibit 50% of cell proliferation.
5-(4-Th io-â-D-r ibofu r a n osyl)fu r a n -3-ca r boxa m id e (1).
Compound 8â (250 mg, 0.86 mmol) was reacted with 30%
ammonium hydroxide (10 mL) and the mixture was stirred at
room temperature for 8 h. Evaporation of the reaction mixture
gave a solid residue which was purified by chromatography
on silica gel column eluting with CHCl3-MeOH-NH4OH (80:
15:5). Compound 1 was obtained as a white solid (190 mg,
83%). Mp: 131-134 °C. TLC (iPrOH-NH4OH, 80:20): Rf 0.6.
1
UV (MeOH): λmax 208 nm (ꢀ 12500), 262 nm (sh, ꢀ 3100). H
NMR (Me2SO-d6): δ 3.20 (m, 1H, H4′); 3.40, 3.60 (2m, 2H,
H5′b, H5′a); 4.05 (m, 1H, H3′); 4.15 (m, 1H, H2′); 4.30 (d, J )
7.3 Hz, 1H, H1′); 5.05 (t, J ) 5.3 Hz, 1H, OH); 5.10 (d, J ) 4.1
Hz, 1H, OH); 5.25 (d, J ) 7.0 Hz, 1H, OH); 6.67 (s, 1H, H4);
7.12, 7.62 (2 brs, 2H, NH2); 8.10 (s, 1H, H2). Anal. C, H, N.
5-(4-Th io-r-D-r ibofu r a n osyl)fu r a n -3-ca r boxa m id e (9).
The title compound was obtained from 8R (400 mg, 1.38 mmol)
as reported for 1 as a white foam (300 mg, 80%). TLC (iPrOH-
NH4OH, 80:20): Rf 0.57. 1H NMR (Me2SO-d6): δ 3.48 (m, 2H,
H4′, H5′b); 3.85 (m, 2H, H5′a, H3′); 4.15 (t, J ) 3.1 Hz, 1H,
H2′); 4.54 (d, J ) 3.4 Hz, 1H, H1′); 4.85 (brs, 1H, OH); 5.18
(brs, 1H, OH); 6.72 (s, 1H, H4); 7.12, 7.65 (2 brs, 2H, NH2);
8.03 (s, 1H, H2). Anal. C, H, N.
Eth yl 2-(2,3,5-Tr i-O-a cetyl-4-th io-R-D-r ibofu r a n osyl)-
th ioph en e-3-car boxylate (12r), Eth yl 2-(2,3,5-Tr i-O-acetyl-
4-th io-â-D-r ibofu r a n osyl)th iop h en e-3-ca r boxyla te (12â),
Eth yl 5-(2,3,5-Tr i-O-a cetyl-4-th io-â-D-r ibofu r a n osyl)th io-
p h en e-3-ca r boxyla te (13â), a n d Eth yl 5-(2,3,5-Tr i-O-
a ce t yl-4-t h io-r-D-r ib ofu r a n osyl)t h iop h e n e -3-ca r b oxy-
la te (13r). To a cooled solution of ethyl thiophene-3-carboxy-
late (10) (1 g, 6.40 mmol) in 60 mL of dry 1,2-dichloroethane
were added 1,2,3,5-tetra-O-acetyl-4-thio-D-ribofuranose (11)
(2.14 g, 6.40 mmol) and then SnCl4 (1.6 mL, 6.40 mmol). The
mixture was reacted at 0 °C for 0.5 h and then at room
temperature for 24 h. After dilution with H2O, the mixture
was neutralized with NaHCO3 and extracted with CH2Cl2 (50
mL × 3). The organic layers were dried (Na2SO4) and concen-
trated in vacuo to obtained a brown oily residue which was
chromatographed on silica gel column eluting with CHCl3-
hexane (55:45). From the first eluate, a mixture of 12R, 12â
and 13â was obtained (800 mg, 31%). TLC (diethyl ether-
hexane, 70:30): Rf 0.50.
From the last fraction 13R was separated as an oil (650 mg,
26%). TLC (diethyl ether-hexane, 70:30): Rf 0.43. 1H NMR
(CDCl3): δ 1.35 (t, J ) 7.1 Hz, 3H, OCH2CH3); 2.05-2.15 (3s,
9H, COCH3); 4.10 (m, 1H, H4′); 4.15 (m, 1H, H5′b); 4.30 (q, J
) 7.0 Hz, 2H, OCH2CH3); 4.42 (m, 1H, H5′a); 5.10 (d, J ) 3.9
Hz, 1H, H1′); 5.26 (dd, J ) 3.1, 8.9 Hz, 1H, H3′); 5.63 (t, J )
3.6 Hz, 1H, H2′); 7.45 (s, 1H, H4); 8.05 (s, 1H, H2). Anal. C,
H.
5-(4-Th io-â-D-r ib ofu r a n osyl)t h iop h en e-3-ca r b oxa m -
id e (2). The mixture of 12R, 12â, and 13â (700 mg, 1.75 mmol)
was treated with methanolic ammonia (30 mL) at room
temperature. A TLC analysis showed complete consumption
of the starting material after 30 h. Evaporation to dryness of
the reaction mixture gave a yellow oily residue which was
treated with 30% ammonium hydroxide (30 mL) at room
temperature for 24 h. Evaporation of the reaction mixture gave
a solid residue which was chromatographed on silica gel
column eluting with CHCl3-MeOH-NH4OH (82:16:2) to give
2 (300 mg, 63%) as a white solid. Mp: 137-141 °C. TLC
(CHCl3-MeOH, 80:20): Rf 0.13. UV (MeOH): λmax 210 nm (ꢀ
IMP Deh yd r ogen a se Assa y. K562 cells (1 × 106 /mL; 10
mL) in logarithmic phase of growth were incubated with saline
or indicated concentrations of compounds for 2 h at 37 °C. The
cells were harvested by centrifugation, lysed and assayed for
IMPDH activity as described.7 In short, cells were extracted
in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.5%
NP-40, and 2 µg/mL aprotinin, and the supernate after
centrifugation was used as the source of IMPDH. The enzyme
activity was measured according to the cited method.14 Briefly,
5-µL aliquots of 0.5 M KCl containing 20 mM allopurinol were
dispensed into the apex of Eppendorf tubes and dried at 25
°C. For the assay, in a total volume of 10 µL, tubes contained
5 µL of the substrate mixture containing 286 µM [2,8-3H]IMP
(200 µCi/mL) and 1 mM NAD. The reaction was initiated by

1270 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Franchetti et al.
(5) Dukhan, D.; De Valette, F.; Marquet, R.; Ehresman, B.; Ehres-
man C.; Morvan, F.; Barascut, J .-L.; Imbach, J .-L. 4′-Thio-
RNA: Synthesis, Base Pairing Properties and Interaction with
Dimerization Initiation Site of HIV-1. Nucleosides Nucleotides
1999, 18, 1423-1424.
(6) Franchetti, P.; Cappellacci, L.; Abu Sheikha, G.; J ayaram, H.
N.; Gurudutt, V. V.; Sint, T.; Schneider, B. P.; J ones, W. D.;
Goldstein, B. M.; Perra, G.; De Montis A.; Loi, A. G.; La Colla,
P.; Grifantini, M. Synthesis, Structure, and Antiproliferative
Activity of Selenophenfurin, an Inosine 5′-Monophosphate De-
hydrogenase Inhibitor Analogue of Selenazofurin. J . Med. Chem.
1997, 40, 1731-1737.
(7) J ayaram, H. N.; Zhen, W.; Gharehbaghi, K. Biochemical Con-
sequences of Resistance to Tiazofurin in Human Myelogenous
Leukemia K562 Cells. Cancer. Res. 1993, 53, 2344-2348.
(8) (a) Frisch et al. Gaussian 98, revision A.6; Gaussian, Inc.:
Pittsburgh, PA, 1998. (b) Frisch, A. E.; Frisch, M. J . Gaussian
98 users reference, 2nd ed.; Gaussian, Inc.: Pittsburgh, PA, 1998.
(9) Reed, A. E.; Curtiss, L. A.; Weinhold, F. Intermolecular Interac-
tions from a Natural Bond Orbital, Donor-acceptor Viewpoint.
Chem. Rev. 1988, 88, 899-926.
(10) Becke, A. D. Density-functional thermochemistry. III. The Role
of Exact Exchange. J . Chem. Phys. 1993, 98, 5648-5652.
(11) Burling, F. T.; Goldstein, B. M. Computational Studies of
Nonbonded Sulfur-oxygen and Selenium-oxygen Interactions
in the Thiazole and Selenazole Nucleosides. J . Am. Chem. Soc.,
1992, 114, 2313-2320.
the addition of a 5-µL aliquot of enzyme extract and incubated
at 37 °C for 30 min, and the reaction was terminated by
heating at 95 °C for 1 min. Tubes were centrifuged, 5 µL of
100% KOH was deposited on the underside of the cap, and
the tubes were closed and incubated at room temperature
overnight (16 h). The caps were cut, and the radioactivity was
determined by scintillation spectrometry. IMP dehydrogenase
activity was expressed as nmol of XMP formed/mg of protein/
h.
Deter m in a tion of th e Con cen tr a tion of In tr a cellu la r
Ribon u cleotid es. Cells in culture were treated with saline
or compounds at the concentrations specified in Table 1 for 2
h at 37 °C, harvested by centrifugation, and washed once with
cold saline. Cells were extracted with cold 10% trichloroacetic
acid and centrifuged for 0.5 min; the supernatant was im-
mediately neutralized with 0.5 M tri-n-octylamine in Freon.
An aliquot of the neutralized extract was analyzed on HPLC
using a Partisil 10-SAX column as described.15
Ack n ow led gm en t. This work was supported by
Ministero dell′Universita` e della Ricerca Scientifica e
Tecnologica (40% funds) and by CNR, Italy.
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J M990257B