Design and Synthesis of 3′- and 5′ -O-(3-Benzenesulfonylfuroxan-4-yl)-2′-deoxyuridines: Biological Evaluation as Hybrid Nitric Oxide Donor-Nucleoside Anticancer Agents

Article abstract of DOI:10.1021/jm030544m

A group of 3′-O- and 5′ -O-(3-benzenesulfonylfuroxan-4-yl)-2′-deoxyuridines possessing a variety of substituents (H, Me, I, F, CF₃) at the C-5 position of the nucleoside moiety were synthesized for evaluation as hybrid anticancer agents that ha

Full text of DOI:10.1021/jm030544m

1840
J . Med. Chem. 2004, 47, 1840-1846
Design a n d Syn th esis of 3′- a n d
5′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-2′-d eoxyu r id in es: Biologica l Eva lu a tion a s
Hybr id Nitr ic Oxid e Don or -Nu cleosid e An tica n cer Agen ts
Sameh Moharram, Aihua Zhou, Leonard I. Wiebe, and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2N8 Canada
Received October 24, 2003
A group of 3′-O- and 5′-O-(3-benzenesulfonylfuroxan-4-yl)-2′-deoxyuridines possessing a variety
of substituents (H, Me, I, F, CF3) at the C-5 position of the nucleoside moiety were synthesized
for evaluation as hybrid anticancer agents that have the ability to simultaneously release
cytotoxic nitric oxide (‚NO). Incubation of these nitric oxide donor-nucleoside conjugates in
the presence of 18 mM L-cysteine released a high percentage of ‚NO (21-48% at 1 h; 37-86%
at 16 h). The release of ‚NO in the absence of the thiol cofactor was negligible. These hybrid
‚NO donor-nucleosides exhibited high cellular toxicity (CC50 ) 10-6-10-8 M range) against a
battery of tumor cell lines (143B-LTK, 143B, EMT-6, KBALB-STK, and KBALB) and normal
human fibroblasts (Hs578Bst). No differences in cytotoxicity between nontransfected (143B,
KBALB) and the corresponding transfected (143B-LTK, KBALB-STK) cancer cell lines
possessing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK+) were observed,
indicating that expression of the viral TK enzyme did not provide a gene therapeutic effect.
In tr od u ction
Ch em istr y
Selective monoprotection of the 5-substituted 2′-
deoxyuridine derivatives (1a -e, R ) H, Me, I, F, CF3)
by reaction with 1 equiv of tert-butyldimethylsilyl
chloride (TBDMSCl) and 4-(dimethylamino)pyridine
(DMAP) in DMF afforded the respective 5′-O-tert-
butyldimethylsilyl derivatives (2a -e) in 82-93% yields
(see Scheme 1).16,17 Coupling of 2a -e with 3,4-bis-
(benzenesulfonyl)furoxan (8a )18 in the presence of 50%
aqueous NaOH in THF11 furnished the corresponding
3′-O-(3-benzenesulfonylfuroxan-4-yl) derivative (3a -e)
in 79-92% yield. Subsequent removal of the 5′-O-
TBDMS protecting group by treatment with 1 N HCl
in THF gave the respective target 3′-O-(3-benzenesulfo-
nylfuroxan-4-yl)-5-substituted-2′-deoxyuridines (4a -e)
in 88-94% chemical yields.
It was anticipated that application of a similar
methodology for the synthesis of the regioisomeric 5′-
O-(3-benzenesulfonylfuroxan-4-yl)-2′-deoxyuridines (7a-
d ) would require selective protection of the 3′-hydroxyl
group. In this regard, after various trials using different
protection-deprotection strategies, it was found that the
best results were obtained by the protection of both the
3′- and 5′-hydroxyl groups of the parent nucleosides
(1a -e) as the TBDMS ethers. Selective cleavage of the
5′-O-TBDMSO ether protecting group under mild acidic
conditions (80% acetic acid/THF, 4:1, v/v) with a pro-
longed reaction time (up to 3 days) afforded the respec-
tive monoprotected 3′-O-TBDMSO ether (5a -e) in 54-
70% yield. The 3′-O-TBDMSO ethers (5a -d ; R ) H, Me,
I, F) were subsequently converted to the respective 5′-
O-(3-benzenesulfonylfuroxan-4-yl) derivatives (7a -d ) as
illustrated in Scheme 1. In contrast, a similar attempt
to convert the 5-trifluoromethyl compound (5e) to the
corresponding 5′-O-(3-benzensulfonylfuroxan-4-yl) de-
rivative (7e) was unsuccessful because of product de-
composition under these reaction conditions.
Nitric oxide (‚NO)is a mediator of many physiological
and pathological processes. Its in vivo synthesis is
catalyzed by neuronal, inducible, and endothelial iso-
forms of nitric oxide synthase (NOS) using L-arginine
as substrate.1 The biological actions of ‚NO are para-
doxical because it serves important regulatory functions
in addition to its potentially useful cytotoxic effects.2-4
‚NO also possesses vasoactive5,6 and neurotransmitter7,8
properties. The production of ‚NO by macrophages,
Kupfer cells, natural killer T-cells, and endothelial cells
can induce cytotoxicity against a variety of tumors.9,10
It is well documented that the 1,2,5-oxadiazole N-oxide
ring (1,2,5-oxadiazole 2-oxide, furoxan ring) and deriva-
tives thereof release ‚NO in the presence of thiol
cofactors.11 Mechanisms for the release of ‚NO from the
1,2,5-oxadiazole N-oxide ring system have been postu-
lated,12 but the structure of the resulting product(s) is
still unclear. A variety of 1,2,5-oxadiazole N-oxides have
been evaluated as vasodilators to treat cardiovascular
disease.13 Recent studies have shown that certain 1,2,5-
oxadiazole N-oxides possessing a C-3 moiety with a good
leaving group that can be displaced by biological nu-
cleophiles exhibit in vitro cytotoxic activity.14,15 It was
therefore of interest to attach the 3-benzenesulfonyl-
furoxan-4-yl ‚NO donor moiety to the 3′-O- and 5′-O-
positions of 2′-deoxyuridine to determine whether this
new type of hybrid ‚NO donor-nucleoside dual prodrug
combination would result in a synergestic, or additive,
cytotoxic effect. We now report the synthesis, ‚NO
release data, and in vitro cytotoxicity for a group of 3′-
O- (4a -e) and 5′-O-(3-benzenesulfonylfuroxan-4-yl)-2′-
deoxyuridines (7a -d ) possessing a variety of 2′-deoxy-
uridine C-5 substituents (H, Me, I, F, CF3).
* To whom correspondence should be addressed. Phone: 780-492-
5993. Fax: 780-492-1217. E-mail: eknaus@pharmacy.ualberta.ca.
10.1021/jm030544m CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/24/2004

Design and Synthesis of Deoxyuridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1841
Sch em e 1a
a
Reagents and conditions: (i) 1 equiv of TBDMSCl, 3 equiv of imidazole, 0.1 equiv of DMAP in DMF, 25 °C, 1 h; (ii) 1.5 equiv of 8a in
THF, 0 °C, aqueous 50% NaOH solution, 1 h; (iii) 1 N HCl, THF, 25 °C, 3 h; (iv) 2.1 equiv of TBDMSCl, 5 equiv of imidazole, 0.3 equiv
of DMAP in DMF, 25 °C, 1 h; (v) AcOH/THF (4:1, v/v), 60 °C, 3 days.
Ta ble 1. In Vitro Percent Nitric Oxide Release for 3′-O-(3-
Resu lts a n d Discu ssion
Benzenesulfonylfuroxan-4-yl)-5-substituted-2′-deoxyuridines
(4a -e), 5′-O-(3-Benzenesulfonylfuroxan-4-yl)-5-substituted-2′-
The unstable nitric oxide (‚NO) radical is readily
deoxyuridines (7a -d ), 3,4-Bis(benzenesulfonyl)furoxan (8a ),
oxidized to the nitrosonium cation (NO+), which is
and 3-Benzenesulfonyl-4-methoxyfuroxan (8b)
moderately stable in aqueous solutions but highly
% NO releasea
(PBS)
% NO releaseb
(PBS + 18 mM L-cysteine)
reactive with nucleophiles or other nitrogen oxides.
Under physiological conditions, ‚NO is readily oxidized
to nitrite and nitrate or it is trapped by thiols as an
S-nitroso adduct. Under aerobic conditions, these reac-
tive nitrogen oxides can be trapped by various amines,
in particular by aromatic amines to form diazonium
salts or by aromatic 1,2-diamines to form benzotriazoles.
The Griess reagent19,20 provides a simple and well-
characterized colorimetric assay for nitrites, and ni-
trates that have been reduced to nitrites, with a
detection limit of about 100 nM. Nitrites react with
sulfanilic acid in acidic solution to form an intermediate
diazonium salt that couples to N-(1-naphthyl)ethylene-
diamine to yield a purple azo derivative that can be
monitored by absorbance at 548 nm.
The percentages of ‚NO released upon incubation of
the 3′-O- (4a -e) and 5′-O-(3-benzenesulfonylfuroxan-
4-yl)-2′-deoxyuridines (7a -d ) in the absence and pres-
ence of 18 mM L-cysteine (thiol cofactor)21-25 are listed
in Table 1. In this regard, incubation in aqueous
phosphate buffer solution generally released negligible
amounts of ‚NO in the absence of L-cysteine (0-9.9%
at 1-16 h). In contrast, in the presence of 18 mM
L-cysteine as thiol cofactor, the percentage of ‚NO
compd
1 h
16 h
1 h
16 h
4a
4b
4c
4d
4e
7a
7b
7c
0.9 ( 0.5
1.4 ( 0.1
1.2 ( 0.4
0.4 ( 0.1
0.3 ( 0.2
1.5 ( 0.4
2.2 ( 0.2
9.8 ( 1.2
0.8 ( 0.2
0.2 ( 0.1
1.1 ( 0.2
3.5 ( 0.2
0.2 ( 0.1
0.4 ( 0.2
0.4 ( 0.3
0.2 ( 0.2
0.4 ( 0.1
0.2 ( 0.1
2.2 ( 0.1
9.9 ( 0.1
0.7 ( 0.1
0.4 ( 0.1
1.0 ( 0.4
24.0 ( 0.2
45.6 ( 1.2
39.5 ( 0.2
39.8 ( 0.4
41.5 ( 1.9
48.3 ( 1.2
46.0 ( 0.6
20.9 ( 0.8
28.3 ( 0.5
41.6 ( 1.1
22.7 ( 0.4
12.3 ( 0.7
1.9 ( 0.1
85.8 ( 0.6
77.3 ( 1.5
74.8 ( 0.7
73.9 ( 0.5
78.5 ( 1.2
77.9 ( 0.5
36.8 ( 1.4
71.9 ( 0.4
68.7 ( 1.9
38.8 ( 1.6
44.8 ( 0.5
2.6 ( 0.6
7d
8a
8b
ISBNc
a
The percent nitric oxide released from the test compound was
determined as the percent of nitrite (NO2-) produced in phosphate-
buffered saline (PBS) as quantitated using the Griess reagent
((SD, n ) 3). The percent nitric oxide released from the test
compound was determined as the percent of nitrite (NO2
b
-
)
produced in the present of L-cysteine (18 mM) as quantitated using
the Griess reagent. c ISDN ) isosorbide dinitrate (ISDN possesses
two ONO2 groups that may release ‚NO, whereas compounds 4
and 7 possess only one ONO2 group that may release ‚NO).
released was high (21-48% at 1 h and 37-86% at 16
h) relative to the reference compound isosorbide dini-

1842 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Moharram et al.
Ta ble 2. In Vitro Cell Cytotoxicity for 3′-O-(3-Benzenesulfonylfuroxan-4-yl)-5-substituted-2′-deoxyuridines (4a -e),
5′-O-(3-Benzenesulfonylfuroxan-4-yl)-5-substituted-2′-deoxyuridines (7a -d ), 3,4-Bis(benzenesulfonyl)furoxan (8a ), and
3-Benzenesulfonyl-4-methoxyfuroxan (8b)
cellular toxicity (CC50, M) toward various cell linesa
compd
4a
4b
4c
4d
4e
7a
7b
KBALBb
KBALB-STKc
143Bd
143B-LTKc
EMT-6e
Hs578Bstf
7.9 × 10-7
5.6 × 10-7
3.8 × 10-7
2.0 × 10-6
3.2 × 10-6
4.6 × 10-6
2.8 × 10-6
9.2 × 10-7
4.8 × 10-8
5.2 × 10-7
4.8 × 10-7
9.7 × 10-5
6.0 × 10-11
9.5 × 10-5
9.7 × 10-4
1.7 × 10-6
1.0 × 10-6
0.4 × 10-6
8.4 × 10-7
2.0 × 10-6
5.4 × 10-6
4.8 × 10-6
9.2 × 10-7
6.0 × 10-8
6.0 × 10-7
4.4 × 10-7
1.0 × 10-5
8.8 × 10-11
1.0 × 10-4
6.5 × 10-4
9.6 × 10-7
7.2 × 10-7
5.6 × 10-7
2.4 × 10-7
5.2 × 10-7
3.3 × 10-6
3.6 × 10-6
5.4 × 10-7
3.2 × 10-6
4.0 × 10-8
7.0 × 10-8
7.0 × 10-3
9.0 × 10-5
2.9 × 10-6
8.8 × 10-7
1.3 × 10-6
8.4 × 10-7
4.8 × 10-7
6.3 × 10-6
5.6 × 10-6
7.1 × 10-7
2.0 × 10-6
2.8 × 10-8
3.2 × 10-7
7.4 × 10-3
1.0 × 10-4
8.0 × 10-8
2.4 × 10-6
8.0 × 10-8
2.0 × 10-6
8.8 × 10-7
7.5 × 10-7
2.0 × 10-6
1.0 × 10-6
6.0 × 10-8
1.0 × 10-7
5.6 × 10-7
3.8 × 10-4
9.0 × 10-12
1.3 × 10-4
4.8 × 10-6
2.0 × 10-6
0.8 × 10-6
4.4 × 10-6
4.4 × 10-6
4.8 × 10-6
6.8 × 10-6
8.0 × 10-7
7.6 × 10-8
1.0 × 10-7
6.8 × 10-7
7c
7d
8a
8b
IUDRg
FUDRh
thymidine
ISDNi
6.0 × 10-4
7.0 × 10-4
a
The molar concentration of the test compound that killed 50% of the cells (or 50% cell survival) upon incubation for 3-5 days at 37
°C in a humidified atmosphere of 95% air and 5% CO2 (mean value, n ) 6) was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay. b Transformed fibroblast sarcoma cell line. c These cells were transfected by, and expressed,
the herpes simplex virus type 1 thymidine kinase (HSV-1 TK) gene. Human osteosarcoma cell line. e Mouse mammary carcinoma cell
d
line. f Human fibroblast cell lines. IUDR ) 5-iodo-2′-deoxyuridine. FUDR ) 5-fluoro-2′-deoxyuridine. Isosorbide dinitrate.
g
h
i
trate (1.9% and 2.6% release at 1 and 16 h) that
possesses two ONO2 ‚NO donor moieties. These ‚NO
release data are consistent with reports that buffers
containing reduced thiols such as L-cysteine or glu-
tathione, which serve as a source of thiols equivalent
to the mercapto groups present in plasma, are required
for the release of ‚NO from a furoxan moiety.6,22
Compounds 4a -e and 7a -d (a , R ) H; b, R ) Me; c,
R ) I; d , R ) F; e, R ) CF3) and the reference
compounds IUDR, FUDR, and thymidine were evalu-
ated for their tumor cell cytotoxicity against a battery
of cancer cell lines (KBALB, KBALB-STK, 143B, 143B-
LTK, EMT-6) using the MTT cytotoxicity assay26 (Table
2). This group of furoxanyl deoxynucleosides generally
exhibited more potent cytotoxicities (CC50 ) 10-6-10-8
cysteine (23% in 1 h; 39% in 16 h) and that 8a was
highly cytotoxic (CC50 ) 10-7-10-8 M range). Similarly,
3-benzenesulfonyl-4-methoxyfuroxan (8b), which is con-
sidered to be a very close structural mimic of the
furoxany-4-yl moiety present in compounds 4a -e and
7a -d , was also highly cytotoxic (CC50 ) 10-7-10-8
M
range). A plausible explanation for these results is that
the high cytotoxicity exhibited by the hybrid nucleo-
side-‚NO donor compounds 4 and 7 is due predomi-
nantly to the furoxan moiety, with a minimal synergistic
or additive contribution from the nucleoside component.
It is expected that the furoxans 8a and 8b exert their
cytotoxic effect by an alternative mechanism such as
DNA alkylation, since 8a ,b cannot serve as nucleotide
substrates for DNA polymerase catalyzed incorporation
into DNA. In this regard, covalent alkylation by the N7-
or O6-atom of a guanine base in DNA could result in
the covalent attachment of DNA to a furoxan-4-yl ring
carbon by nucleophilic displacement of the 3-benzene-
sulfonyl moiety present in compounds 4a -e, 7a -d , and
8a ,b. To obtain further evidence to support this postu-
late, attempts were made to isolate the byproduct(s)
produced after ‚NO release. Accordingly, when com-
pound 4a was incubated with 18 mM L-cysteine in
phosphate buffer for 16 h, thin-layer chromatography
(TLC) showed complete disappearance of 4a and the
appearance of a more polar compound with a different
TLC Rf value than the parent nucleoside 2′-deoxyuri-
dine. Several attempts to isolate this incubation product
were unsuccessful because a progressive decomposition
occurred during both the isolation and attempts to
purify the product. Irrespective of the mechanism(s) by
which ‚NO is released from the furoxanyl ring system,
the carbon component of the furoxan ring system would
be retained at the C-3′ (4) or C-5′ (7) position of the
nucleoside. The product(s) produced after elimination
of ‚NO likely exhibits negligible cytotoxic activity be-
cause 3-benzenesulfonyl-4-methoxyfuroxan (8b) exhibits
cytotoxic activity comparable to that of the nucleoside
analogues 4 and 7. The 3′-O- (4a -d ) and 5′-O-(3-
benzenesulfonylfuroxan-4-yl) (7a -d ) compounds are
M range) than the reference compounds IUDR (CC50
)
M
10-3-10-5 M range), FUDR (CC50 ) 10-4-10-12
range), and thymidine (CC50 ) 10-4-10-5 M range). The
only exceptions to this general observation was FUDR,
which exhibited a greater cytotoxicity against KBALB,
KBALB-STK, and EMT-6 cells than the furoxanyl
compounds 4 and 7. It is anticipated that the potent
cytotoxicities exhibited by the 5-substituted compounds
4a (R ) H) and 4b (R ) Me) are predominantly, or
exclusively, due to the furoxanyl moiety because the 2′-
deoxyuridine (4a ) and thymidine (4b) nucleoside moi-
eties are expected to make little, or no, contribution to
the overall cytotoxic effect. The observation that com-
pounds 4a -e and 7a -d exhibited similar cytotoxicity
against nontransfected (KBALB, 143B) and the corre-
sponding transfected (KBALB-STK, 143B-LTK) cancer
cell lines possessing the herpes simplex virus type 1
(HSV-1) thymidine kinase gene (TK+) indicates that
expression of the viral TK enzyme did not provide a gene
therapeutic effect.
The high amount of ‚NO release, and potent cytotox-
icities exhibited, by these furoxanyl deoxynucleosides
prompted us to further investigate the role of the
furoxan moiety with respect to the cytotoxic effect. It
was found that 3,4-bis(benzenesulfonyl)furoxan (8a )
released ‚NO readily in the presence of 18 mM L-

Design and Synthesis of Deoxyuridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1843
also cytotoxic (CC50 ) 10-6-10-8 M range) against
human fibroblast cells (Hs578Bst cell line). This obser-
vation indicates that any drug design study incorporat-
ing a 3-benzenesulfonylfuroxan moiety should examine
their toxicity against normal cells.
9.70 (br s, 1H, NH), 7.54 (s, 1H, H-6), 6.43 (dd, J ) 8.1, 5.5
Hz, 1H, H-1′), 4.43-4.45 (m, 1H, H-3′), 4.01 (d, J ) 2.2 Hz,
1H, H-4′), 3.84 (dd, J ) 11.4, 2.2 Hz, 1H, H-5′), 3.79 (dd, J )
11.4, 2.2 Hz, 1H, H-5′), 2.34-2.44 and 2.06-2.16 (two m, 1H
each, H-2′), 1.85 (s, 3H, CH3), 0.86 (s, 9H, t-Bu), 0.11 (s, 3H,
SiCH3), 0.10 (s, 3H, SiCH3); 13C NMR (CDCl3) δ 163.93 (C-4
CdO), 150.59 (C-2 CdO), 135.44 (C-6), 110.90 (C-5), 87.36 (C-
4′), 85.06 (C-1′), 72.48 (C-3′), 63.62 (C-5′), 41.12 (C-2′), 25.94
[C(CH3)3], 18.36 [C(CH3)3], 12.53 (C-5 CH3), -5.38 [d, J Si,CH3
) 6.6 Hz, Si(CH3)2]. Anal. (C16H28N2O5Si) C, H, N.
5′-O-(ter t-Bu t yld im et h ylsilyl)-5-iod o-2′-d eoxyu r id in e
(2c): white solid; yield, 89%; mp 218-219 °C; 1H NMR (CDCl3)
δ 10.66 (s, 1H, NH), 8.02 (s, 1H, H-5), 6.22 (dd, J ) 8.1, 5.9
Hz, 1H, H-1′), 4.45 (br d, J ) 3.3 Hz, 1H, OH), 4.20-4.30 (m,
1H, H-3′), 3.95-3.99 (m, 1H, H-4′), 3.79 (dd, J ) 11.4, 2.2 Hz,
1H, H-5′), 3.70 (dd, J ) 11.4, 2.2 Hz, 1H, H-5′), 2.24-2.34 and
1.88-2.00 (two m, 1H each, H-2′), 0.84 (s, 9H, t-Bu), 0.06 (s,
3H, SiCH3), 0.04 (s, 3H, SiCH3); 13C NMR (CDCl3) δ 160.28
(C-4 CdO), 149.89 (C-2 CdO), 144.03 (C-6), 87.68 (C-4′), 85.56
(C-1′), 71.70 (C-3′), 68.33 (C-5), 63.37 (C-5′), 41.47 (C-2′), 25.96
[C(CH3)3], 18.26 [C(CH3)3], -5.35 [d, J Si,CH3 ) 10.9 Hz, Si-
(CH3)2]. Anal. (C15H25IN2O5Si) C, H, N.
Con clu sion s
A group of 3′- (4) and 5′-O-(3-benzenesulfonylfuroxan-
4-yl)-2′-deoxyuridines (7) were designed for evaluation
as hybrid nucleoside-‚NO donor conjugates. Biological
evaluation showed that these compounds are effective
‚NO donor agents in the presence of 18 mM L-cysteine
and exhibit cytotoxic activity against a battery of cancer
cell lines (KBALB, KBALB-STK, 143B, 143B-LTK,
EMT-6) as well as a contraindicated cytotoxic effect
against normal nonmalignant human fibroblast cells.
The observation that the non-nucleoside ‚NO donor (8b)
exhibits cytotoxicity similar to that of the 3′-O- and 5′-
O-furoxan-4-yl deoxynucleosides (4 and 7) suggests that
cytoxicity is due predominantly to the effect of the
3-benzenesulfonylfuroxan-4-yl moiety that may alkylate
DNA.
5′-O-(t er t -Bu t yld im e t h ylsilyl)-5-flu or o-2′-d e oxyu r i-
1
d in e (2d ): white solid; yield, 82%; mp 201-202 °C; H NMR
(CD3OD) δ 8.10 (d, J ) 6.2 Hz, 1H, H-6), 7.8 (br s, 1H, NH),
6.37 (dd, J ) 7.2, 5.9 Hz, 1H, H-1′), 4.43-4.58 (m, 1H, H-3′),
4.08-4.12 (m, 1H, H-4′), 3.93 (dd, J ) 11.5, 1.7 Hz, 1H, H-5′),
3.82 (dd, J ) 11.5, 1.7 Hz, 1H, H-5′), 2.40-2.50 and 2.00-
2.16 (two m, 1H each, H-2′), 0.90 (s, 9H, t-Bu), 0.11 (s, 3H,
SiCH3), 0.10 (s, 3H, SiCH3); 13C NMR (CD3OD) δ 157.47 (d,
J CCF ) 26.4 Hz, C-4 CdO), 149.34 (C-2 CdO), 140.53 (d, J CF
) 236.2 Hz, C-5), 124.43 (d, J CCF ) 39.5 Hz, C-6), 87.87 (C-4′),
85.73 (C-1′), 72.03 (C-3′), 63.48 (C-5′), 41.32 (C-2′), 25.86
Exp er im en ta l Section
Gen er a l. Melting points were determined with a Thomas-
Hoover capillary apparatus and are uncorrected. Nuclear
magnetic resonance (1H NMR, 13C NMR) spectra were recorded
on a Bruker AM-300 spectrometer. Proton chemical shifts (δ)
are given relative to internal TMS (δ 0), and the assignment
of exchangeable protons (NH, OH) was confirmed by addition
of D2O. 13C NMR spectra were acquired using the J modulated
spin-echo technique where methyl and methine carbon reso-
nances appear as positive peaks and methylene and quater-
nary carbon resonances appear as negative peaks. Carbon
chemical shifts (δ) are given relative to CDCl3 (δ 77). Elemental
analyses were performed by the MicroAnalysis Service Labo-
ratory, Department of Chemistry, University of Alberta, and
the results were within (0.4% of theoretical values for all
elements listed. Silica gel 60A (Silicycle Co., 230-400 mesh)
was used for all flash column chromatography separations. 3,4-
Bis(benzenesulfonyl)furoxan (8a )18 and 3-benzenesulfonyl-4-
methoxyfuroxan (8b)11 were prepared according to literature
procedures. All other reagents were purchased from Aldrich
Chemical (Milwaukee, WI).
[C(CH3)3], 18.35 [C(CH3)3], -5.60 [Si(CH3)2]. Anal. (C15H25
FN2O5Si) C, H, N.
-
5′-O-(ter t-Bu tyld im eth ylsilyl)-5-tr iflu or om eth yl-2′-d e-
oxyu r id in e (2e): white solid; yield, 93%; mp 105-107 °C; 1H
NMR (CD3OD) δ 8.07 (s, 1H, H-6), 6.14 (dd, J ) 7.3, 5.9 Hz,
1H, H-1′), 4.31-4.37 (m, 1H, H-3′), 4.03-4.09 (m, 1H, H-4′),
3.90 (dd, J ) 11.7, 2.2 Hz, 1H, H-5′), 3.84 (dd, J ) 11.7, 2.2
Hz, 1H, H-5′), 2.43 (ddd, J ) 13.5, 5.9, 2.2 Hz, 1H, H-2′), 2.13
(ddd, J ) 13.5, 7.3, 2.2 Hz, 1H, H-2′), 0.89 (s, 9H, t-Bu), 0.09
[s, 6H, Si(CH3)2]; 13C NMR (CD3OD) δ 160.94 (C-4 CdO),
151.03 (C-2 CdO), 142.55 (q, J CCCF ) 3.3 Hz, C-6), 123.78 (q,
J CF ) 268.1 Hz, CF3), 105.09 (q, J CCF ) 33.0 Hz, C-5), 89.82
(C-4′), 88.31 (C-1′), 72.84 (C-3′), 64.60 (C-5′), 42.62 (C-2′), 26.44
[C(CH3)3], 19.28 [C(CH3)3], -5.42 (d, J Si,CH3 ) 5.5 Hz, Si(CH3)2].
Anal. (C16H25F3N2O5Si) C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of 5′-O-(ter t-
Bu tyld im eth ylsilyl)-5-su bstitu ted -2′-d eoxyu r id in es (2a -
e). To a stirred solution of a compound selected from the group
1a -e (1 mmol), imidazole (206 mg, 3 mmol), and DMAP (12.2
mg, 0.1 mmol) in DMF (5 mL) was added TBDMSCl (158 mg,
1.05 mmol) at 0 °C, and the mixture was allowed to warm to
room temperature and stirred for 1 h. The reaction mixture
was quenched using a saturated aqueous solution of am-
monium chloride (10 mL) and extracted with ethyl acetate (3
× 5 mL). The combined organic extracts were dried (Na2SO4)
and concentrated in vacuo to give an oil that was purified by
flash chromatography using EtOAc/hexane (3:1, v/v) as eluant
to give the respective title product listed below.
5′-O-(ter t-Bu tyldim eth ylsilyl)-2′-deoxyu r idin e (2a): white
solid; yield, 90%; mp 127-129 °C; 1H NMR (CDCl3) δ 9.70 (br
s, 1H, NH), 7.94 (d, J ) 8.2 Hz, 1H, H-6), 6.37 (t, J ) 6.4 Hz,
1H, H-1′), 5.7 (d, J ) 8.2 Hz, 1H, H-5), 4.46-4.50 (m, 1H, H-3′),
4.03-4.10 (m, 1H, H-4′), 3.89 (dd, J ) 11.9, 1.8 Hz, 1H, H-5′),
3.84 (dd, J ) 11.9, 1.8 Hz, 1H, H-5′), 2.38-2.48 and 2.06-
2.16 (two m, 1H each, H-2′), 0.90 (s, 9H, t-Bu), 0.09 [s, 6H,
Si(CH3)2]; 13C NMR (CDCl3) δ 163.57 (C-4 CdO), 150.45 (C-2
CdO), 140.26 (C-6), 102.23 (C-5), 87.43 (C-4′), 85.37 (C-1′),
71.94 (C-3′), 63.30 (C-5′), 41.50 (C-2′), 25.88 [C(CH3)3], 18.34
Gen er a l Meth od for th e P r ep a r a tion of 3′-O-(3-Ben -
zen esu lfon ylfu r oxa n -4-yl)-5′-O-(ter t-bu tyld im eth ylsilyl)-
5-su bstitu ted -2′-d eoxyu r id in es (3a -e). To a stirred solu-
tion of the compound selected from the group 2a -e (1 mmol)
and 8a (550 mg, 1.5 mmol) in THF (5 mL) at 0 °C was added
a 50% aqueous sodium hydroxide solution (3 mmol). The
mixture was stirred for 1 h at 25 °C, diluted with water (5
mL), and extracted with ethyl acetate (3 × 5 mL). The
combined organic extracts were dried (Na2SO4), and the
solvent was removed in vacuo to give an oil that was purified
by flash chromatography using EtOAc/hexane (1:1, v/v) as
eluant to give the respective title product listed below.
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5′-O-(ter t-bu tyl-
d im eth ylsilyl)-2′-d eoxyu r id in e (3a ): white solid; yield, 82%;
mp 188-190 °C; 1H NMR (CDCl3) δ 8.90 (br s, 1H, NH), 8.08
(d, J ) 7.7 Hz, 2H, o-Ph), 7.88 (d, J ) 8.1 Hz, 1H, H-6), 7.80
(t, J ) 7.7 Hz, 1H, p-Ph), 7.68 (t, J ) 7.7 Hz, 2H, m-Ph), 6.42
(dd, J ) 8.8, 5.5 Hz, 1H, H-1′), 5.74 (d, J ) 8.1 Hz, 1H, H-5),
5.35-5.41 (m, 1H, H-3′), 4.40-4.43 (m, 1H, H-4′), 3.90-3.98
(m, 2H, H-5′), 2.75 (dd, J ) 14.6, 5.5 Hz, 1H, H-2′), 2.28 (ddd,
J ) 14.6, 8.8, 6.2 Hz, 1H, H-2′), 0.92 (s, 9H, t-Bu), 0.13 [s, 6H,
Si(CH3)2]. Anal. (C23H30N4O9SSi) C, H, N, S.
[C(CH3)3], -5.50 [d, J Si,CH3 ) 6.6 Hz, Si(CH3)2]. Anal. (C15H26
-
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5′-O-(ter t-bu tyl-
d im eth ylsilyl)-2′-d eoxyth ym id in e (3b): white solid; yield,
86%; mp 146-148 °C; 1H NMR (CDCl3) δ 9.37 (br s, 1H, NH),
8.08 (d, J ) 7.3, 2H, o-Ph), 7.80 (t, J ) 7.3 Hz, 1H, p-Ph), 7.68
N2O5Si) C, H, N.
5′-O-(ter t-Bu tyld im eth ylsilyl)-2′-d eoxyth ym id in e (2b):
1
white solid; yield, 84%; mp 198-200 °C; H NMR (CDCl3) δ

1844 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Moharram et al.
(t, J ) 7.3, 2H, m-Ph), 7.51 (s, 1H, H-6), 6.38 (dd, J ) 9.2, 5.2
Hz, 1H, H-1′), 5.34-5.40 (m, 1H, H-3′), 4.34-4.39 (m, 1H,
H-4′), 3.90-4.00 (m, 2H, H-5′), 2.67 (dd, J ) 14.0, 5.2 Hz, 1H,
H-2′), 2.24 (ddd, J ) 14.0, 9.2, 5.5 Hz, 1H, H-2′), 1.92 (s, 3H,
C-5 CH3), 0.92 (s, 9H, t-Bu), 0.134 (s, 3H, SiCH3), 0.130 (s,
3H, SiCH3). Anal. (C24H32N4O9SSi) C, H, N, S.
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5-iod o-2′-d eoxy-
u r id in e (4c): white solid; yield, 89%; mp 140-141 °C; 1H NMR
(CDCl3) δ 11.37 (s, 1H, NH), 8.42 (s, 1H, H-6), 7.98 (d, J )
7.3, 2H, o-Ph), 7.71 (t, J ) 7.3 Hz, 1H, p-Ph), 7.62 (t, J ) 7.3,
2H, m-Ph), 6.27 (dd, J ) 8.8, 5.5 Hz, 1H, H-1′), 5.40-5.45 (m,
1H, H-3′), 4.25-4.33 (m, 1H, H-4′), 3.72-3.82 (m, 2H, H-5′),
2.54 (dd, J ) 14.6, 5.5 Hz, 1H, H-2′), 2.35 (ddd, J ) 14.6, 8.8,
5.5 Hz, 1H, H-2′). Anal. (C17H15IN4O9S) C, H, N, S.
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5′-O-(ter t-bu tyl-
d im eth ylsilyl)-5-iod o-2′-d eoxyu r id in e (3c): viscous pale-
yellow oil; yield, 79%; 1H NMR (CDCl3) δ 8.02 (d, J ) 7.6 Hz,
2H, o-Ph), 7.91 (s, 1H, H-6), 7.74 (t, J ) 7.6 Hz, 1H, p-Ph),
7.62 (t, J ) 7.6 Hz, 2H, m-Ph), 6.34 (t, J ) 4.9 Hz, 1H, H-1′),
5.28-5.38 (m, 1H, H-3′), 4.32-4.40 (m, 1H, H-4′), 3.88-3.98
(m, 2H, H-5′), 2.65-2.80 (m, 1H, H-2′), 2.00-2.20 (m, 1H, H-2′),
0.92 (s, 9H, t-Bu), 0.14 (s, 3H, SiCH3), 0.13 (s, 3H, SiCH3). Anal.
(C23H29IN4O9SSi) C, H, N, S.
3′-O-(3-Ben zen esu lfon ylfu r oxan -4-yl)]-5-flu or o-2′-deoxy-
u r id in e (4d ): white solid; yield, 90%; mp 206-208 °C; 1H
NMR (CDCl3) δ 11.92 (d, J ) 4.7 Hz, 1H, NH), 7.73-8.24 (m,
6H, H-6, C6H5), 6.20 (dd, J ) 8.7, 5.8 Hz, 1H, H-1′), 5.37-5.45
(m, 1H, H-3′), 4.26-4.50 (m, 1H, H-4′), 3.62-3.77 (m, 2H, H-5′),
2.30-2.66 (m, 2H, H-2′); 13C NMR (CDCl3) δ 157.70 (furoxan
C-4), 156.83 (d, J CCF ) 26.4 Hz, C-4 CdO), 148.92 (C-2 CdO),
140.00 (d, J CF ) 230.7 Hz, C-5), 136.93 (phenyl C-1), 136.05
(phenyl C-4), 129.85 and 128.51 (phenyl C-2, C-3), 123.60 (J CCF
) 39.5 Hz, C-6), 110.71 (furoxan C-3), 84.29, 84.12, and 82.69
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5′-O-(ter t-bu tyl-
d im eth ylsilyl)-5-flu or o-2′-d eoxyu r id in e (3d ): white solid;
1
yield, 87%; mp 194-195 °C; H NMR (CDCl3) δ 11.98 (d, J )
(C-4′, C-3′, C-1′), 61.10 (C-5′), 36.53 (C-2′). Anal. (C17H15
-
4.8 Hz, 1H, NH), 7.73-8.05 (m, 6H, C6H5, H-6), 6.18 (t, J )
5.9 Hz, 1H, H-1′), 5.30-5.37 (m, 1H, H-3′), 4.35-4.42 (m, 1H,
H-4′), 3.90 (dd, J ) 11.7, 2.9 Hz, 1H, H-5′), 3.81 (dd, J ) 11.7,
2.9 Hz, 1H, H-5′), 2.64 (dd, J ) 14.3, 5.9 Hz, 1H, H-2′), 2.32
(ddd, J ) 14.3, 5.9, 5.5 Hz, 1H, H-2′), 0.86 (s, 9H, t-Bu), 0.08
[s, 6H, Si(CH3)2]. Anal. (C23H29FN4O9SSi) C, H, N, S.
FN4O9S) C, H, N, S.
3′-O-(3-Ben zen esu lfon ylfu r oxan -4-yl)-5-tr iflu or om eth yl-
2′-d eoxyu r id in e (4e): white solid; yield, 88%; mp 115-116
°C; 1H NMR (CD3OD) δ 8.76 (s, 1H, H-6), 8.07 (d, J ) 7.6 Hz,
2H, o-Ph), 7.83 (t, J ) 7.6 Hz, 1H, p-Ph), 7.70 (t, J ) 7.6 Hz,
2H, m-Ph), 6.34 (dd, J ) 8.4, 5.9 Hz, 1H, H-1′), 5.48-5.54 (m,
1H, H-3′), 4.40-4.45 (m, 1H, H-4′), 3.83-3.88 (m, 2H, H-5′),
2.71 (dd, J ) 14.3, 5.9 Hz, 1H, H-2′), 2.47 (ddd, J ) 14.3, 8.4,
5.5 Hz, 1H, H-2′). Anal. (C18H15F3N4O9S) C, H, N, S.
Gen er a l Meth od for th e P r ep a r a tion of 3′-O-(ter t-
Bu tyld im eth ylsilyl)-5-su bstitu ted -2′-d eoxyu r id in es (5a -
e). To a stirred solution of the compound selected from the
group 1a -e (1 mmol), imidazole (344 mg, 5 mmol), and DMAP
(37 mg, 0.3 mmol) in DMF (10 mL) was added TBDMSCl (330
mg, 2.2 mmol) at 0 °C, and the mixture was allowed to warm
to 25 °C and stirred for 1 h. The reaction was quenched using
a saturated aqueous solution of ammonium chloride, and the
mixture was extracted with ethyl acetate (3 × 5 mL). The
combined organic extracts were dried (Na2SO4), and the
solvent was removed in vacuo to give a viscous oil. The crude
oil was dissolved in THF (15 mL), 80% acetic acid in THF (4:
1, v/v) was added, and the mixture was stirred at 60 °C for 3
days. The mixture was extracted with ethyl acetate (3 × 5 mL),
the combined organic extracts were dried (Na2SO4), and the
solvent was removed in vacuo to afford an oil that was purified
by flash chromatography with EtOAc/hexane (3:1, v/v) as
eluant to give the respective title product listed below.
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5′-O-(ter t-bu tyl-
d im et h ylsilyl)-5-t r iflu or om et h yl-2′-d eoxyu r id in e (3e):
1
white solid; yield, 92%; mp 225-226 °C; H NMR (CDCl3) δ
8.25 (s, 1H, H-6), 8.04 (d, J ) 7.9 Hz, 2H, o-Ph), 7.82 (t, J )
7.9 Hz, 1H, p-Ph), 7.68 (t, J ) 7.9 Hz, 2H, m-Ph), 6.20 (dd, J
) 8.4, 5.9 Hz, 1H, H-1′), 5.40-5.46 (m, 1H, H-3′), 4.46-4.52
(m, 1H, H-4′), 3.92-3.99 (m, 2H, H-5′), 2.82 (dd, J ) 14.3, 5.9
Hz, 1H, H-2′), 2.32 (ddd, J ) 14.3, 8.4, 5.5 Hz, 1H, H-2′), 0.88
(s, 9H, t-Bu), 0.10 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3). Anal.
(C24H29F3N4O9SSi) C, H, N, S.
Gen er a l Meth od for th e P r ep a r a tion of 3′-O-(3-Ben -
z e n e s u lfo n y lfu r o x a n -4-y l)-5-s u b s t i t u t e d -2′-d e o x y -
u r id in es (4a -e). To a stirred solution of a compound selected
from the group 3a -e (1 mmol) in THF (10 mL) was added 1
N HCl solution (3 mL), and the mixture was stirred at room
temperature for 3 h. The mixture was extracted with ethyl
acetate (3 × 5 mL), the combined organic extracts were dried
(Na2SO4), and the solvent was removed in vacuo to afford an
oil that was purified by flash chromatography using EtOAc/
hexane (3:1, v/v) as eluant to give the respective title product
listed below.
3′-O-(3-Be n ze n e su lfon ylfu r oxa n -4-yl)-2′-d e oxyu r i-
d in e (4a ): white solid; yield, 94%; mp 206-207 °C; H NMR
3′-O-(ter t-Bu tyldim eth ylsilyl)-2′-deoxyu r idin e (5a): white
solid; yield, 62%; mp 205-207 °C; 1H NMR (CDCl3) δ 9.20 (br
s, 1H, NH), 7.57 (d, J ) 8.1 Hz, 1H, H-6), 6.18 (t, J ) 6.6 Hz,
1H, H-1′), 5.73 (d, J ) 8.1 Hz, 1H, H-5), 4.49 (dd, J ) 8.8, 4.8
Hz, 1H, H-3′), 3.94-3.91 (m, 2H, H-4′, H-5′), 3.73-3.77 (m,
1H, H-5′), 2.70 (br s, 1H, OH), 2.29 (t, J ) 6.6 Hz, 2H, H-2′),
0.89 (s, 9H, t-Bu), 0.13 [s, 6H, Si(CH3)2]; 13C NMR (CDCl3) δ
163.29 (C-4 CdO), 150.19 (C-2 CdO), 140.98 (C-6), 102.40 (C-
5), 87.59 (C-4′), 86.63 (C-1′), 71.40 (C-3′), 61.81 (C-5′), 40.93
(C-2′), 25.73 [C(CH3)3], 17.99 [C(CH3)3], -4.72 [d, J Si,CH3 ) 12.1
Hz, Si(CH3)2]. Anal. (C15H26N2O5Si) C, H, N.
3′-O-(ter t-Bu tyld im eth ylsilyl)-2′-d eoxyth ym id in e (5b):
white solid; yield, 67%; mp 93-94 °C; 1H NMR (CDCl3) δ 8.60
(br s, 1H, NH), 7.36 (s, 1H, H-6), 6.13 (t, J ) 6.9 Hz, 1H, H-1′),
4.5 (dt, J ) 6.9, 3.7 Hz, 1H, H-3′), 3.90-3.94 (m, 2H, H-4′,
H-5′), 3.73-3.79 (m, 1H, H-5′), 2.60 (br s, 1H, OH), 2.37 (dd,
J ) 13.5, 6.9 Hz, 1H, H-2′), 2.25 (ddd, J ) 13.5, 6.9, 3.7 Hz,
1H, H-2′), 1.93 (s, 3H, C-5 CH3), 0.90 (s, 9H, t-Bu), 0.09 [s,
6H, Si(CH3)2]; 13C NMR (CDCl3) δ 163.57 (C-4 CdO), 150.11
(C-2 CdO), 136.73 (C-6), 110.73 (C-5), 87.36 (C-4′), 86.59 (C-
1′), 71.36 (C-3′), 61.75 (C-5′), 40.32 (C-2′), 25.54 [C(CH3)3], 17.78
[C(CH3)3], 12.31 (C-5 CH3), -4.92 [d, J Si,CH3 ) 12.1 Hz,
Si(CH3)2]. Anal. (C16H28N2O5Si) C, H, N.
1
(DMSO-d6) δ 11.40 (s, 1H, NH), 8.00 (d, J ) 7.7 Hz, 2H, o-Ph),
7.90 (t, J ) 7.7 Hz, 1H, p-Ph), 7.88 (d, J ) 8.0 Hz, 1H, H-6),
7.75 (t, J ) 7.7 Hz, 2H, m-Ph), 6.21 (dd, J ) 8.8, 5.9 Hz, 1H,
H-1′), 5.69 (d, J ) 8.0 Hz, 1H, H-5), 5.38-5.43 (m, 1H, H-3′),
5.32 (t, J ) 5.1 Hz, 1H, OH), 4.22-4.31 (m, 1H, H-4′), 3.36-
3.51 (m, 2H, H-5′), 2.56 (dd, J ) 14.6, 5.9 Hz, 1H, H-2′), 2.37
(ddd, J ) 14.6, 8.8, 5.1 Hz, 1H, H-2′); 13C NMR (DMSO-d6) δ
163.03 (C-4 CdO), 157.80 (furoxan C-4), 150.42 (C-2 CdO),
140.19 (C-6), 136.97 (phenyl C-1), 136.21 (phenyl C-4), 130.00
and 128.47 (phenyl C-2, C-3), 110.81 (furoxan C-3), 102.26 (C-
5), 84.05 and 82.81 (C-4′, C3′, C-1′), 61.20 (C-5′), 36.54 (C-2′).
Anal. (C17H16N4O9S) C, H, N, S.
3′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-2′-d eoxyt h ym i-
1
d in e (4b): white solid; yield, 91%; mp 138-139 °C; H NMR
(DMSO-d6) δ 8.88 (br s, 1H, NH), 8.07 (d, J ) 7.9 Hz, 2H, o-Ph),
7.80 (t, J ) 7.6 Hz, 1H, p-Ph), 7.67 (t, J ) 7.6 Hz, 2H, m-Ph),
7.46 (s, 1H, H-6), 6.21 (dd, J ) 8.5, 5.8 Hz, 1H, H-1′), 5.52-
5.58 (m, 1H, H-3′), 4.37-4.42 (m, 1H, H-4′), 3.90-4.05 (m, 2H,
H-5′), 2.9 (br s, 1H, OH), 2.73 (ddd, J ) 14.6, 8.5, 5.8 Hz, 1H,
H-2′), 2.62 (dd, J ) 14.6, 5.8 Hz, 1H, H-2′), 1.94 (s, 3H, CH3);
13C NMR (DMSO-d6) δ 164.15 (C-4 CdO), 158.10 (furoxan C-4),
150.79 (C-2 CdO), 137.22 (phenyl C-1), 136.65 and 136.21 (C-
6, phenyl C-4), 130.40 and 128.76 (phenyl C-2, C-3), 111.08
and 110.43 (C-5, furoxan C-3), 84.22, 84.15, and 83.10 (C-4′,
C-3′, C-1′), 61.47 (C-5′), 36.63 (C-2′), 12.60 (CH3). Anal.
(C18H18N4O9S) C, H, N, S.
3′-O-(ter t-Bu t yld im et h ylsilyl)-5-iod o-2′-d eoxyu r id in e
(5c): white solid; yield, 55%; mp 187-188 °C; 1H NMR (CDCl3)
δ 11.66 (br s, 1H, NH), 8.34 (s, 1H, H-6), 6.07 (t, J ) 6.2 Hz,
1H, H-1′), 5.18 (br s, 1H, OH), 4.40 (ddd, J ) 6.2, 3.7, 2.6 Hz,
1H, H-3′), 3.77 (dd, J ) 6.6, 3.7 Hz, 1H, H-4′), 3.50-3.64 (m,

Design and Synthesis of Deoxyuridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1845
0.92 (s, 9H, t-Bu), 0.14 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3); 13
2H, H-5′), 2.22 (dd, J ) 13.2, 6.2 Hz, 1H, H-2′), 2.08 (ddd, J )
13.2, 6.2, 2.6 Hz, 1H, H-2′), 0.86 (s, 9H, t-Bu), 0.07 [s, 6H, Si-
(CH3)2]; 13C NMR (CDCl3) δ 160.25 (C-4 CdO), 149.93 (C-2
CdO), 144.73 (C-6), 87.50 (C-4′), 84.51 (C-1′), 71.57 (C-3′),
69.40 (C-5), 60.40 (C-5′), 25.62 [C(CH3)3], 17.63 [C(CH3)3],
-4.87 (d, J Si,CH3 ) 5.5 Hz, Si(CH3)2]. Anal. (C15H25IN2O5Si) C,
H, N.
C
NMR (DMSO-d6) δ 158.55 (furoxan C-4), 156.77 (d, J CCF ) 26.4
Hz, C-4 CdO), 148.93 (C-2 CdO), 140.65 (J CF ) 237.3 Hz, C-5),
136.65 (phenyl C-1), 135.89 (phenyl C-4), 129.63 and 128.74
(phenyl C-2 and C-3), 127.20 (d, J CCF ) 33.0 Hz, C-6), 110.60
(furoxan C-3), 85.08 and 84.45 (C-4′, C-3′), 71.87 (C-3′), 69.86
(C-5′), 40.39 (C-2′), 25.72 [C(CH3)3], 17.96 [C(CH3)3], -4.76 [d,
J Si,CH3 ) 6.7 Hz, Si(CH3)2]. Anal. (C23H29FN4O9SSi) C, H, N,
S.
3′-O-(t er t -Bu t yld im e t h ylsilyl)-5-flu or o-2′-d e oxyu r i-
d in e (5d ): white solid; yield, 70%; mp 165-166 °C; H NMR
1
(CDCl3) δ 9.13 (br s, 1H, NH), 7.97 (d, J ) 6.2 Hz, 1H, H-6),
6.23 (t, J ) 6.2 Hz, 1H, H-1′), 4.48 (ddd, J ) 6.2, 4.0, 2.2 Hz,
1H, H-3′), 3.98-3.96 (m, 2H, H-4′, H-5′), 3.95-3.81 (m, 1H,
H-5′), 2.31-2.35 (m, 2H, H-2′), 0.90 (s, 9H, t-Bu), 0.10 [s, 6H,
Si(CH3)2]; 13C NMR (CDCl3) δ 156.73 (d, J CCF ) 26.4 Hz, C-4
CdO), 148.67 (C-2 CdO), 140.44 (d, J CF ) 236.2 Hz, C-5),
124.89 (d, J CCF ) 34.0 Hz, C-6), 87.61 (C-4′), 86.27 (C-1′), 71.39
(C-3′), 61.82 (C-5′), 41.27 (C-2′), 25.75 [C(CH3)3], 18.00 [C(CH3)3],
-4.71 [d, J Si,CH3 ) 13.2 Hz, Si(CH3)2]. Anal. (C15H25FN2O5Si)
C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of 5′-O-(3-Ben -
ze n e s u lfo n y lfu r o x a n -4-y l)]-5-s u b s t it u t e d -2′-d e o x y -
u r id in es (7a -d ). To a stirred solution of a compound selected
from the group of 6a -d (1 mmol) in THF (10 mL) was added
a 1 N HCl solution (3 mL), and the mixture was stirred at 25
°C for 3 h. The mixture was extracted with ethyl acetate (3 ×
5 mL), the combined organic extracts were dried (Na2SO4), and
the solvent was removed in vacuo to give an oil that was
purified by flash chromatography using EtOAc/hexane (3:1,
v/v) as eluant to give the respective title product listed below.
3′-O-(ter t-Bu tyld im eth ylsilyl)-5-tr iflu or om eth yl-2′-d e-
oxyu r id in e (5e): white solid; yield, 54%; mp 225-226 °C; 1H
NMR (CDCl3) δ 9.80 (br s, 1H, NH), 8.58 (s, 1H, H-6), 6.20 (t,
J ) 6.1 Hz, 1H, H-1′), 4.45-4.53 (m, 1H, H-3′), 3.93-3.98 (m,
2H, H-4′, H-5′), 3.77-3.81 (m, 1H, H-5′), 2.20-2.42 (m, 2H,
H-2′), 0.87 (s, 9H, t-Bu), 0.07 [s, 6H, Si(CH3)2]; 13C NMR
(CDCl3) δ 158.96 (C-4 CdO), 149.50 (C-2 CdO), 142.18 (q,
J CCCF ) 5.5 Hz, C-6), 121.93 (q, J CF ) 270.3 Hz, CF3), 104.84
(d, J CCF ) 33.0 Hz, C-5), 88.03 (C-4′), 86.78 (C-1′), 71.13 (C-
3′), 61.29 (C-5′), 41.75 (C-2′), 25.64 [C(CH3)3], 17.90 [C(CH3)3],
-4.87 [d, J Si,CH3 ) 14.2 Hz, Si(CH3)2]. Anal. (C16H25F3N2O5Si)
C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of 5′-O-(3-Ben -
zen esu lfon ylfu r oxa n -4-yl)-3′-O-(ter t-bu tyld im eth ylsilyl)-
5-su bstitu ted -2′-d eoxyu r id in es (6a -d ). To a stirred solu-
tion of a compound selected from the group 5a -d (1 mmol)
and 8a (550 mg, 1.5 mmol) in THF (5 mL) at 0 °C was added
a 50% aqueous sodium hydroxide solution (3 mmol). The
mixture was stirred for 1 h, diluted with water, and extracted
with ethyl acetate (3 × 5 mL). The combined organic extracts
were dried (Na2SO4), and the solvent was removed in vacuo
to give an oil that was purified by flash chromatography using
EtOAc/hexane (1:1, v/v) to give the respective title product
listed below.
5′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-3′-O-(ter t-bu tyl-
d im eth ylsilyl)-2′-d eoxyu r id in e (6a ): white solid; yield, 83%;
mp 132-134 °C; 1H NMR (CDCl3) δ 9.06 (br s, 1H, NH), 7.80
(d, J ) 8.0, 2H, o-Ph), 7.77 (t, J ) 8.0 Hz, 1H, p-Ph), 7.74 (d,
J ) 8.1 Hz, 1H, H-6), 7.64 (t, J ) 8.0 Hz, 2H, m-Ph), 6.46 (t,
J ) 6.9 Hz, 1H, H-1′), 5.84 (d, J ) 8.1 Hz, 1H, H-5), 4.57-
4.72 (m, 3H, H-3′, H-5′), 4.14-4.20 (m, 1H, H-4′), 2.34-2.44
(m, 2H, H-2′), 0.92 (s, 9H, t-Bu), 0.13 (s, 3H, SiCH3), 0.11 (s,
3H, SiCH3); 13C NMR (DMSO-d6) δ 162.97 (C-4 CdO), 158.59
(furoxan C-4), 150.30 (C-2 CdO), 140.13 (C-6), 137.11 (phenyl
C-1), 135.89 (phenyl C-4), 129.70 and 128.56 (phenyl C-2 and
C-3), 110.23 (furoxan C-3), 103.25 (C-5), 84.36 and 83.84 (C-
4′, C-1′), 70.87 (C-3′), 69.18 (C-5′), 40.64 (C-2′), 25.69 [C(CH3)3],
17.96 [C(CH3)3], -4.70 [d, J Si,CH3 ) 18.6 Hz, Si(CH3)2]. Anal.
(C23H30N4O9SSi) C, H, N, S.
5′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-3′-O-(ter t-bu tyl-
d im eth ylsilyl)-2′-d eoxyth ym id in e (6b): white solid; yield,
86%; mp 110-112 °C; 1H NMR (CDCl3) δ 8.80 (br s, 1H, NH),
8.00 (d, J ) 7.7 Hz, 2H, o-Ph), 7.79 (t, J ) 7.7 Hz, 1H, p-Ph),
7.63 (t, J ) 7.7 Hz, 2H, m-Ph), 7.46 (s, 1H, H-6), 6.47 (t, J )
7.0 Hz, 1H, H-1′), 4.56-4.70 (m, 3H, H-3′, H-5′), 4.16-4.22
(m, 1H, H-4′), 2.26-2.43 (m, 2H, H-2′), 1.92 (s, 3H, C-5 CH3),
0.92 (s, 9H, t-Bu), 0.14 (s, 3H, SiC-H3), 0.12 (s, 3H, SiC-H3).
Anal. (C24H32N4O9SSi) C, H, N, S.
5′-O-(3-Be n ze n e su lfon ylfu r oxa n -4-yl)-2′-d e oxyu r i-
d in e (7a ): white solid; yield, 80%; mp 163-164 °C; H NMR
1
(DMSO-d6) δ 11.29 (s, 1H, NH), 7.64-8.01 (m, 6H, C6H5, H-6),
6.28 (t, J ) 6.7 Hz, 1H, H-1′), 5.50 (d, J ) 7.9 Hz, 1H, H-5),
4.56-4.65 (m, 2H, H-5′), 4.40-4.48 (m, 1H, H-3′), 4.12-4.19
(m, 1H, H-4′), 2.20-2.45 (m, 2H, H-2′); 13C NMR (DMSO-d6) δ
163.18 (C-4 CdO), 158.83 (furoxan C-4), 150.41 (C-2 CdO),
140.54 (C-6), 136.84 (phenyl C-1), 136.13 (phenyl C-4), 129.91
and 128.29 (phenyl C-2 and C-3), 110.71 (furoxan C-3), 102.13
(C-5), 84.47 and 83.55 (C-4′ and C-1′), 70.87 (C-5′), 70.24 (C-
3′), 56.25 (C-2′). Anal. (C17H16N4O9S) C, H, N, S.
5′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)]-2′-d eoxyth ym i-
1
d in e (7b): white solid; yield, 85%; mp 245-246 °C; H NMR
(DMSO-d6) δ 11.35 (s, 1H, NH), 8.00 (d, J ) 7.7 Hz, 2H, o-Ph),
7.88 (t, J ) 7.7 Hz, 1H, p-Ph), 7.73 (t, J ) 7.7 Hz, 2H, m-Ph),
7.51 (s, 1H, H-6), 6.29 (t, J ) 6.6 Hz, 1H, H-1′), 5.54 (d, J )
4.4 Hz, 2H, OH), 4.56-4.66 (m, 2H, H-5′), 4.40-4.49 (m, 1H,
H-3′), 4.10-4.19 (m, 2H, H-4′), 2.11-2.40 (m, 2H, H-2′), 1.75
(s, 3H, CH3); 13C NMR (DMSO-d6) δ 163.94 (C-4 CdO), 159.07
(furoxan C-4), 150.66 (C-2 CdO), 136.84 (phenyl C-1), 136.30
and 136.04 (phenyl C-4, C-6), 130.17 and 128.40 (phenyl C-2,
C-3), 110.87 and 110.17 (furoxan C-3, C-5), 84.05 and 83.5o
(C-4′, C-1′), 71.22 (C-5′), 70.54 (C-3′), ∼38-39 (C-2′, buried
under solvent peaks), 12.21 (CH3). Anal. (C18H18N4O9S) C, H,
N, S.
5′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-5-iod o-2′-d eoxy-
u r id in e (7c): white solid; yield, 89%; mp 213-215 °C; 1H NMR
(DMSO-d6) δ 11.75 (s, 1H, NH), 8.00 (d, J ) 7.7 Hz, 2H, o-Ph),
7.98 (s, 1H, H-6), 7.88 (t, J ) 7.7 Hz, 1H, p-Ph), 7.72 (t, J )
7.7 Hz, 2H, m-Ph), 6.18 (t, J ) 7.0 Hz, 1H, H-1′), 5.51 (d, J )
4.0 Hz, 2H, OH), 4.67 (dd, J ) 10.9, 2.9 Hz, 1H, H-5′), 4.60
(dd, J ) 10.9, 5.1 Hz, 1H, H-5′), 4.36-4.45 (m, 1H, H-3′), 4.15-
4.22 (m, 1H, H-4′), 2.31-2.43 (m, 1H, H-2′), 2.07-2.25 (m, 2H,
H-2′). Anal. (C17H15FN4O9S) C, H, N, S.
5′-O-(3-Ben zen esu lfon ylfu r oxan -4-yl)-5-flu or o-2′-deoxy-
u r id in e (7d ): white solid; yield, 79%; mp 198-200 °C; 1H
NMR (DMSO-d6) δ 11.88 (s, 1H, NH), 7.69-8.01 (m, 6H, C6H5,
H-6), 6.25 (dd, J ) 7.6, 5.9 Hz, 1H, H-1′), 4.57-4.66 (m, 2H,
H-5′), 4.39-4.47 (m, 1H, H-3′), 4.13-4.20 (m, 1H, H-4′), 2.14-
2.38 (m, 2H, H-2′); 13C NMR (DMSO-d6) δ 158.71 (furoxan C-4),
156.75 (d, J CCF ) 26.4 Hz, C-4 CdO), 148.81 (C-2 CdO), 139.90
(d, J CF ) 231.8 Hz, C-5), 136.71 (phenyl C-1), 135.93 (phenyl
C-4), 129.71 and 128.15 (phenyl C-2, phenyl C-3), 124.48 (d,
J CCF ) 33.0 Hz, C-6), 110.58 (furoxan C-3), 84.58 and 83.94
(C-4′, C-1′), 70.93 (C-5′), 70.13 (C-3′), ∼38-39 (C-2′, buried
under solvent peaks). Anal. (C17H15IN4O9S) C, H, N, S.
In Vitr o Nitr ic Oxid e Relea se Assa ys. 1. In cu ba tion
w ith 18 m M L-Cystein e in P h osp h a te Bu ffer (p H 7.4). In
vitro nitric oxide release was assayed using a modification of
the previously reported procedure.25 Briefly, a solution of the
test compound (1 mL of a 0.2 mM solution in 0.1 M phosphate
buffer, pH 7.4) was mixed thoroughly with a freshly prepared
solution of L-cysteine (1 mL of a 3.6 mM solution in 0.1 M
phosphate buffer, pH 7.4), and the mixture was incubated at
5′-O-(3-Ben zen esu lfon ylfu r oxa n -4-yl)-3′-O-(ter t-bu tyl-
d im eth ylsilyl)-5-flu or o-2′-d eoxyu r id in e (6d ): white solid;
1
yield, 96%; mp 225-226 °C; H NMR (DMSO-d6) δ 11.29 (br
s, 1H, NH), 8.04 (d, J ) 7.9 Hz, 2H, o-Ph), 7.81 (t, J ) 7.9 Hz,
1H, p-Ph), 7.75 (d, J ) 7.1 Hz, 1H, H-6), 7.63 (t, J ) 7.9 Hz,
2H, m-Ph), 6.45 (t, J ) 6.9 Hz, 1H, H-1′), 4.58-4.70 (m, 3H,
H-3′, H-5′), 4.21-4.26 (m, 1H, H-4′), 2.30-2.45 (m, 2H, H-2′),

1846 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Moharram et al.
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furoxans. Biochem. Pharmacol. 1992, 44, 1149-1157.
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37 °C for 1 and 16 h in the absence of air. After exposure to
air for 10 min at 25 °C, an aliquot of the Griess reagent (1
mL) [freshly prepared by mixing equal volumes of 1.0%
sulfanilamide and 0.1% N-naphthylethylenediamine dihydro-
chloride in water] was added to an equal volume (1 mL) of
each test compound’s incubation solution with mixing. After
10 min had elapsed, absorbance was measured at 540 nm
using a Philips PU 8740 UV-vis scanning spectrophotometer.
Solutions of 0-100 µM sodium nitrite were used to prepare a
nitrite absorbance versus concentration curve under the same
experimental conditions. The percent nitric oxide released
(quantitated as nitrite ion) was calculated ((SEM, n ) 3) from
the standard nitrite versus concentration curve.
2. In cu ba tion w ith P h osp h a te Bu ffer (p H 7.4). This
assay was performed as described under procedure 1 above
except that a solution of the test compound (2 mL of a 2 mM
solution in 0.1 M phosphate buffer pH 7.4) was used and no
L-cysteine was added.
In Vitr o Cell Cytotoxicity (MTT Assa y). Human 143B
and human 143B-LTK cells were cultured in complete MEME
with 10% fetal bovine serum (FBS). Murine KBALB, KBALB-
STK, and Hs578Bst human fibroblast cells were cultured in
complete DMEM medium supplemented with 10% FBS, and
murine EMT-6 cells were cultured in complete WAYMOUTH
medium with 12.5% FBS. Exponentially growing cells were
trypsinized, centrifuged, and resuspended in growth medium,
and the cell number was readjusted to 8 × 103 cells/mL. Cells
were seeded into 96-well plates at 8 × 102 cells/well and
incubated at 37 °C in a humidified 5% CO2 atmosphere for 24
h.
The test compound was dissolved in the medium indicated
above, and 100 µL of this solution was added to the cells in
96-well plates to produce the preselected test compound
concentration. Complete growth medium (100 µL) was added
to control wells. The plates were incubated for 3 days at 37 °C
in a humidified atmosphere consisting of 95% air and 5% CO2.
At the end of the incubation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT, Sigma) was dissolved
in phosphate-buffered saline (PBS) to produce a concentration
of 5 mg/mL, filtered through a 0.45 µm membrane filter, and
diluted (1:5) with prewarmed medium. An aliquot of this
solution (50 µL) was added to each well, and the plates were
incubated at 37 °C for 4 h. The medium was removed from
the wells, dimethyl sulfoxide (150 µL) was added to each well,
and the plates were placed on a shaker for 15 min to dissolve
the formazan crystals. The absorbance at 540 nm (A540) was
measured immediately in each well using a scanning multiwell
spectrophotometer (ELISA reader). A540 values, corrected for
the absorbance in medium blanks, reflected the concentration
of viable cells. The CC50 values reported refer to the test drug
concentrations that reduced the A540 to 50% of the control value
(mean value, n ) 6). This assay,26 which depends on the
metabolic reduction of MTT to colored formazan, measures
cytostatic and cytotoxic effects of the test drug.
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Ghigo, D.; Bosia, A. NO donor and biological properties of
different benzofuroxans. Pharm. Res. 1999, 16, 956-960.
(23) Gasco, A.; Fruttero, R.; Sorba, G. NO-donors: an emerging class
of compounds in medicinal chemistry. Farmaco 1996, 51, 617-
635.
Ack n ow led gm en t. We are grateful to the Alberta
Cancer Board (Grant No. RI-124) for financial support
of this research.
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