Synthesis of 3′- and 5′-nitrooxy pyrimidine nucleoside nitrate esters: "Nitric oxide donor" agents for evaluation as anticancer and antiviral agents

Article abstract of DOI:10.1021/jm020299r

A group of 3′-O-nitro-2′-deoxyuridines, 3′-O -nitro-2′-deoxycytidines and 5′-O-nitro-2′-deoxyuridines possessing a variety of substituents (H, Me, F, I) at the C-5 position were synthesized for evaluation as anticancer/antiviral agents that have the abili

Full text of DOI:10.1021/jm020299r

J . Med. Chem. 2003, 46, 995-1004
995
Syn th esis of 3′- a n d 5′-Nitr ooxy P yr im id in e Nu cleosid e Nitr a te Ester s: “Nitr ic
Oxid e Don or ” Agen ts for Eva lu a tion a s An tica n cer a n d An tivir a l Agen ts
Ebrahim Naimi,† Aihua Zhou,† Panteha Khalili,† Leonard I. Wiebe,† J an Balzarini,§ Erik De Clercq,§ and
Edward E. Knaus*,†
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada, and
Rega Institute for Medical Research, Minderbroedersstraat 10, Leuven B-3000, Belgium
Received J uly 10, 2002
A group of 3′-O-nitro-2′-deoxyuridines, 3′-O-nitro-2′-deoxycytidines, and 5′-O-nitro-2′-deox-
yuridines possessing a variety of substituents (H, Me, F, I) at the C-5 position were synthesized
for evaluation as anticancer/antiviral agents that have the ability to concomitantly release
cytotoxic nitric oxide (•NO). Although these compounds generally released a greater percent
of •NO than the reference drug isosorbide dinitrate upon incubation in the presence of L-cysteine,
or serum, their cytotoxicity (CC50 ) 10-3 to 10-6 M range) was comparable to 5-iodo-2′-
deoxyuridine, but weaker than 5-fluoro-2′-deoxyuridine, against a variety of cancer cell lines.
No differences in cytotoxicity against nontransfected (KBALB, 143B), and the corresponding
transfected (KBALB-STK, 143B-LTK) 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. These nitrate esters were inactive
antiviral agents except for 5-iodo-3′-O-nitro-2′-deoxyuridine that showed modest activity against
HSV-1, HSV-2, and vaccinia virus.
In tr od u ction
via a •NO-dependent, nonapoptotic mechanism.14 Sev-
eral antiviral acyclic nucleotide analogues activate
expression of cytokine genes such as TNF-R and IL-10
in macrophages, and IFN-γ in splenocytes, resulting in
an enhanced production of •NO.15 A group of glyco-S-
nitrosothiols were found to be cytotoxic to DU-145
human prostate cancer cells in vitro.16 Induction of •NO
production by cytokines indicated that high levels of
•NO may contribute to the induction of apoptosis and
inhibition of pancreatic tumor growth.17 A correlation
exists between the intracellular levels of •NO with the
cytotoxicity of hydroxyguanidine in HL60 cells, where
a higher toxicity was observed in hypoxic cells relative
to oxic cells.18 The hypoxic cell radiosensitization effects
of the •NO-donor Et2N[N(NO)]- Na+, coupled with the
vasodilatory effect of •NO on tumor vasculature, suggest
that an agent of this type opens a new avenue in
radiation oncology treatment.19 The rate and extent of
•NO release from •NO/nucleophile adducts, which re-
lease •NO spontaneously in solution, correlated with
inhibition of DNA synthesis in A375 human melanoma
cells.9 Tamoxifen has been shown to induce iNOS,
producing •NO that is cytotoxic.20 •NO-induced apop-
tosis in macrophages activated with cisplatin and IFN-γ
requires activation of an endonuclease.21 •NO-donor
agents have been shown to enhance melphalan cytox-
icity in breast cancer MCF-7 cells, possibly by delaying
entry into the S-phase and a G2/M block.22 Using a
number of aromatic N-nitroso compounds, it was shown
that the extent of •NO generation was the reciprocal of
ID50 growth inhibition (cytotoxicity).23 These data col-
lectively indicate that •NO exhibits a cytotoxic effect
and/or enhances the cytotoxic effect of anticancer drugs.
However, there are a few instances where •NO is
detrimental to anticancer activity. For example, •NO
generated from a •NO-donor drug was shown to prevent
Since the discovery that nitric oxide (•NO) is biosyn-
thesized in mammalian cells, •NO has become a mol-
ecule of immense interest in medical research. In
mammalian systems, •NO is synthesized via an enzy-
matic oxidation of L-arginine by nitric oxide synthase
isozymes [neuronal (nNOS), inducible (iNOS), endo-
thelial (eNOS)] that are differentially expressed and
play different physiological roles.1-4 •NO production in
response to immune activation, or inflammatory reac-
tion, has been shown to serve as part of the host defense
system against cancerous cells, and intracellular mi-
crobes and parasites.5 In this regard activated macro-
phages, that produce •NO, have been shown to inhibit
DNA synthesis in tumor cells.6 Pretreatment of various
cell lines with a •NO-donor led to inhibition of the
replication of poliovirus, picornavirus, and rhinovirus.3
In addition, IFN-γ treated cells produce •NO that
inhibits HSV-1 (herpesvirus).3 Other studies indicate
that the antitumor activity of IL-1R may be mediated
by the production of •NO from tumor-derived endothe-
lial cells,7 and that macrophage •NO synthesis delays
progression of ultraviolet light-induced murine skin
cancer through to an antitumor immune response.8
•NO plays an important role in the action of a number
of anticancer drugs due to its ability to influence various
aspects of tumor biology including modulation of cell
growth,9 apoptosis,10 differentiation,11 metastatic capa-
bility,12 and tumor-induced immunosuppression.13 Thus,
adriamycin stimulates •NO production in EMT-6 cells,
and in vivo adriamycin inhibits tumorigenesis partially
* To whom correspondence should be addressed. Phone: 780-492-
5993. Fax: 780-492-1217. E-mail: eknaus@pharmacy.ualberta.ca
†University of Alberta.
§ Rega Institute.
10.1021/jm020299r CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/12/2003

996 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Naimi et al.
Sch em e 1a
F igu r e 1. Structures of glycerol trinitrate (1), 5′-O-nitro-2′-
deoxyuridine (2), 3′,5′-di-O-nitro-2′-deoxyuridine (3), 3′-O-nitro-
2′-deoxycytidine (4), nitrara-C (5), and nitrara-A (6) nitrate
esters.
taxol-induced apoptosis, and this was attributed to
inhibition of an IL-1 beta converting enzyme-like pro-
tease cascade (human neuroblastoma cell line, NB-39-
nu).24 A related study showed that the protective effect
of •NO on taxol-induced apoptosis may be partially
caused by inhibiting entrance of the cells into the G2/M
phase (human leukemia HL-60 cells).25 These data
indicate that the efficacy of an •NO-donor anticancer
drug may be dependent upon the specific cell line.
a
Reagents and conditions: (a) C6H5COOH, Ph3P, DIAD, DMF,
25 °C; (b) NH4NO3, DMF, 110 °C; (c) ICl, NaN3, MeCN, 0 °C f 25
°C; (d) NaOMe, MeOH, 25 °C.
Ch em istr y
Organic nitrates (RONO2), which are nitric acid esters
of mono- or polyhydric alcohols, represent the oldest
class of •NO-donor compounds that include the classical
drug glycerol trinitrate (1) that is used to treat angina
pectoris.26 Although some 5′-O-nitro-2′-deoxyuridine
(2),27,28 3′,5′-di-O-nitro-2′-deoxyuridine (3),29-31 and 3′-
O-nitro-2′-deoxycytidine (4)32 compounds have been
synthesized, no biological data were reported (see
structures in Figure 1). On the other hand, the 2′-O-
nitro derivatives of arabinocytidine (Nitrara-C, 5), which
resists enzymatic deamination, exhibited antileukemic
activity in vitro and in vivo.33 The related 2′-O-nitro
derivative of arabinoadenine (Nitrara-A, 6), that is
deaminated about 25-fold slower than Ara-A, inhibited
the proliferation of human T-lymphoblasts (CCRF-
CEM cell line) in vitro.34 The design of tumor selective
•NO-donor/nucleoside hybrid drugs, that release both
cytotoxic •NO and a nucleoside drug in a controlled
fashion, constitutes an attractive method to develop a
novel class of double-barreled anticancer drugs. The
nucleoside component of these hybrid drugs, as the 5′-
monophosphate, could act as an irreversible inhibitor
of thymidylate synthase (TS), whereas the 5′-triphos-
phate could serve as an unnatural substrate for, or
inhibitor of, DNA polymerase to elicit a cytotoxic
anticancer/antiviral effect. Accordingly, we now report
the syntheses, in vitro •NO release data, and anticancer
and antiviral activities, for a group of 3′-O-nitro-2′-
deoxyuridines (10a -d ), 3′-O-nitro-2′-deoxycytidines
(13a -c), and 5′-O-nitro-2′-deoxyuridines (15a -c).
A group of 2,3′-anhydro-5′-O-benzoyl-2′-deoxyuridines
8a -d possessing a variety of C-5 substituents (R ) H,
Me, F, I) were prepared from the 2′-deoxyuridine
precursors 7a -d by a one-pot tandem Mitsunobu reac-
tion,35,36 using triphenylphosphine, diisopropyl azodi-
carboxylate, and benzoic acid in 81%, 74%, 90%, and
82% yield, respectively. Ring opening of the 2,3′-anhydro
derivatives 8a -d proceeded readily upon reaction with
ammonium nitrate (NH
4NO3) in dimethyl formamide to
form the respective 3′-O-nitrate esters 9a -d in 31%,
34%, 50%, and 2.8% yield, respectively. Since conversion
of the 5-iodo compound 8d to 9d was low (2.8% yield)
in this reaction, compound 9d was prepared by iodina-
tion of 9a using iodine monochloride (ICl) and sodium
azide (NaN3) in acetonitrile37 in 82.5% yield (see Scheme
1). Deprotection of compounds 9a -d using NaOMe in
MeOH afforded the target 5-substituted-3′-O-nitro-2′-
deoxyuridines 10a -d in 91-95% yield.
The configuration at the C-3′ carbon, and the rotamer
orientation of the uracil (10a ) and thymine (10b) ring,
was analyzed by nuclear Overhauser enhancement
(NOE) 1H NMR difference spectroscopy (Figure 2).
Selective irradiation of the H-3′ signal resulted in an
enhancement of the arabino (up) H-2′ signal of 10a
(2.2%), and of 10b (1.9%), indicating that the C-3′
nitrooxy substituent is in the ribo (down) position. No
NOE effect was observed between H-3′ and either H-2′
or H-1′ of 10a ,b. The observation that selective irradia-
tion of the H-4′ signal resulted in enhancement of the
H-1′ signal of 10a (3.1%), and of 10b (2.5%), indicates

Nitrooxy Pyrimidine Nucleoside Nitrate Esters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 997
Sch em e 2a
F igu r e 2. Some NOE measurements to determine the rota-
meric orientation of the uracil base moiety, and the conforma-
tion of the nitrooxy substituent, for the 3′-O-nitro-2′-deoxyuri-
dine compounds 10a ,b in DMSO-d6 at 22 °C.
that these two hydrogen atoms are on the same face of
the sugar moiety which is indicative of the â-configu-
ration.38 Selective irradiation of the H-6 signal of 10a
produced an enhancement of the arabino (up) H-2′
signal (2.6%), whereas a similar irradiation for 10b
showed enhancement for the arabino H-2′ (1.3%), and
the H-3′ (1.9%), signals. These NOE data indicate the
C-6 hydrogen of the uracil moiety of 10a , or the thymine
moiety of 10b, is oriented in the direction of the sugar
ring.
A related group of 3′-O-nitro-2′-deoxycytidines 13a -c
having a C-5 hydrogen, methyl, or iodo substituent were
prepared using the three-step reaction sequence il-
lustrated in Scheme 2. Thus, reaction of the 5′-O-
benzoyl-3′-O-nitro-2′-deoxyuridines 9a ,b with 1,2,4-
triazole and 4-chlorophenyl dichlorophosphate in pyri-
dine39-41 afforded the corresponding 4-(1,2,4-triazolo)
derivatives 11a and 11b in 79 and 71% yield, respec-
tively. Subsequent treatment of 11a , or 11b, with
aqueous ammonia in dioxane39-41 yielded the 2′-deoxy-
cytidine compound 12a (49%), or 12b (36%), which on
deprotection with NaOMe in MeOH to remove the 5′-
O-benzoyl moiety, afforded the target 3′-O-nitro-2′-
deoxycytidine 13a (95%), or 5-methyl-3′-O-nitro-2′-
deoxycytidine 13b (94%), respectively. Iodination of 13a
with iodic acid and iodine42,43 yielded 5-iodo-3′-O-nitro-
2′-deoxycytidine 13c (56%). The procedure described
above for the synthesis of 13a is superior to the reported
method involving nitration of 2′-deoxycytidine 5′-O-
monophosphate using nitronium tetrafluoroborate, which
gave a mixture of the 5′-O-monophosphates of 5-nitro-
3′-O-nitro-2′-deoxycytidine, 5-nitro-2′-deoxycytidine, and
3′-O-nitro-2′-deoxycytidine in a ratio of 12:1:3 that were
subsequently dephosphorylated by the action of alkaline
phosphatase to the free nucleosides.32
a
Reagents and conditions: (a) 4-chlorophenyl dichlorophos-
phate, 1,2,4-triazole, pyridine, 0 °C f 25 °C; (b) NH4OH, dioxane,
25 °C; (c) NaOMe, MeOH, 25 °C; (d) iodine, iodic acid, acetic acid,
H2O, CCl4, 25 °C.
Sch em e 3a
a
Reagents and conditions: (a) LiNO3, DMF, 100-110 °C,
overnight; (b) ICl, NaN3, MeCN, 0 °C f 25 °C, 48 h.
ing displacement of either a 5′-O-P(dS)OEt3, or 5′-I,
substituent using AgNO3.28 Electrophilic iodination of
15a using ICl and NaN3 in MeCN gave the 5-iodo
product 15c (65%).
The 5′-O-nitro-2′-deoxyuridines 15a -c possessing a
C-5 H, Me, or I substituent were also prepared (see
Scheme 3). Accordingly, nucleophilic displacement of the
5′-O-tosylate moiety in 14a , or 14b, using LiNO3 in
DMF at 110 °C, afforded the respective 5′-O-nitro
product 15a (73%), or 15b (71%). This synthesis of 15a
was superior to nitration of 2′-deoxyuridine with 90%
nitric acid at -70 °C which gave a mixture of 15a (20%)
and 3′,5′-di-O-nitro-2′-deoxyuridine (13%).27 Synthesis
of 15b by displacement of the 5′-O-tosylate moiety in
14b, was equally effective to a reported method involv-
Resu lts a n d Discu ssion
Organic nitrate esters are generally stable in neutral
or mild acidic aqueous solution, but nucleophilic hy-
drolysis to an alcohol and nitrate occur under strongly
alkaline conditions. •NO release from organic nitrates
may occur as a result of either nonenzymatic or enzy-
matic biotransformation that proceeds via a three-
electron reduction. Accordingly, cellular thiols may play
a role in the nonenzymatic production of •NO from
glycerol trinitrate (1, GTN) whereas, a NADPH-depend-

998 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Naimi et al.
Ta ble 1. In Vitro Percent Nitric Oxide Release for
3′-O-Nitro-2′-deoxyuridines (10a -d ),
3′-O-Nitro-2′-deoxycytidines (13a -c), and
5′-O-nitro-2′-deoxyuridines (15a -c)
in serum. Compounds 10a -d , 13a -c, and 15a -c all
exhibited a greater release of •NO in the presence of
serum (2.9-47.1% range at 1 h, and 4.4-59.5% range
at 16 h) than the reference drug isosorbide dinitrate
(1.9% at 1 h and 2.6% at 16 h).
% NO releasea
(18 mM L-cysteine)
% NO releaseb (serum)
Compounds 10a -d , 13a -c, 15a -c, and the reference
compounds 5-iodo-2′-deoxyuridine (IUDR), 5-fluoro-2′-
deoxyuridine (FUDR), and thymidine, were evaluated
for their tumor cell cytotoxicity using the MTT cytotox-
icity assay46 (see data in Table 2). These 3′- and 5′-O-
nitro pyrimidine nucleosides exhibited comparable cy-
totoxicity (CC50 ) 10-3 to 10-6 M range) to 5-iodo-2′-
deoxyuridine, but weak cytotoxicity in comparison to
5-fluoro-2′-deoxyuridine, against KBALB, KBALB-
STK, 143B, 143B-LTK, and EMT-6. 5-Fluoro-3′-O-nitro-
2′-deoxyuridine (10c) was the most cytotoxic compound
compd
1 hour
16 hours
1 hour
16 hours
10a
10b
10c
10d
13a
13b
13c
15a
15b
15c
ISDNc
4.9 ( 0.0
4.9 ( 0.1
0.0 ( 0.0
3.9 ( 0.1
1.5 ( 0.0
3.8 ( 0.0
4.0 ( 0.1
5.4 ( 0.1
5.0 ( 0.0
3.7 ( 0.1
3.5 ( 0.2
51.0 ( 0.1
53.6 ( 0.7
3.8 ( 0.1
7.7 ( 0.7
7.4 ( 0.9
4.0 ( 0.9
12.3 ( 1.1
18.8 ( 0.9
47.1 ( 3.1
12.5 ( 0.5
2.9 ( 0.8
3.6 ( 0.4
3.9 ( 0.5
1.9 ( 0.1
9.0 ( 1.5
7.4 ( 0.3
6.6 ( 0.7
13.2 ( 0.3
20.2 ( 0.9
59.5 ( 1.5
17.6 ( 1.7
4.4 ( 1.0
4.8 ( 1.1
4.7 ( 1.0
2.6 ( 0.6
37.6 ( 0.1
17.1 ( 0.2
50.3 ( 0.7
47.2 ( 0.0
59.9 ( 1.7
59.8 ( 0.2
36.4 ( 0.4
24.0 ( 0.2
against this group of cancer cell lines (CC50 ) 10-6
M
a
The percent nitric oxide released from the test compound was
determined as the percent of nitrite (NO2-) produced in the
presence of L-cysteine (18 mM) as quantitated using the Griess
range). Elaboration of the 3′-OH group of IUDR to a 3′-
O-NO2 group (10d ) decreased cytotoxicity by 2.5- to 26-
fold in all cell lines except for EMT-6 cells where
cytotoxicity was enhanced by about 6-fold. The position
of the O-NO2 substituent was not a determinant of
anticancer activity, since 5-iodo-3′-O-nitro-2′-deoxyuri-
dine (10d ) and 5-iodo-5′-O-nitro-2′-deoxyuridine (15c)
were approximately equipotent. The observation that
compounds 10c-d and 13c exhibited similar cytotox-
icity against nontransfected (KBALB, 143B), and the
corresponding 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. This observation was also
made for 10a and 10b that did not show increased
cytostatic activity in murine mammary (FM3A) and
human osteosarcoma cells transfected with the HSV-1
TK gene when compared with their parental counter-
parts (data not shown). The •NO drug ISDN exhibited
comparable cytotoxicity (CC50 ) 10-4 M range) against
all cell lines tested (Table 2).
b
reagent ((SD, n ) 3). The percent nitric oxide released from the
test compound was determined as the percent of nitrite (NO2
-
)
produced in the presence of serum as quantitated using the Griess
reagent ((SD, n ) 3). c ISDN ) isosorbide dinitrate (ISDN
possesses two ONO2 groups which may release •NO, whereas
compounds 10, 13, and 15 possess only one ONO2 group that may
release •NO).
ent cytochrome P450 pathway and specific isozymes of
the glutathione-S-transferase group are thought to be
operative in the biotransformation/bioactivation of or-
ganic nitrates.26 Although the sole product from reaction
of •NO with oxygen in water is nitrite, •NO reacts
rapidly with superoxide anion to initially produce a
cytotoxic peroxynitrite anion (ONOO-) species that can
damage DNA.5
The percentages of •NO released, quantitated as
nitrite (NO2-) using the Griess reaction,44 upon incuba-
tion of the 3′-O-nitro derivatives of 2′-deoxyuridine
(10a -d ) and 2′-deoxycytidine (13a -c), and the 5′-O-
nitro derivatives of 2′-deoxyuridine (15a -c) in the
presence of 18 mM L-cysteine or serum are listed in
Table 1. All three groups of compounds (10, 13, 15) that
possess a single ONO2 moiety, with the exception of
5-fluoro-3′-O-nitro-2′-deoxyuridine (10c), exhibited com-
parable or in most instances superior nitric oxide release
(1.5-5.4% range at 1 h, and 17.1-59.9% range at 16 h)
upon incubation in the presence of L-cysteine compared
to the reference drug isosorbide dinitrate (3.5 and 24%
release at 1 h and 16 h) that contains two ONO2
moieties. These results are consistent with the observa-
tion that thiols enhance in vitro release of •NO in
aqueous buffer solution for •NO-donor compounds al-
though intact cells and tissues show that conversion of
organic nitrate esters (RONO2) to •NO is not free-thiol
dependent.45 In contrast, release of •NO from 10, 13,
and 15 was negligible in the absence of L-cysteine
(<0.05% at 1 h and <1.0% at 16 h). A similar study to
determine the release of •NO upon incubation in the
presence of serum showed that 3′-O-nitro-2′-deoxycyti-
dine compounds (13) generally provided a greater
percent •NO release than the related compounds 10 and
15. The unusually high release of •NO from 5-methyl-
3′-O-nitro-2′-deoxycytidine (13b, 47.1%) upon incubation
with serum at 1 h, relative to that in the presence of 18
mM L-cysteine (3.8% release), indicates that •NO must
be released from 13b by a thiol-independent mechanism
Compounds 10a -d were evaluated for their antiviral
activity in a wide variety of assay systems.47 Antiviral
activities against herpes simplex virus type 1 (KOS),
herpes simplex virus type 2 (G), thymidine kinase-
deficient (TK-) herpes simplex virus type 1 (KOS,
ACVr), vaccinia virus, and vesicular stomatitis virus in
E6SM cells were determined. These compounds were
generally inactive or exhibited negligible activity in
these antiviral assay systems (at concentrations up to
400 µg/mL). 5-Iodo-3′-O-nitro-2′-deoxyuridine (10d )
showed some inhibitory activity against HSV-1 (KOS),
HSV-2 (G), and vaccinia virus (IC50 ) 48 µg/mL),
relative to the reference drug (E)-5-(2-iodovinyl)-2′-
deoxyuridine (IC50 ) 0.0051, 80.0, and 2.3 µg/mL,
respectively). In addition, compounds 10a -d did not
reduce cytopathicity induced by parainfluenza-3 virus,
reovirus-1, Sindbis virus, Coxsakie B4 virus, or Punta
Toro virus in Vero cell cultures (IC50 > 80 µg/mL), or
respiratory syncytial virus in HeLa cell cultures (IC50
> 80 µg/mL).
The modest anticancer, and near absence of antiviral,
efficacy for this novel class of •NO-donor nucleosides
could be due to a number of factors. For example, it is
possible that the sugar moiety does not undergo phos-
phorylation by thymidine kinase (TK) to the 5′-mono-

Nitrooxy Pyrimidine Nucleoside Nitrate Esters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 999
Ta ble 2. In Vitro Cell Cytotoxicity for 3′-O-Nitro-2′-deoxyuridines (10a -d ), 3′-O-Nitro-2′-deoxycytidines (13a -c), and
5′-O-Nitro-2′-deoxyuridines (15a -c)
cellular toxicity (CC50, M) toward various cell linesa
compd
10a
10b
10c
10d
13a
13b
13c
KBALBb
KBALB-STKc
143-Bd
143B-LTKc
EMT-6e
-
-
-
-
8.5 × 10-4
-
-
1.0 × 10-3
8.0 × 10-6
2.4 × 10-4
-
7.0 × 10-3
3.5 × 10-6
2.6 × 10-4
-
-
2.4 × 10-6
7.4 × 10-5
4.1 × 10-4
2.0 × 10-4
1.0 × 10-3
2.0 × 10-4
-
6.2 × 10-6
5.8 × 10-4
2.3 × 10-5
-
-
-
-
-
-
1.0 × 10-3
1.0 × 10-3
-
2.5 × 10-4
2.0 × 10-4
3.4 × 10-4
15a
15b
15c
-
-
-
-
-
-
3.0 × 10-4
2.5 × 10-4
1.0 × 10-5
8.8 × 10-11
1.0 × 10-4
6.5 × 10-4
-
3.4 × 10-4
9.7 × 10-5
6.0 × 10-11
9.5 × 10-5
9.7 × 10-4
3.4 × 10-4
7.0 × 10-3
9.0 × 10-5
-
2.1 × 10-5
7.4 × 10-3
1.0 × 10-4
-
IUDRf
FUDRg
thymidine
ISDNh
3.8 × 10-4
9.0 × 10-12
1.3 × 10-4
-
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. bTransformed 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 IUDR ) 5-iodo-2′-deoxyuridine. FUDR ) 5-fluoro-2′-deoxyuridine. ISDN ) isosorbide dinitrate.
g
h
on a Bruker AM-300 spectrometer. Proton chemical shifts (δ)
phosphate (5′-MP). Support for this explanation is based
on the observations that there are generally negligible
differences in anticancer/antiviral activities between
nontransfected (KBALB, 143B) and viral TK-transfected
(KBALB-STK, 143B-LTK) cell lines. Alternatively, this
group of nucleoside nitrate esters may be devoid of
anticancer/antiviral activity due to the fact that they
are not inhibitors of thymidylate synthase (TS). Cre-
dence for this possibility is based on the belief that the
anticancer activity exhibited by 5-fluoro-2′-deoxyuridine
is primarily due to inhibition of DNA biosynthesis by
blocking TS, the enzyme which catalyzes the methyla-
tion of 2′-deoxyuridine-5′-monophosphate to 2′-deoxy-
thymidine-5′-monophosphate.48,49 Furthermore, inhibi-
tion of TS is the mechanism by which certain nucleosides
such as (E)-5-(2-iodovinyl)-2′-deoxyuridine and 5-(1-
azidovinyl)-2′-deoxyuridine seem to exhibit their cyto-
static effect.50
are given relative to internal TMS (δ 0), and the assignment
of exchangeable protons (NH, OH) was confirmed by addition
of D2O. The nuclear Overhauser enhancement (NOE) studies
were performed under steady-state conditions using the
Bruker NOE DIFF.AU software program (signal:noise ratio
of 136 for a single pulse). DMSO-d6 was dried using molecular
sieves (type 3A, 1.6-mm pellets) and degassed by passage of
dry argon gas at 22 °C just prior to use. Molecular tumbling
time was not altered. 13C NMR spectra were acquired using
the J modulated spin-echo technique where methyl and
methine carbon resonances appear as positive peaks, meth-
ylene and quaternary carbon resonances appear as negative
peaks, and carbon chemical shifts (δ) are given relative to
CDCl3 (δ 77). Elemental analyses were performed by the
MicroAnalysis Service Laboratory, 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. 5′-O-Tosyl-2′-deoxyuridine (14a)51
and 5′-O-tosyl-2′-deoxythymidine (14b)52 were prepared ac-
cording to literature procedures. All other reagents were
purchased from Aldrich Chemical (Milwaukee, WI).
Con clu sion s
Gen er a l Meth od for th e P r ep a r a tion of 5-Su bstitu ted -
2,3′-a n h yd r o-5′-O-ben zoyl-2′-d eoxyu r id in es (8a -d ). A so-
lution containing either 7a , 7b, 7c, or 7d (65.7 mmol) and
triphenylphosphine (Ph3P, 25.9 g, 98.6 mmol) in DMF (90 mL)
was prepared, a solution comprised of diisopropyl azodicar-
boxylate (DIAD, 19.5 mL, 98.6 mmol) and benzoic acid (12.1
g, 98.6 mmol) in DMF (40 mL) was added dropwise with
stirring at 25 °C, and the reaction was allowed to proceed for
30 min at 25 °C. An additional aliquot of Ph3P (98.6 mmol)
and DIAD (98.6 mmol) was added, the reaction was allowed
to proceed for 1 h, Et2O (400 mL) was added, and the mixture
was stirred for 10 min at 25 °C. The resulting suspension was
cooled to ice-bath temperature, and the white crystalline
precipitate was collected by filtration and washed with cold
Et2O to give the respective product 8a , 8b, 8c, or 8d . Physical
and spectral data for 8a -d are listed below.
2,3′-An h yd r o-5′-O-ben zoyl-2′-d eoxyu r id in e (8a ). White
solid; yield, 81%; mp 225-226 °C; 1H NMR (DMSO-d6): δ 7.93
(d, J ) 7.8 Hz, 2H, o-benzoyl hydrogens), 7.66 (d, J ) 7.5 Hz,
1H, H-6), 7.63-7.68 (m, 1H, p-benzoyl hydrogen), 7.47-7.52
(m, 2H, m-benzoyl hydrogens), 5.97 (d, J ) 3.3 Hz, 1H, H-1′),
5.78 (d, J ) 7.5 Hz, 1H, H-5), 5.45 (br s, 1H, H-3′), 4.50-4.60
(m, 2H, H-5′), 4.37 (dd, J ) 11.7, 6.3 Hz, 1H, H-4′), 2.52-2.88
(m, 2H, H-2′); 13C NMR (DMSO-d6): δ 170.05, 165.14, 153.50,
140.57, 133.34, 129.06, 128.92, 128.55, 107.88, 86.94, 81.86,
77.28, 62.48, 32.68. Anal. (C16H14N2O5) C, H, N.
A group of nitric oxide donor 3′-O-nitro derivatives of
2′-deoxyuridine (10) and 2′-deoxycytidine (13), and 5′-
O-nitro-2′-deoxyuridines (15), were designed to inves-
tigate the concept whether the concomitant release of
cytotoxic nitric oxide may enhance the anticancer/
antiviral efficacy of pyrimidine nucleosides. This group
of compounds generally exhibits comparable and in most
instances superior nitric oxide release, upon incubation
with phosphate buffer containing 18 mM L-cysteine or
in the presence of serum, compared to the reference drug
isosorbide dinitrate. This group of 3′- and 5′-O-nitrate
esters exhibit comparable in vitro cytotoxicity to 5-iodo-
2′-deoxyuridine, but weaker cytotoxicity than 5-fluoro-
2′-deoxyuridine, in a variety of cancer cell lines. These
nitrate esters were devoid of antiviral activity except
for 5-iodo-3′-O-nitro-2′-deoxyuridine, which showed mar-
ginal activity against HSV-1, HSV-2 and vaccinia virus
in cell cultures.
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

1000 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Naimi et al.
2,3′-An h ydr o-5′-O-ben zoyl-2′-deoxyth ym idin e (8b). White
solid; yield, 74%; mp 245-246 °C; 1H NMR (DMSO-d6): δ 7.90
(dd, J ) 6.9, 1.2 Hz, 2H, o-benzoyl hydrogens), 7.63-7.68 (m,
1H, p-benzoyl hydrogen), 7.57 (s, 1H, H-6) 7.46-7.57 (m, 2H,
m-benzoyl hydrogens), 5.90 (d, J ) 3.6 Hz, 1H, H-1′), 5.42 (br
s, 1H, H-3′), 4.50-4.61 (m, 2H, H-5′), 4.36 (dd, J ) 11.7, 5.4
Hz, 1H, H-4′), 2.53-2.88 (m, 2H, H-2′), 1.72 (s, 3H, CH3); 13C
NMR (DMSO-d6): δ 170.63, 165.14, 153.13, 136.32, 133.30,
129.04, 128.70, 128.37, 116.00, 86.81, 81.80, 77.07, 62.30,
32.71, 12.85. Anal. (C17H16N2O5) C, H, N.
CdO), 149.82 (d, J ) 12.8 Hz, C-2 CdO), 145.28 (d, J CF ) 242.8
Hz, C-5), 136.47 (d, J CCF ) 25.3 Hz, C-6), 133.36, 129.02, 128.53
and 128.38 (phenyl carbons), 82.61 (C-1′), 78.92 (C-3′), 77.57
(C-4′), 62.16 (C-5′), 31.75 (C-2′). Anal. (C16H14FN3O8) C, H, N.
5-Iod o-3′-O-n it r o-5′-O-b en zoyl-2′-d eoxyu r id in e (9d ).
1
Meth od A: White foam; yield, 2.8%; mp 85-86 °C; H NMR
(CDCl3): δ 9.40 (s, 1H, NH), 8.02-8.11 (m, 2H, o-benzoyl
hydrogens), 7.89 (s, 1H, H-6), 7.60-7.70 (m, 1H, p-benzoyl
hydrogens), 7.43-7.52 (m, 2H, m-benzoyl hydrogens), 6.21 (dd,
J ) 6.6, 5.7 Hz, 1H, H-1′), 5.64 (d, J ) 6.6 Hz, 1H, H-3′), 4.68-
4.76 (m, 2H, H-5′), 4.58 (br s, 1H, H-4′), 2.81 (dd, J ) 14.4, 5.1
Hz, 1H, H-2′R), 2.37 (ddd, J ) 14.7, 7.5, 7.2 Hz, 1H, H-2′â);
13C NMR (CDCl3): δ 165.87, 159.53, 149.47, 143.43, 133.84,
129.60, 128.87, 128.81, 85.92, 82.60, 81.52, 69.03, 63.91, 36.97.
Anal. (C16H14IN3O8) C, H, N.
5-Flu or o-2,3′-an h ydr o-5′-O-ben zoyl-2′-deoxyu r idin e (8c).
White solid; yield, 90%; mp 235-236 °C; 1H NMR (DMSO-
d6): δ 8.10 (d, J ) 5.4 Hz, 1H, H-6), 7.89-7.91 (m, 2H,
o-benzoyl hydrogens), 7.62-7.68 (m, 1H, p-benzoyl hydrogen),
7.46-7.51 (m, 2H, m-benzoyl hydrogens), 5.92 (d, J ) 3.9 Hz,
1H, H-1′), 5.48 (br s, 1H, H-3′), 4.54-4.65 (m, 2H, H-5′), 4.43
(dd, J ) 11.7, 6.3 Hz, 1H, H-4′), 2.67-2.71 (m, 1H, H-2′R), 2.56
Meth od B: A mixture of 9a (1.0 g, 2.65 mmol) and NaN3
(0.69 g, 10.6 mmol) in dry MeCN (50 mL) was cooled in an
ice-bath, a solution of ICl (1.07 g, 6.6 mmol) in MeCN (5 mL)
was added dropwise during 5 min, and the reaction was
allowed to proceed at 25 °C for 48 h with stirring under an
argon atmosphere. Removal of the solvent in vacuo gave a
residue that was purified via flash silica gel column chroma-
tography using hexanes-EtOAc (1:1, v/v) as eluent to afford
9d (1.1 g, 82.5%) as a white foam, which was identical (mp,
1H NMR) to 9d described above under Method A.
Gen er a l Meth od for th e P r ep a r a tion of 5-Su bstitu ted -
3′-O-n itr o-2′-d eoxyu r id in es (10a -d ). A solution of NaOMe
in MeOH (12.75 mL of 0.5 M) was added to either 9a , 9b, 9c,
or 9d (3.18 mmol) and the reaction was allowed to proceed at
25 °C for 1 h with stirring. The reaction was quenched by
addition of NH4Cl (0.5 g), and the solvent was removed in
vacuo. Purification of the residue by flash silica gel column
chromatography using MeOH-CH2Cl2 (1:9, v/v) as eluent
yielded the respective product 10a , 10b, 10c, or 10d . Physical
and spectral data for 10a -d are listed below.
(dt, J ) 13.5, 2.7 Hz, 1H, H-2′â); 13C NMR (DMSO-d6):
δ
165.11 (CO2), 162.16 (d, J CCF ) 16.5 Hz, C-4 CO), 151.22 (C-
2), 144.31 (d, J CF ) 248.3 Hz, C-5), 133.32, 128.99 and 128.51
(phenyl CH), 128.90 (phenyl C-1), 125.34 (d, J CCF ) 33.0 Hz,
C-6), 87.45 (C-1′), 82.11 (C-3′), 77.70 (C-4′), 62.21 (C-5′), 32.46
(C-2′). Anal. (C16H13FN2O5) C, H, N.
5-Iod o-2,3′-a n h yd r o-5′-O-ben zoyl-2′-d eoxyu r id in e (8d ).
White solid; yield, 82%; mp 224-225 °C; 1H NMR (DMSO-
d6): δ 8.30 (s, 1H, H-6), 7.86-7.94 (m, 2H, o-benzoyl hydro-
gens), 7.60-7.67 (m, 1H, p-benzoyl hydrogen), 7.46-7.51 (m,
2H, m-benzoyl hydrogens), 5.98 (d, J ) 3.6 Hz, 1H, H-1′), 5.46
(br s, 1H, H-3′), 4.54-4.61 (m, 2H, H-5′), 4.42 (ddd, J ) 11.7,
6.3, 5.4 Hz, 1H, H-4′), 2.55-2.88 (m, 2H, H-2′); 13C NMR
(DMSO-d6): δ 166.57, 165.14, 153.80, 145.05, 133.34, 128.99,
128.88, 128.55, 87.15, 81.99, 80.68, 77.64, 62.16, 32.48. Anal.
(C16H13IN2O5) C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of 5-Su bstitu ted -
3′-O-n itr o-5′-O-ben zoyl-2′-d eoxyu r id in es (9a -d ). A mix-
ture containing either 8a , 8b, 8c, or 8d (26.1 mmol) and
NH4NO3 (31.3 g, 391.5 mmol) in dry DMF (120 mL) was stirred
at 110-120 °C for 12 h under argon. The solvent was removed
in vacuo, and the residue was purified via silica gel flash
column chromatography using hexanes-EtOAc (1:3, 1:2, 1:7
or 1:1, v/v, respectively) as eluent to give the corresponding
product 9a , 9b, 9c, or 9d . Physical and spectral data for 9a -d
are listed below.
3′-O-Nitr o-2′-d eoxyu r id in e (10a ) was obtained directly as
white crystals; yield, 91%; mp 188-190 °C; 1H NMR (DMSO-
d6): δ 11.35 (br s, 1H, NH), 7.88 (d, J ) 8.4 Hz, 1H, H-6), 6.13
(dd, J ) 8.7, 5.7 Hz, 1H, H-1′), 5.67 (d, J ) 8.4 Hz, 1H, H-5),
5.58 (d, J ) 6.0 Hz, 1H, H-3′), 5.25 (br s, 1H, 5′-OH), 4.21 (d,
J ) 1.8 Hz, 1H, H-4′), 3.60-3.74 (m, 2H, H-5′), 2.45-2.56 (m,
1H, H2′R), 2.40 (ddd, J ) 14.7, 8.7, 6.3 Hz, 1H, H-2′â); 13C
NMR (DMSO-d6): δ 162.86, 150.25, 140.12, 102.13, 84.59,
83.84, 82.46, 61.26, 35.23. Anal. (C9H11N3O7) C, H, N.
3′-O-Nitr o-2′-d eoxyth ym id in e (10b). White crystals (CH2-
3′-O-Nitr o-5′-O-ben zoyl-2′-deoxyu r idin e (9a). White crys-
1
tals (CH2Cl2-hexane); yield, 31%; mp 168-170 °C; H NMR
1
Cl2-hexane); yield, 92%; mp 137-139 °C; H NMR (DMSO-
(DMSO-d6): δ 11.43 (s, 1H, NH), 7.99 (d, J ) 7.8 Hz, 2H,
o-benzoyl hydrogens), 7.67 (d, J ) 7.8 Hz, 1H, H-6), 7.65-
7.70 (m, 1H, p-benzoyl hydrogen), 7.52-7.57 (m, 2H, m-benzoyl
hydrogens), 6.13 (dd, J ) 7.8, 6.3 Hz, 1H, H-1′), 5.76 (dd, J )
5.4, 1.2 Hz, 1H, H-3′), 5.60 (d, J ) 7.8 Hz, 1H, H-5), 4.48-
4.61 (m, 3H, H-4′, H-5′), 2.56-2.74 (m, 2H, H-2′); 13C NMR
(DMSO-d6): δ 165.32, 162.77, 150.15, 140.44, 133.48, 129.15,
129.06, 128.72, 102.06, 84.78, 83.29, 79.17, 64.04, 34.24. Anal.
(C16H15N3O8) C, H, N.
d6): δ 11.36 (s, 1H, NH), 7.72 (s, 1H, H-6), 6.14 (dd, J ) 8.4,
6.0 Hz, 1H, H-1′), 5.59 (d, J ) 5.7 Hz, 1H, H-3′), 5.31 (t, J )
5.4 Hz, 1H, 5′-OH), 4.18 (d, J ) 1.8 Hz, 1H, H-4′), 3.67 (dd, J
) 5.1, 3.3 Hz, 2H, H-5′), 2.37-2.52 (m, 2H, H-2′), 1.78 (s, 3H,
CH3); 13C NMR (DMSO-d6): δ 163.50 (C-4 CdO), 150.31 (C-2
CdO), 135.66 (C-6), 109.76 (C-5), 84.54 (C-1′), 83.45 (C-3′),
82.23 (C-4′), 61.28 (C-5′), 34.91 (C-2′), 12.24 (CH3). Anal.
(C10H13N3O7) C, H, N.
5-F lu or o-3′-O-n itr o-2′-d eoxyu r id in e (10c) was obtained
directly as white crystals; yield, 95%; mp 195-197 °C; 1H NMR
(DMSO-d6): δ 11.20 (br s, 1H, NH), 7.76 (d, J ) 6.0 Hz, 1H,
H-6), 6.49 (d, J ) 2.7 Hz, 1H, H-1′), 5.28 (d, J ) 1.2 Hz, 1H,
H-3′), 5.02 (br s, 1H, 5′-OH), 4.20 (dt, J ) 6.3, 2.4 Hz, 1H,
H-4′), 3.42-3.58 (m, 2H, H-5′), 2.45-2.56 (m, 2H, H-2′); 13C
NMR (DMSO-d6): δ 153.31 (d, J CCF ) 26.4 Hz, C-4 CdO),
150.12 (C-2 CdO), 145.17 (d, J CF ) 241.7 Hz, C-5), 136.04 (d,
J CCF ) 29.7 Hz, C-6), 85.85 (C-1′), 78.62 (C-3′), 77.26 (C-4′),
59.28 (C-5′), 31.68 (C-2′). Anal. (C9H10FN3O7) C, H, N.
5-Iod o-3′-O-n itr o-2′-d eoxyu r id in e (10d ) was obtained
directly as white crystals; yield, 92%; mp 170-172 °C; 1H NMR
(DMSO-d6): δ 11.74 (s, 1H, NH), 8.36 (s, 1H, H-6), 6.09 (dd, J
) 8.4, 6.0 Hz, 1H, H-1′), 5.59 (d, J ) 5.7 Hz, 1H, H-3′), 5.40 (t,
J ) 5.1 Hz, 1H, 5′-OH), 4.23 (br d, J ) 1.8 Hz, 1H, H-4′), 3.62-
3.80 (m, 2H, H-5′), 2.40-2.56 (m, 2H, H-2′); 13C NMR (DMSO-
d6): δ 160.13, 149.87, 144.51, 84.34, 84.18, 82.69, 69.76, 61.10,
35.54. Anal. (C9H10IN3O7) C, H, N.
3′-O-Nitr o-5′-O-ben zoyl-2′-d eoxyth ym id in e (9b). White
crystals (CH2Cl2-hexane); yield, 34%; mp 109-110 °C; 1H
NMR (DMSO-d6): δ 11.40 (s, 1H, NH), 8.02 (dd, J ) 8.4, 1.5
Hz, 2H, o-benzoyl hydrogens), 7.66-7.71 (m, 1H, p-benzoyl
hydrogen), 7.52-7.57 (m, 2H, m-benzoyl hydrogens), 7.44 (s,
1H, H-6), 6.17 (dd, J ) 8.4, 6.9 Hz, 1H, H-1′), 5.80 (br d, J )
6.6 Hz, 1H, H-3′), 4.62 (dt, J ) 7.8, 2.1 Hz, 1H, H-4′), 4.49-
4.57 (m, 2H, H-5′), 2.57-2.72 (m, 2H, H-2′), 1.59 (s, 3H, CH3);
13C NMR (DMSO-d6): δ 165.30, 163.35, 150.16, 135.43, 133.49,
129.14, 129.06, 128.74, 109.89, 83.99, 83.32, 78.97, 64.03,
34.21, 11.79. Anal. (C17H17N3O8) C, H, N.
5-F lu or o-3′-O-n itr o-5′-O-ben zoyl-2′-d eoxyu r id in e (9c).
White crystals (EtOAc-hexane); yield, 50%; mp 168-170 °C;
1H NMR (DMSO-d6): δ 11.15 (br s, 1H, NH), 7.86 (dd, J )
8.4, 1.5 Hz, 2H, o-benzoyl hydrogens), 7.76 (d, J ) 6.0 Hz, 1H,
H-6), 7.63-7.68 (m, 1H, p-benzoyl hydrogen), 7.47-7.52 (m,
2H, m-benzoyl hydrogen), 6.55 (d, J ) 3.3 Hz, 1H, H-1′), 5.46
(br d, J ) 1.5 Hz, 1H, H-3′), 4.52-4.62 (m, 2H, H-5′), 4.43 (dd,
J ) 11.7, 6.0 Hz, 1H, H-4′), 2.56-2.63 (m, 2H, H-2′); 13C NMR
(DMSO-d6): δ 165.11 (CO2), 153.34 (d, J CCF ) 26.4 Hz, C-4
Gen er a l
Meth od
for
th e
P r ep a r a tion
of
5-Su b st it u t ed -4-(1,2,4-t r ia zolo)-4-d eoxy-3′-O-n it r o-5′-O-

Nitrooxy Pyrimidine Nucleoside Nitrate Esters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1001
ben zoyl-2′-d eoxyu r id in e (11a ,b). 4-Chlorophenyl dichloro-
phosphate (0.88 mL, 5.32 mmol), and then 1,2,4-triazole (0.74
g, 10.64 mmol), was added to a solution of either 9a or 9b (5.32
mmol) in pyridine (20 mL) at 0 °C with stirring. The reaction
was allowed to proceed at 25 °C for 4 days with stirring, and
the solvent was removed in vacuo. The residue was dissolved
in CH2Cl2 (200 mL), this solution was washed with water (2
× 50 mL) and aqueous NaHCO3 (50 mL), the organic fraction
was dried (Na2SO4), and the solvent was removed in vacuo.
Purification of the residue by flash silica gel column chroma-
tography using hexanes-EtOAc (1:3 and 1:5, v/v, respectively)
gave the respective product 11a or 11b. Physical and spectral
data for 11a ,b are listed below.
165.16, 154.66, 137.54, 133.48, 129.06, 128.73, 101.81, 84.98,
83.87, 79.08, 64.22, 35.04, 12.90. Anal. (C17H18N4O7) C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of 5-Su bstitu ted -
3′-O-n itr o-2′-d eoxycytid in es (13a ,b). A solution of NaOMe
in MeOH (3.7 mL of 0.5 M) was added to either 12a or 12b
(0.93 mmol), the mixture was stirred at 25 °C for 15 min, and
the solvent was removed in vacuo. Purification of the residue
by flash silica gel column chromatography using MeOH:CH2-
Cl2 (1:4, v/v) as eluent afforded the respective products 13a
and 13b. Physical and spectral data for 13a ,b are listed below.
3′-O-Nitr o-2′-d eoxycytid in e (13a ). White foam; yield,
1
95%; mp 65-66 °C (lit.32 mp 177-178 °C); H NMR (DMSO-
d6): δ 7.80 (d, J ) 7.8 Hz, 1H, H-6), 7.20 and 7.26 (two s, 1H
each, NH2), 6.14 (dd, J ) 8.7, 5.4 Hz, 1H, H-1′), 5.76 (d, J )
7.8 Hz, 1H, H-5), 5.59 (d, J ) 6.0 Hz, 1H, H-3′), 5.25 (t, J )
5.4 Hz, 1H, 5′-OH), 4.19 (br d, J ) 1.8 Hz, 1H, H-4′), 3.62-
3.66 (m, 2H, H-5′), 2.45-2.52 (m, 1H, H-2′R), 2.29 (ddd, J )
14.7, 6.0, 2.7 Hz, 1H, H-2′â); 13C NMR (DMSO-d6): δ 163.67,
152.34, 141.70, 94.37, 85.02, 84.80, 82.78, 61.09, 35.88. Anal.
(C9H12N4O6) C, H, N.
4-(1,2,4-Tr ia zolo)-4-d eoxy-3′-O-n it r o-5′-O-b en zoyl-2′-
d eoxyu r id in e (11a ) was obtained directly as white crystals
that were washed with cold ether; yield, 79%; mp 85-87 °C;
1H NMR (CDCl3): δ 9.20 (s, 1H, triazole hydrogen), 8.23 (d, J
) 7.2 Hz, 1H, H-6), 8.09 (s, 1H, triazole hydrogen), 7.89 (dd,
J ) 8.7, 1.2 Hz, 2H, o-benzoyl hydrogens), 7.49-7.58 (m, 1H,
p-benzoyl hydrogen), 7.32-7.42 (m, 2H, m-benzoyl hydrogens),
6.94 (d, J ) 7.2 Hz, 1H, H-5), 6.20 (dd, J ) 7.5, 5.7 Hz, 1H,
H-1′), 5.65 (d, J ) 6.9 Hz, 1H, H-3′), 4.81 (dd, J ) 12.0, 3.3
Hz, 1H, H-5′a), 4.72 (dd, J ) 5.1, 3.0 Hz, 1H, H-4′), 4.66 (dd,
J ) 12.0, 3.6 Hz, 1H, H-5′b), 3.18 (ddd, J ) 15.3, 7.2, 1.5 Hz,
1H, H-2′R), 2.33-2.43 (m, 1H, H-2′â); 13C NMR (CDCl3): δ
165.72 (CO2), 159.42 (C-2 CdO), 153.88 (C-4), 145.19 (C-6),
143.18 (phenyl C-1), 133.78, 129.34, 128.77 and 128.68 (phenyl
and triazole CH), 94.74 (C-5), 88.47 (C-1′), 82.78 (C-3′), 82.46
(C-4′), 63.85 (C-5′), 38.01 (C-2′). Anal. (C18H16N6O7) C, H, N.
5-Meth yl-3′-O-n itr o-2′-d eoxycytid in e (13b). Colorless oil,
which was crystallized from EtOAc-hexane to give white
1
crystals; yield, 94%; mp 141-143 °C; H NMR (DMSO-d6): δ
7.62 (s, 1H, H-6), 7.35 and 6.90 (two s, 1H each, NH2), 6.14
(dd, J ) 9.0, 5.7 Hz, 1H, H-1′), 5.57 (d, J ) 5.1 Hz, 1H, H-3′),
5.35 (br s, 1H, 5′-OH), 4.16 (d, J ) 2.1 Hz, 1H, H-4′), 3.60-
3.70 (m, 2H, H-5′), 2.41-2.49 (m, 1H, H-2′R), 2.25-2.36 (m,
1H, H-2′â), 1.84 (s, 3H, CH3); 13C NMR (DMSO-d6): δ 165.40
(C-2 C)O), 155.10 (C-4), 138.12 (C-6), 101.96 (C-5), 84.88 and
84.79 (C-1′ and C-3′), 82.36 (C-4′), 61.45 (C-5′), 35.70 (C-2′),
13.30 (CH3). Anal. (C10H14N4O6) C, H, N.
4-(1,2,4-Tr ia zolo)-4-d eoxy-3′-O-n it r o-5′-O-b en zoyl-2′-
d eoxyth ym id in e (11b). White foam; yield, 71%; mp 59-60
°C; 1H NMR (DMSO-d6): δ 9.29 (s, 1H, triazole hydrogen), 8.35
(s, 1H, triazole hydrogen), 8.24 (s, 1H, H-6), 7.89 (dd, J ) 8.4,
1.5 Hz, 2H, o-benzoyl hydrogens), 7.57-7.62 (m, 1H, p-benzoyl
hydrogen), 7.43-7.48 (m, 2H, m-benzoyl hydrogens), 6.15 (dd,
J ) 7.2, 6.6 Hz, 1H, H-1′), 5.85 (dd, J ) 4.8, 2.1 Hz, 1H, H-3′),
4.83 (br dd, J ) 6.3, 4.2 Hz, 1H, H-4′), 4.73 (dd, J ) 12.3, 3.9
Hz, 1H, H-5′a), 4.60 (dd, J ) 12.3, 4.5 Hz, 1H, H-5′b), 2.91
(ddd, J ) 15.3, 6.6, 1.5 Hz, 1H, H-2′R), 2.68-2.80 (m, 1H,
H-2′â), 2.10 (s, 3H, CH3); 13C NMR (DMSO-d6): δ 165.24,
157.85, 152.71, 147.35, 147.29, 133.39, 129.08, 128.96, 128.92,
128.58, 104.66, 87.76, 83.64, 80.95, 64.14, 36.32, 15.76. Anal.
(C19H18N6O7) C, H, N.
5-Iod o-3′-O-n it r o-2′-d eoxycyt id in e (13c). A mixture of
13a (0.144 g, 0.53 mmol), iodine (0.135 g, 0.53 mmol), and iodic
acid (0.093 g, 0.53 mmol) in acetic acid, H2O, and CCl4 (8:3:2,
v/v/v, 26 mL) was stirred at 25 °C for 14 h. Removal of the
solvents in vacuo, coevaporation of toluene (2 × 20 mL) from
the residue, dissolution of the residue in CH2Cl2 (100 mL),
filtration, washing with aqueous NaHCO3 (50 mL), and drying
the organic fraction (Na2SO4) were carried out consecutively.
The solvent was removed in vacuo to give a residue that was
purified via flash silica gel column chromatography using
MeOH:CH2Cl2 (1:9, v/v) as eluent to afford 13c (0.118 g, 56%)
1
as a white foam: mp 81-82 °C; H NMR (DMSO-d6): δ 8.23
(s, 1H, H-6), 7.95 and 6.75 (two br s, 1H each, NH2), 6.09 (dd,
J ) 8.7, 5.7 Hz, 1H, H-1′), 5.59 (d, J ) 6.0 Hz, 1H, H-3′), 5.36
(t, J ) 5.4 Hz, 1H, 5′-OH), 4.20-4.28 (m, 1H, H-4′), 3.62-3.76
(m, 2H, H-5′), 2.49-2.56 (m, 1H, H-2′R), 2.36 (ddd, J ) 15.0,
8.4, 6.6 Hz, 1H, H-2′â); 13C NMR (DMSO-d6): δ 163.58, 153.61,
147.07, 85.11, 84.83, 82.63, 61.13, 57.04 (C-5), 36.10. Anal.
(C9H11IN4O6) C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of 5-Su bstitu ted -
3′-O-n itr o-5′-O-ben zoyl-2′-d eoxycytid in es (12a ,b). A solu-
tion of either 11a or 11b (3.74 mmol) in NH4OH/dioxane (1:3,
v/v, 80 mL) was stirred at 25 °C for 5 h prior to removal of the
solvent in vacuo. The residue was purified via flash silica gel
column chromatography using MeOH:CH2Cl2 (1:9, v/v) as
eluent to give the respective products 12a and 12b. Physical
and spectral data for 12a ,b are listed below.
Gen er a l Meth od for th e P r ep a r a tion of 5-Su bstitu ted -
5′-O-n itr o-2′-d eoxyu r id in es (15a ,b). A mixture of either 14a
or 14b (6.54 mmol) and LiNO3 (4.48 g, 65 mmol) in dry DMF
(30 mL) was stirred at 100 °C for 14 h under argon. Removal
of the solvent in vacuo gave a residue, toluene (20 mL) was
added, and the solvent was removed in vacuo. Purification of
the residue via flash silica gel column chromatography using
a gradient of 5% f10% MeOH in CH2Cl2 as eluent gave a pale
yellow oil which was recrystallized from MeOH to yield the
respective product 15a or 15b. Physical and spectral data for
15a ,b are listed below.
5′-O-Nitr o-2′-d eoxyu r id in e (15a ). Yield, 73%; mp 170-
172 °C (lit.27 mp 175 °C); 1H NMR (DMSO-d6): δ 11.34 (s, 1H,
NH), 7.65 (d, J ) 8.1 Hz, 1H, H-6), 6.16 (dd, J ) 7.2, 6.6 Hz,
1H, H-1′), 5.65 (d, J ) 8.1 Hz, 1H, H-5), 5.53 (d, J ) 3.9 Hz,
1H, 3′-OH), 4.79 (dd, J ) 11.4, 3.6 Hz, 1H, H-5′a), 4.66 (dd, J
) 11.4, 6.9 Hz, 1H, H-5′b), 4.20-4.27 (m, 1H, H-3′), 3.95-4.00
(m, 1H, H-4′), 2.09-2.28 (m, 2H, H-2′); 13C NMR (DMSO-d6) δ
162.81, 150.19, 140.65, 101.98, 84.59, 81.76, 73.05 (C-5′), 70.10,
38.19. Anal. (C9H11N3O7) C, H, N.
3′-O-Nitr o-5′-O-ben zoyl-2′-d eoxycytid in e (12a ) was ob-
tained directly as white crystals, which were washed with cold
ether; yield, 49%; mp 139-141 °C; 1H NMR (DMSO-d6): δ 7.98
(dd, J ) 8.4, 1.2 Hz, 2H, o-benzoyl hydrogens), 7.68 (ddt, J )
14.7, 7.5, 1.2 Hz, 1H, p-benzoyl hydrogen), 7.62 (d, J ) 7.8
Hz, 1H, H-6), 7.51-7.56 (m, 2H, m-benzoyl hydrogens), 7.23
(s, 2H, NH2), 6.13 (dd, J ) 7.8, 6.0 Hz, 1H, H-1′), 5.77 (d, J )
4.8 Hz, 1H, H-3′), 5.68 (d, J ) 7.8 Hz, 1H, H-5), 4.51-4.60 (m,
3H, H-4′, H-5′), 2.52-2.61 (m, 2H, H-2′); 13C NMR (DMSO-
d6): δ 165.80, 165.43, 165.24 and 154.57 (quaternary carbons),
140.68 (C-6), 133.34, 129.05, and 128.60 (phenyl CH), 94.30
(C-5), 85.60 (C-1′), 83.77 (C-3′), 79.21 (C-4′), 64.19 (C-5′), 35.04
(C-2′). Anal. (C16H16N4O7) C, H, N.
5-Meth yl-3′-O-n itr o-5′-O-ben zoyl-2′-deoxycytidin e (12b)
was obtained directly as white crystals, which were washed
with cold ether; yield, 36%; mp 158-160 °C; 1H NMR (DMSO-
d6): δ 8.15 (d, J ) 8.1 Hz, 2H, o-benzoyl hydrogens), 7.76-
7.85 (m, 1H, p-benzoyl hydrogen), 7.59-7.71 (m, 2H, m-benzoyl
hydrogens), 7.55 (br s, 1H, NHa), 7.49 (s, 1H, H-6), 7.00 (br s,
1H, NHb), 6.32 (dd, J ) 8.1, 6.0 Hz, 1H, H-1′), 5.90-5.96 (m,
1H, H-3′), 4.63-4.79 (m, 3H, H-4′, H-5′), 2.60-2.70 (m, 2H,
H-2′), 1.77 (s, 3H, CH3); 13C NMR (DMSO-d6): δ 165.30, 165.22,
5′-O-Nitr o-2′-d eoxyth ym id in e (15b). Yield, 71%; mp 183-
185 °C (lit.,28 mp 189 °C); 1H NMR (DMSO-d6): δ 11.32 (s,
1H, NH), 7.46 (s, 1H, H-6), 6.19 (t, J ) 6.9 Hz, 1H, H-1′), 5.51
(d, J ) 3.3 Hz, 1H, 3′-OH), 4.81 (dd, J ) 11.4, 3.9 Hz, 1H,

1002 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Naimi et al.
H-5′a), 4.69 (dd, J ) 11.4, 6.6 Hz, 1H, H-5′b), 4.28-4.30 (m,
1H, H-3′), 3.96 (ddd, J ) 6.6, 3.9, 3.6 Hz, 1H, H-4′), 2.25 (ddd,
J ) 14.1, 7.2, 6.9 Hz, 1H, H-2′R), 2.11 (ddd, J ) 14.1, 6.3, 3.9
Hz, 1H, H-2′â), 1.78 (s, 3H, CH3); 13C NMR (DMSO-d6): δ
163.50, 150.28, 135.99, 109.79, 84.08, 81.69, 73.07 (C-5′), 70.26,
38.08, 12.07. Anal. (C10H13N3O7) C, H, N.
In Vit r o Cell Cyt ot oxicit y (MTT a ssa y). KBALB,
KBALB-STK, human 143B, and human 143B-LTK cells were
cultured in complete DMEM medium supplemented with 10%
fetal bovine serum (FBS), and EMT-6 cells were cultured in
complete WAYMOUTH medium in 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 DMEM medium, and
100 µL of this solution was added to cells in 96-well plates to
produce the preselected test compound concentration. DMEM
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 bro-
mide (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
DMEM 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,46 which depends on the metabolic reduction of
MTT to colored formazan, measures cytostatic and cytotoxic
effects of the test drug.
An tivir a l Activity Assa ys. The antiviral assays were
based on an inhibition of virus-induced cytopathicity in either
E6SM, HeLa or Vero cell cultures, following previously estab-
lished procedures.47 Herpes simplex virus type 1 (HSV-1)
(KOS), HSV-2 (G), vaccinia virus, vesicular stomatitis virus
(VSV), and the thymidine kinase-deficient HSV-1 TK- KOS
(ACVr) strains were propagated in E6SM cell cultures, parain-
fluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie B4 virus
and Punta Toro virus in Vero cell cultures, and respiratory
syncytial virus in HeLa cell cultures. Briefly, confluent cell
cultures in microtiter plates were exposed to 100 CCID50 of
virus, 1 CCID50 being the virus dose required to infect 50% of
the cell cultures. After a 1 h virus adsorption period, residual
virus was removed, and the cell cultures were incubated in
the presence of varying concentrations (400, 100, etc., µg/mL)
of the test compounds. Viral cytopathicity was recorded as soon
as it reached completion in the control virus-infected cell
cultures.
5-Iod o-5′-O-n itr o-2′-d eoxyu r id in e (15c). A mixture of 15a
(0.2 g, 0.74 mmol) and NaN3 (0.19 g, 2.94 mmol) in dry MeCN
(20 mL) was cooled in an ice-bath. A solution of ICl (0.36 g,
2.2 mmol) in MeCN (3 mL) was added dropwise during 5 min,
and the reaction was allowed to proceed at 25 °C for 48 h with
stirring under argon. The solvent was removed in vacuo and
the residue was purified via silica gel flash column chroma-
tography using a gradient of 5% f 10% MeOH in CH2Cl2 as
eluent to give 15c (0.19 g, 65%) as white crystals: mp 181-
183 °C; 1H NMR (DMSO-d6): δ 11.80 (s, 1H, NH), 8.10 (s, 1H,
H-6), 6.18 (dd, J ) 6.9, 3.9 Hz, 1H, H-1′), 5.59 (d, J ) 4.2 Hz,
1H, 3′-OH), 4.75-4.94 (m, 2H, H-5′), 4.33-4.37 (m, 1H, H-3′),
4.06 (ddd, J ) 6.9, 6.6, 3.6 Hz, 1H, H-4′), 2.40 (ddd, J ) 14.1,
7.2, 6.9 Hz, 1H, H-2′R), 2.19 (ddd, J ) 14.1, 6.6, 3.9 Hz, 1H,
H-2′â); 13C NMR (DMSO-d6): δ 160.21(C-4 CdO), 149.86 (C-2
CdO), 144.80 (C-6), 85.18 (C-1′), 82.08 (C-3′), 72.85 (C-5′),
70.13 (C-4′), 69.78 (C-5), 38.32. Anal. (C9H10IN3O7) C, H, N.
In Vitr o Nitr ic Oxid e Relea se Assa ys. 1. In cu ba tion
With 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.44 Briefly, a solution of the
test compound (1 mL of a 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 36 mM solution in 0.1 M
phosphate buffer, pH 7.4), and the mixture was incubated at
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 (prepared and stored in aqueous 5% phosphoric
acid) and 0.1% N-naphthylethylenediamine dihydrochloride 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
0-60 µM sodium nitrite were used to prepare a nitrite
absorbance versus concentration curve under the same ex-
perimental 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 1 mM
solution in 0.1 M phosphate buffer pH 7.4) was used, and no
L-cysteine was added.
3. In cu ba tion w ith Ra t Ser u m . Nitric oxide release was
measured by modification of the reported procedure for in vivo
serum samples.53 Briefly, rat serum was prepared by filtration
with a Millipore UL trafree-15 centrifugal filter. Stock solu-
tions of test compounds (1 × 10-2 M) were prepared, and 10
µL of each test compound solution was mixed with filtered
serum (90 µL), which corresponds to the reaction sample. Each
reaction sample was incubated for either 1 h or 16h at 37 °C
in the presence of 0.2 U/mL Aspergillus nitrate reductase, 50
mM HEPES buffer, 5 µM FAD, and 0.1 mM NADPH to provide
a total volume of 500 µL that was comprised of the following
composition for the blank and the reaction sample (in brack-
ets), respectively: water 300 µL (290 µL), 1 M HEPES 25 µL
(25 µL), serum 90 µL (100 µL), 0.1 mM FAD 25 µL (25 µL), 1
mM NADPH 50 µL (50 µL), 10U/mL Nase 10 µL (10 µL).
Following the incubation, 5 µL of lactate dehydrogenase (1500
U/mL) and 50 µL of 100 mM pyruvic acid were added to each
tube to oxidize any unreacted NADPH, and samples were
incubated for 10 min. Greiss reagent (550 µL) was then added
to each tube, the mixture was allowed to stand at 25 °C for 10
min, absorbance for each sample was determined at 540 nm,
and % nitric oxide release was calculated as indicated under
Procedure 1 described above.
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. The experiments done at the Rega
Institute were supported by the Fonds voor Weten-
schappelijk Onderzoek (FWO)-Vlaanderen Krediet no.
9.0104.98, the Geconcerteerde Onderzoeksacties (GOA)-
Vlaamse Gemeenschap contract no. 2000/12 and the
Belgian Federation Against Cancer (to J . B.). We thank
Ann Absillis, Anita Camps, Frieda De Meyer, Lizette
van Berckelaer, Lies Vandenheurck, and Anita Van
Lierde for excellent technical assistance.
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