5-Alkynyl analogs of arabinouridine and 2′-deoxyuridine: Cytostatic activity against herpes simplex virus and varicella-zoster thymidine kinase gene-transfected cells

Article abstract of DOI:10.1021/jm0701472

A group of arabinouridines (TMSEAU, EAU, IEAU-TA) and 2′- deoxyuridines (TMSEDU, EDU, IEDU) having a variety of substituents at the uracil C-5 position (trimethylsilylethynyl, TMSE; ethynyl, E; or iodoethynyl, IE), and the sugar C-2′ position (2′-arabino

Full text of DOI:10.1021/jm0701472

J. Med. Chem. 2007, 50, 2851-2857
2851
5-Alkynyl Analogs of Arabinouridine and 2′-Deoxyuridine: Cytostatic Activity against Herpes
Simplex Virus and Varicella-Zoster Thymidine Kinase Gene-Transfected Cells
†
†
‡
‡
‡
‡
Walter A. Cristofoli, Leonard I. Wiebe, Erik De Clercq, Graciela Andrei, Robert Snoeck, Jan Balzarini, and
Edward E. Knaus*,†
Faculty of Pharmacy and Pharmaceutical Sciences, UniVersity of Alberta, Edmonton, Alberta, Canada T6G 2N8, and Rega Institute for
Medical Research, Minderbroedersstraat 10, LeuVen B-3000, Belgium
ReceiVed February 8, 2007
A group of arabinouridines (TMSEAU, EAU, IEAU-TA) and 2′-deoxyuridines (TMSEDU, EDU, IEDU)
having a variety of substituents at the uracil C-5 position (trimethylsilylethynyl, TMSE; ethynyl, E; or
iodoethynyl, IE), and the sugar C-2′ position (2′-arabino OH in arabinouridine, AU; or 2′-deoxyribo H in
2′-deoxyuridine, DU) were prepared to acquire antiviral structure-activity relationships. A broad-spectrum
viral panel screen showed that these 5-alkynylarabino/deoxy-uridines exhibit moderate anti-HSV-1 activity,
with no difference in potency between arabinouridines and 2′-deoxyuridines. The 2′-deoxyuridines TMSEDU,
EDU, and IEDU, unlike the arabinouridines, exhibited potent antiviral activity against cytomegalovirus, but
they were also highly cytostatic. The abilities of the 5-alkynylarabino/deoxy-uridines to inhibit nontransfected
-
(
wild-type or thymidine kinase-deficient, tk ) and viral gene transfected (HSV-1, HSV-2, or VZV thymidine
+
kinase-positive, tk ) FM3A and OST (osteosarcoma) cells were determined. This group of 5-alkynylarabino/
deoxy-uridines showed an enhanced ability to inhibit cells transfected with a viral thymidine kinase gene
+
+
+
-
(
HSV-1tk , HSV-2tk , VZVtk ) relative to wild-type or thymidine kinase-deficient (tk ) cells.
Introduction
yuridine is less potent and approximately equiactive against
HSV-1 and HSV-2.
1
,2
The design of arabinouridines and 2′-deoxyuridines possess-
ing novel two-carbon substituents at the C-5 position, which
are potent and selective antivirals, represent an important area
The rational variation of substituents on the uracil base moiety
of pyrimidine nucleosides can confer new properties (oral
7
bioavailability, metabolic stability, pharmacokinetics) that the
of antiviral drug development. The discovery of (E)-5-(2-
bromovinyl)-2′-deoxyuridine (BVDU,a Brivudin), (E)-5-(2-
natural bases lack and, when incorporated into nucleic acids
by enzymatic processes, can further alter the structure and/or
function of these biopolymers. For example, 2′-deoxyuridine
derivatives, which possess a C-5 substituent two-carbon atoms
in length, usually exhibit antiviral, but not anticancer, activity.
Structure-activity relationship (SAR) correlations for a group
of 5-olefinic 2′-deoxyuridines indicated that optimum inhibition
of HSV-1 in vitro occurred when the 5-substituent was unsatur-
ated and conjugated with the uracil ring, was not longer than
four carbon atoms, had the (E)-stereochemistry, and included a
hydrophobic electronegative functionality such as (E)-CHd
bromovinyl)arabinouridine (BVAU), and 5-(prop-1-ynyl)-
arabinouridine (PAU) significantly boosted interest and contributed
to the concept of a specific antiviral therapy. First generation
antiherpetics, such as 5-iodo- and 5-trifluoromethyl-2′-deox-
yuridine, exhibited little if any selectivity in their antiviral action.
In contrast, BVDU, BVAU, and PAU are highly specific
inhibitors of herpes simplex virus (HSV-1) and varicella-zoster
virus (VZV) replication. This selectivity is attributed to specific
phosphorylation by virus-encoded thymidine kinase (TK) in
virus-infected, but not in uninfected, host cells.1-4 Within the
8
CHsI). 5-Alkynyl pyrimidine nucleosides subsequently re-
large group of 5-substituted pyrimidine nucleosides that have
been investigated, (E)-5-[2-halo (iodo, IVDU; bromo, BVDU;
ceived considerable attention to treat VZV infections.
1,2
Although 5-ethynyl-2′-deoxyuridine (5-CtCH) was highly
active against HSV-1, CMV, and VZV in vitro, it was also
cytotoxic. On the other hand, 5-(sCtCsCH3) derivatives of
arabinouridine and 2′-deoxyuridine exhibited highly selective
anti-VZV activity and they were 2-3 orders of magnitude less
cytotoxic. In this regard, changing the C-5 substituent from
chloro, CVDU]-vinyl and 5-(2-chloroethyl)-2′-deoxyuridine
5
(
CEDU) are among the most potent and selective in their action
against HSV-1. In this regard, CEDU showed efficacy against
systemic HSV-1 infection and HSV-1 encephalitis in mice at a
6
5
-15-fold lower concentration than BVDU. 5-Ethyl-2′-deox-
-
CtCH to -CtCsCH3 greatly increased VZV selectivity and
*
To whom correspondence should be addressed. Phone: 780-492-5993.
decreased cytotoxicity, and these effects could be attributed to
the inability of the -CtCsCH3 analogs to act as efficient
substrates for cellular TK or to decreased affinity of the
Fax: 780-492-1217. E-mail: eknaus@pharmacy.ualberta.ca.
†
University of Alberta.
Rega Institute for Medical Research.
Abbreviations: BVAU, (E)-5-(2-bromovinyl)arabinouridine; BVDU,
‡
a
9
monophosphate for thymidylate synthase (TS). The methyl
(
E)-5-(2-bromovinyl)-2′-deoxyuridine; CEDU, 5-(2-chloroethyl)-2′-deox-
yuridine; CMV, cytomegalovirus; CVDU, (E)-5-(2-chlorovinyl)-2′-deox-
yuridine; EAU, 5-ethynylarabinouridine; EDU, 5-ethynyl-2′deoxyuridine;
HEL, human embryonic lung; HSV-1, herpes simplex virus type-1; HSV-
(2.0 Å) and iodine (2.15 Å) substituents are often considered
to be isosteres in view of their similarities in size (Van der
Waals’ radius). For example, 5-iodo-2′-deoxyuridine is an
antimetabolite of the natural DNA substrate 5-methyl-2′-
deoxyuridine (2′-deoxythymidine). A structural comparison of
the linear 5-(CtCsI) substituent in 5-(2-iodoethynyl)-2′-
deoxyuridine (IEDU) with the (E)-5-(CHdCHsI) substituent
in (E)-5-(2-iodovinyl)-2′-deoxyuridine (IVDU) is illustrated in
2, herpes simplex virus type-2; IEAU-TA, 5-(2-iodoethynyl)arabinouridine-
2′,3′,5′-tri-O-acetate; IEDU, 5-(2-iodoethynyl-2′-deoxyuridine; IVDU, (E)-
5-(2-iodovinyl)-2′-deoxyuridine; PAU, 5-(prop-1-ynyl)arabinouridine; TK,
thymidine kinase; TMSEAU, 5-(2-trimethylsilylethynyl)arabinouridine;
TMSEAU-TA, 5-(2-trimethylsilylethynyl)arabinouridine-2′,3′,5′-tri-O-
acetate; TMSEDU, 5-(2-trimethylsilylethynyl)-2′-deoxyuridine; TS, thymidy-
late synthase; VZV, varicella-zoster virus.
1
0.1021/jm0701472 CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/23/2007

2
852 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Cristofoli et al.
Scheme 2a
Figure 1. Calculated bond angles and distances in IEDU and IVDU.
Scheme 1a
a
Reagents and conditions: (a) Me3SisCtCH, MeCN, Et3N, (PPh3)2PdCl2,
CuI, 50 °C, 3 h; (b) MeOH, 0.05 N NaOMe, 25 °C, 2 h; (c) AgNO3,
N-iodosuccinimide, DMF, 25 °C, 3 h (protected from light).
and CuI in Et3N and MeCN afforded TMSEDU (6, 79%), as
illustrated in Scheme 2. Although we and others have protected
1
7,20
21
the sugar hydroxyl groups as acetates,
p-chlorobenzoates,
9
or p-toluoates when a Sonogashira coupling reaction is
employed, the reaction works equally well with the unprotected
5
-iodo-2′-deoxyuridine (5). The TMS group in TMSEDU was
readily removed by treatment with 0.05 N NaOMe to yield the
9
previously reported EDU (7, 87%). The AgNO3-catalyzed
iodination of TMSEDU with N-iodosuccinimide in DMF yielded
IEDU (5, 40%). This two-step reaction that was developed in
1
a
Reagents and conditions: (a) 0.05 N NaOMe, MeOH, 25 °C, 1h; (b)
K2CO3, MeOH, benzene, 25 °C, 6 h; (c) AgNO3, N-iodosuccinimide, DMF,
5 °C, 3 h (protected from light).
996 in our research program is shorter than a recently reported20
2
synthesis of IEDU that involved the iodination of 3′,5′-di-O-
acetyl-5-ethynyl-2′-deoxyuriidne using iodonium di-syn-collidine
perchlorate.
Figure 1. The calculated interspatial distances between the iodine
atom and the C-5 carbon center (4.5 Å in IEDU and 4.3 Å in
IVDU) and the C-C5-C6 bond angles (120.2° in IEDU and
1
18.2° in IVDU) are relatively similar. It was, therefore,
Biological Results and Discussion
anticipated in 1996, when this investigation was carried out,
that arabinouridines and 2′-deoxyuridines possessing a linear
A group of arabinouridines (TMSEAU, EAU, IEAU-TA) and
2
′-deoxyuridines (TMSEDU, EDU, IEDU) having a variety of
5
-(CtCsI) substituent may act as antiviral agents due to their
10
substituents at the uracil C-5 position (trimethylsilylethynyl,
TMSE; ethynyl, E; or iodoethynyl, IE) and the sugar C-2′
position (2′-arabino OH in arabinouridine, AU; or 2′-deoxyribo
H in 2′-deoxyuridine, DU) were prepared (see structures in
Schemes 1 and 2) to acquire an antiviral structure-activity
relationship (SAR). This type of information is useful for the
design of chemotherapeutic agents to treat viral diseases and
selective phosphorylation by virus-infected cells, as radiop-
11,12
harmaceutical agents for imaging,
or chemotherapeutic
agents for treating13 herpes simplex virus type-1 thymidine
+
kinase positive (HSV-1 TK ) gene-transfected tumors (gene
therapy of cancer).1
4-16
We now report the synthesis, antiviral
activities, and cellular toxicities for a group of structurally related
-alkynyl analogs of arabinouridine and 2′-deoxyuridine.
5
radiopharmaceutical agents to monitor viral gene expression that
Chemistry
11-16
is beneficial in the gene therapy of cancer.
Accordingly,
Treatment of TMSEAU-TA (1), prepared according to the
method of Robins et al.,17 with 0.05 N NaOMe selectively
cleaved the acetate groups to afford the deprotected compound
TMSEAU (2) in 81% yield (see Scheme 1). Subsequent reaction
of TMSEAU with K2CO3 in MeOH removed the TMS moiety
to furnish EAU (3, 70%). A similar synthesis of EAU, using
the para-toluoyl ester analog of TMSEAU-TA, has been
biological evaluations were performed using an extensive battery
of antiviral assays (see Tables 1-5) and specific cell lines, both
wild-type and those that were transfected with a viral thymidine
+
+
+
kinase gene (HSV-1tk , HSV-2tk , or VZVtk ). The 2′,3′,5′-
tri-O-acetyl (TA) derivative (IEAU-TA) of 5-(2-iodoethynyl)-
arabinouridine (IEAU) was originally prepared with the view that
IEAU-TA would then serve as a prodrug that undergoes in vivo
cleavage by plasma esterases to release the parent compound.
The antiviral activities exhibited by the group of 5-alkyny-
larabino(deoxy)uridines was determined against a broad spec-
trum of viruses (see data in Table 1). This group of compounds,
except for IEAU-TA, exhibited moderate anti-HSV-1 inhibitory
activity (EC50 ) 0.7 to 2 µg/mL range) relative to the reference
drug BVDU. Antiviral activity against HSV-2 was maintained,
9
described. Reaction of TMSEAU-TA with N-iodosuccinimide
in the presence of AgNO3 catalyst in DMF, according to a
method originally developed by Isobe et al.18 for the elaboration
of RsCtCsTMS f RsCtCsI, furnished IEAU-TA (4,
7
8%).
The Sonogashira19 coupling reaction of 5-iodo-2′-deoxyuri-
dine (5) with Me3SisCtCH in the presence of (PPh3)2PdCl2

5
-Alkynyl Analogs of Arabinouridine and 2′-Deoxyuridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2853
Table 1. Cytotoxicity and Antiviral Activity of 5-Ethynyl Analogs of Arabino/deoxy-uridine in E6SM Cell Cultures
b
minimum inhibitory concentration: EC50 (µg/mL)
MCCa
(µg/mL)
HSV-1
(KOS)
HSV-2
(G)
vaccinia
virus
HSV-1
TK B2006
HSV-1
TK VMW1837
-
-
cmpd
TMSEAU
EAU
IEAU-TA
TMSEDU
EDU
400
400
40
>400
>400
g100
g400
400
0.7
0.7
20
2
0.7
2
0.02
70
0.002
0.02
2
7
7
7
2
7
7
>10
150
70
>10
4
0.7
2
40
20
150
>200
>10
7
0.2
0.1
0.2
0.2
4
7
40
70
0.02
7
IEDU
brivudin
ribavirin
7
c
150
150
0.007
0.07
d
20
>100
>400
e
ganciclovir
>100
>400
0.7
40
f
acyclovir
a
b
Minimum compound concentration required to cause a microscopically detectable alteration of normal cell morphology. Minimum inhibitory compound
concentration required to reduce virus-induced cytopathogenicity by 50%. (E)-5-(2-Bromovinyl)-2′-deoxyuridine. d 1-(â-D-ribofuranosyl)-1,2,4-thiazole-
-carboxamide. 9-[(1,3-Dihydroxy-2-propoxy)methyl]guanine. 9-[2-Hydroxyethoxy)methyl]guanine.
c
e
f
3
Table 2. Antiviral Activity of 5-Ethynyl Analogs of Arabino/
deoxy-uridine against Cytomegalovirus in Human Embryonic Lung Cells
1.0 µM range). In sharp contrast to the arabinouridines,
TMSEAU and EAU, the 2′-deoxyuridines TMSDU, EDU, and
IEDU retained appreciable antiviral activity against the TK
VZV strains 07/1 (EC50 ) 0.69 to 0.89 µM range) and YS/R
(EC50 ) 0.75 to 0.90 µM range).
a
-
antiviral activity EC50
cytoxic/cytostatic
activity
(µM)
cell
morphology
(MCC)
cell
b
growthc
(CC50)
Antiviral activity against cytomegalovirus (CMV) [strains
AD-169 and Davis] was determined using a cytopathicity (CPE)
reduction assay (Table 2). In these anti-CMV assays, the arabino
compounds TMSEAU, EAU (EC50s > 50 µM), and IEAU-TA
AD-169
davis
TMSEAU
EAU
IEAU-TA
TMSEDU
EDU
>50
>50
>20
1.2
>50
200
200
50
200
>200
200
127
>200
20
0.18
2.5
0.76
121
>50
>20
(EC50 > 20 µM) were all inactive. In contrast, the 2′-deoxyribo
0.75
1.2
2.5
0.66
6.3
0.85
0.58
0.66
2.75
compounds TMSEDU, EDU, and IEDU exhibited potent anti-
CMV activities against AD-169 (IC50 ) 1.2 to 2.5 µM range)
and Davis (IC50 ) 0.58 to 0.85 µM range) strains similar to the
reference drug cidofovir (IC50 ) 0.66 µM). The 2′-deoxyribo
compounds were virtually not cytotoxic (as their arabino
counterparts) (MCC g 50 µM) but markedly more cytostatic
(CC50 ) 0.18 to 2.5 µM range) than the arabino compounds
(CC50 ) 20 to >200 µM range). These anti-CMV and cell
cytotoxicity (CC50) data are consistent with previous studies
IEDU
cidofovir
d
ganciclovir
>200
a
Inhibitory concentration required to reduce virus plaque formation by
b
5
0%. Virus input was 100 plaque forming units (PFU). Minimum cytotoxic
concentration that causes a microscopically detectable alteration of cell
c
morphology. Cytotoxic compound concentration required to reduce cell
d
growth by 50%. 1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine.
9
showing that (i) EDU is more potent than EAU against CMV,
but at a lower potency range (EC50 ) 2-7 µg/mL). No distinct
differences in activities against HSV-1 or HSV-2 between the
arabinouridine (TMSEAU, EAU) and 2′-deoxyuridine (TM-
SEDU, EDU, IEDU) subclasses of compounds were observed.
On the other hand, there was a distinct difference in antiviral
9
(
ii) EDU is more cytostatic than EAU, (iii) IEDU is a more
potent anti-CMV agent than ribavirin, and (iv) IEDU is very
cytostatic to PBM and CEM cells. The high cytostatic activity
of the 5-alkynyl-2′-deoxyuridines may likely be due to inhibition
of TS and subsequent inhibition of cellular DNA synthesis.
2
0
20
-
activity against thymidine kinase-deficient HSV-1 tk (B2006
Other antiviral assays (data not shown) were performed to
determine the ability of these 5-alkynylarabino/deoxyuridines
to reduce virus-induced cytopathicity in HeLa cell cultures. All
compounds were inactive at the highest test compound con-
centration employed against vesicular stomatitis virus (>40 to
>400 µg/mL range; ribavirin IC50 ) 20 µg/mL), Coxsackie
virus B4 (>40 to >400 µg/mL range; ribavirin IC50 ) 20 µg/
mL), and respiratory syncytial virus (>40 to >400 µg/mL range;
ribavirin IC50 ) 2 µg/mL). Other antiviral assays (data not
shown) carried out using Vero cell cultures also indicated that
this group of arabino/deoxyuridines failed to reduce virus-
induced cytopathicity by 50% at the highest test compound
concentration employed against parainfluenza-3 virus (20 to
>400 µg/mL range), reovirus-1 (>40 to >400 µg/mL range,
ribavirin IC50 ) 0.7 µg/mL), Sindbis virus (>40 to >400 µg/
mL), and Punta Toro virus (20 to >400 µg/mL range, ribavirin
IC50 ) 20 µg/mL).
and VMW1837 strains) cells. In this regard, the arabinouridines
(TMSEAU, EAU), like BVDU, were inactive as expected, but
the 2′-deoxyuridines (TMSEDU, EDU, IEDU) retained similar
antiviral activity (IC50 ) 0.7-4 µg/mL, B2006 strain) or slightly
reduced potency (IC50 ) 4-7 µg/mL, VMW1837 strain) against
-
HSV-1 tk strains. The 2′-deoxyuridines (TMSEDU, EDU,
IEDU), which showed high potencies against vaccinia virus
(IC50 ) 0.1-0.2 µg/mL range) similar to the reference drug
BVDU (IC50 ) 0.2 µg/mL), were markedly more potent than
the arabinouridines (TMSEAU, EAU, IC50 ) 7 µg/mL). None
of the drugs showed appreciable cytotoxicity to the monolayer
E6SM cell cultures.
Additional antiviral studies using VZV-infected human
+
+
embryonic lung (HEL) cells [TK VZV (OKA), TK VZV
-
-
(
YS), TK-deficient VZV/TK (07/1) and TK -deficient VZV/
-
TK (YS/R)] were carried out to determine the inhibitory test
compound concentration required to reduce viral plaque forma-
tion by 50% (EC50, µM) for a viral input of 20 plaque-forming
units (PFU) against these VZV strains (see data in Table 3). In
these studies, the arabinouridines, TMSEAU and EAU, and the
The abilities of the 5-alkynylarabino/deoxyuridines to inhibit
-
nontransfected (wild-type and thymidine kinase deficient, tk )
and viral gene transfected (HSV-1, HSV-2 or VZV thymidine
+
2
′-deoxyuridines, TMSEDU, EDU and IEDU, all showed
kinase positive, tk ) FM3A (Table 4), and OST (osteosarcoma;
+
pronounced antiviral activity against the TK OKA strain
Table 5) cells have been investigated. The arabinouridines
TMSEAU and EAU, as expected, did not inhibit (IC50 g
(
IC50 ) 0.48 to 1.0 µM) and TK+ YS strain (IC50 ) 0.29 to

2
854 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Cristofoli et al.
Table 3. Activity of 5-Ethynyl Analogs of Arabino/deoxy-uridine against Varicella-Zoster Virus in Human Embryonic Lung Cells
antiviral activitya
cytotoxicity/cytostatic
activity (µM)
EC50 (µM)
TK+ VZV
OKA strain
TK- VZV
cell morphologyb
cell growthc
(CC50)
cmpd
YS strain
07/1 strain
YS/R strain
(MCC)
TMSEAU
EAU
IEAU-TA
TMSEDU
EDU
0.48
1.0
8.5
0.26
0.16
0.18
0.003
0.0001
0.33
0.29
3.1
0.15
0.11
0.11
0.005
0.0001
>50/>50
>50/>50
>20
>50/>50
>50/>50
>20
200
200
50
>200
>200
200
127
>200
20
0.18
2.5
0.76
>100
>50
0.69
0.90
0.81
0.89
92
>57
0.75
0.78
115
>57
IEDU
brivudin
d
>100
>50
d,e
sorividine
a
Inhibitory compound concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque forming units (PFU). Data from two
b
c
separate experiments are listed. Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. Cytotoxic
concentration required to reduce cell growth by 50%. Data taken from the literature.
d
22 e
(E)-5-(2-Bromovinyl)uridine.
-
+
Table 4. Inhibitory Effects of 5-Ethynyl Analogs of Arabino/
deoxy-uridine on the Proliferation of Murine Mammary Carcinoma
transfected cell lines (OSTtk /VZVtk -a, IC50 ) 0.71 to
-
+
0
.75 µM range and OSTtk /VZVtk -b, IC50 ) 1.9 to 2.2 µM
range). The 2′-deoxyuridines TMSEDU, EDU, and IEDU were
weak inhibitors of thymidine kinase-deficient cells (OSTtk ,
IC50 ) 22 to 34 µM range), as expected, but they are extremely
potent inhibitors of the tk gene-transfected cell lines OSTtk /
(
FM3A) Cell Lines Transfected with the HSV-1 or HSV-2 Thymidine
a
Kinase Gene
-
-
-
FM3Atk /
FM3Atk /
FM3Atk-
HSV-1tk+
HSV-2tk+
cmpd
FM3A/O
-
TMSEAU
EAU
IEAU-TA
TMSEDU
EDU
g250
g250
g250
g250
20 ( 3.4
g250
g250
87 ( 37
0.19 ( 0.15
3.8 ( 0.81
3.9 ( 1.64
0.29 ( 0.07
0.30 ( 0.06
0.96 ( 0.32
0.48 ( 0.14
0.39 ( 0.16
0.35 ( 0.17
0.002 ( 0.001
+
-
VZVtk -a (IC50 ) 0.041 to 0.083 µM range) and OSTtk /
+
18 ( 0.06
0.23 ( 0.04
0.18 ( 0.02
0.30 ( 0.08
13 ( 0.1
5.4 ( 0.02
VZVtk -b (IC50 ) 0.12 to 0.20 µM range). These latter data
0.16 ( 0.02
0.13 ( 0.02
0.16 ( 0.02
0.004 ( 0.001
are consistent with an earlier study that showed that EDU is a
9
more potent inhibitor than EAU of VZV-infected cells. The
IEDU
brivudinb
observation that IEDU-TA is more cytostatic to E6SM, HEL,
and FM3A/O cells than TMSEAU and EAU could be due to
its greater lipophilic character that may enhance its ability to
cross the cell membrane by passive diffusion. The fact that
IEAU-TA exhibits similar inhibitory effects against wild-type
a
IC50 (µM), 50% inhibitory compound concentration required to inhibit
b
FM3A cell proliferation by 50%; mean value ((s.d., n g 3). Data taken
2
3
from the literature.
Table 5. Cytostatic Activity of 5-Ethynyl Analogs of Arabino/
+
+
-
-
FM3A, transfected FM3A/HSV-1tk or FM3A/HSV-2tk cells
deoxy-uridine Against the Osteosarcoma Cell Lines OSTtk , OSTtk /
VZVtk -a and OSTtk /VZVtk -b Cells
+
-
+
a
-
-
+
(Table 4), and OSTtk and OSTtk /VZVtk cells (Table 5) is
in agreement with the belief that the high cellular toxicity may
be due to its high lipophilic character and lack of significant
further intracellular or extracellular metabolization by HSV-1,
HSV-2, or VZV TK.
OSTtk-
-
+
-
+
cmpd
OSTtk /VZVtk -a
OSTtk /VZVtk -b
TMSEAU
EAU
IEAU-TA
TMSEDU
EDU
IEDU
brivudin
sorivudine
g176
g250
0.75 ( 0.12
0.71 ( 0.13
3.6 ( 0.22
0.041 ( 0.044
0.083 ( 0.016
0.063 ( 0.016
0.60 ( 0.33
0.15 ( 0.09
1.9 ( 0.33
2.2 ( 0.93
3.9 ( 0.13
0.20 ( 0.13
0.16 ( 0.04
0.12 ( 0.02
0.35 ( 0.15
0.20 ( 0.09
5.40 ( 1.00
23 ( 6.02
23 ( 5.62
34 ( 22.86
g250
The differences in cytostatic and cytotoxic activity of the
arabinouridine versus the 2′-deoxyuridine derivatives are strik-
ing. Both series of compounds were not cytotoxic. That means
that in metabolically poorly active (resting, confluent) cell
cultures, the compounds are not converted to potentially toxic
metabolites and the compounds are not incorporated into DNA
due to lack of any significant DNA synthesis in these cells.
However, in actively growing (S-phase) thymidine kinase-
competent cell cultures, the arabinouridines were poorly cyto-
static, whereas the 2′-deoxyuridine counterparts were highly
cytostatic. These findings suggest that the 2′-deoxyuridines are
good substrates for cytosolic TK-1 and efficiently converted to
their active metabolites in the proliferating cells. Targets for
cytostatic activity may be TS and incorporation into DNA.
Indeed, it has been demonstrated earlier that EDU-5′-mono-
phosphate is a potent inhibitor of TS, resulting in efficient
g250
a
IC50 (µM), compound concentration required to inhibit cell proliferation
-
+
-
by 50%; mean value ((s.d., n g 3). OSTtk /VZVtk -a and OSTtk /
VZVtk -b are two different, independently obtained, VZVtk gene-transfected
OSTtk cell lines.
+
+
50 µM) wild-type FM3A/O or FM3Atk- (cells deficient in
2
-
cytosol tk) cells, but they did inhibit proliferation of FM3Atk /
+
-
HSV-1tk (IC50 ) 3.8 to 4.0 µM range) and FM3Atk /HSV-
+
2
tk (0.29 to 0.30 µM range) cells (Table 4). The 2′-
deoxyuridines TMSEDU, EDU, and IEDU did not appreciably
-
inhibit FM3Atk cell proliferation (IC50 ) 87 to g250 µM
-
+
+
range), but they markedly did inhibit FM3Atk /HSV-1tk
-
(
(
IC50 ) 0.13 to 0.16 µM range) and FM3Atk /HSV-2tk
2
4
inhibition of cell proliferation. TMSEDU and IEDU may have
a similar mechanism of antiproliferative action. Our data also
revealed that the arabinouridines are likely a poor substrate for
cytosolic TK-1, for one of the next phosphorylating (nucleotide
kinase) enzymes, and poorly inhibit their eventual cytostatic
target (i.e., TS or DNA synthesis). Instead, they are well-
recognized by the viral TKs, resulting in highly selective
antivirals that can also be of use as radiopharmaceuticals to
monitor viral tk gene expression in combined gene/chemo-
therapy of cancer.
0.35 to 0.48 µM range) cell proliferation. However, in contrast
with the arabinouridines, the 2′-deoxyribosides TMSDU, EDU,
and IEDU were also highly cytostatic to wild-type FM3A/O
cells (CC50 ) 0.18 to 0.30 µM range). A similar antiproliferative
activity was also observed for HEL cells (see Tables 2 and 3;
CC50 ) 0.18 to 2.5 µM). Similar cytostatic activity profiles were
observed using osteosarcoma cells transfected with the VZVtk
gene (see Table 5). For example, the arabinouridines TMSEAU
-
and EAU were inactive against OSTtk cells, but they were
potent inhibitors of cell proliferation of two VZVtk gene-

5
-Alkynyl Analogs of Arabinouridine and 2′-Deoxyuridine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2855
Conclusions
1H, J ) 4.4, 3.9 Hz, H-3′), 3.73 (br q, J ) 5.4, 4.4 Hz, 1H, H-4′),
3.59 (br m, 2H, H-5′).
A group of 5-alkynyl analogs of arabinouridine and 2′-
deoxyuridine were synthesized to evaluate their ability to inhibit
viral infection and their potential application for imaging/
monitoring viral gene thymidine kinase expression in the gene
therapy of cancer. In vitro SARs indicate that (i) high cellular
toxicity is a deterrent to the potential use of the highly potent
1
-(2,3,5-Tri-O-acetyl-â-D-arabinofuranosyl)-5-(2-iodoethynyl)-
uracil (4, IEAU-TA). AgNO (15 mg, 0.086 mmol) and N-
3
iodosuccinimide (136 mg, 0.60 mmol) were added to a solution of
TMSEAU-TA (200 mg, 0.43 mmol) in anhydrous DMF (4 mL).
The reaction flask was covered with aluminum foil and the reaction
was allowed to proceed at 25 °C with stirring for 3 h. The reaction
flask was then immersed in an ice bath, distilled water (15 mL)
was added, and the mixture was stirred for 5 min. This mixture
was extracted with chloroform (4 × 15 mL), the combined
chloroform extracts were washed with brine (3 × 50 mL), and the
5
-alkynyl-2′-deoxyuridines (TMSEDU, EDU) to treat CMV
infection and (ii) the high potency of the 5-trimethylsilylethynyl
analogs of arabinouridine (TMSEAU) against osteosarcoma or
+
+
mammary carcinoma VZVtk and HSV tk gene-transfected
2 4
chloroform fraction was dried (Na S0 ). Removal of the solvent in
-
-
cells, relative to VZVtk or HSV tk cells, suggests that these
precursors may be suitable candidates for radioiodination using
electrophilic radioiodine to prepare the radiopharmaceuticals
I]-IEAU and [ ]-IEDU to monitor viral gene expression
during gene therapy cancer protocols.
vacuo gave IEAU-TA (4), which was recrystallized from MeOH
l
to give yellow crystals (176 mg, 78%), mp 182-183 °C; H NMR
(
3
CDCl ) δ 8.56 (s, 1H, NH), 7.79 (s, 1H, H-6), 6.28 (d, 1H, J )
1
24
124
[
4.5 Hz, H-1′), 5.46 (m, 1H, H-2′), 5.17 (dd, 1H, J ) 4.2, 2.8 Hz,
25
H-3′), 4.44 (dd, J ) 11.2, 4.2 Hz, 1H, H-5′), 4.41 (dd, J ) 11.2,
5
3
.7 Hz, 1H, H-5′′), 4.22 (dd, J ) 9.8, 4.2 Hz, 1H, H-4′), 2.19 (s,
17
H, OAc), 2.17 (s, 3H, OAc), 2.15 (s, 3H, OAc). Anal. (C17H -
Experimental Section
IN
2
O
9
) C, H, N.
General. All chemicals and solvents used in this study were
purchased from Aldrich Chemical. 1-(2,3,5-Tri-O-acetyl-â-D-ara-
binofuranosyl)-5-[2-(trimethylsilyl)ethynyl]uracil (1) was prepared
according to a literature procedure.17 Reactions requiring anhydrous
conditions were performed under an atmosphere of argon. All
solvents were dried by standard methods26 and distilled just prior
to use or were purchased as anhydrous solvents in Sure-Sealed
bottles. Tetrahydrofuran was dried over sodium and benzophenone.
Dichloromethane, triethylamine, and acetonitrile were dried over
calcium hydride. Melting points were determined with a Thomas-
Hoover capillary apparatus. Flash column chromatography was
performed on silica gel 60 (E. Merck, 230-400 mesh). All columns
were dry packed and eluted using positive air pressure, as
1
-(2-Deoxy-â-D-ribofuranosyl)-5-[2-(trimethylsilyl)ethynyl]u-
racil (6, TMSEDU). 5-Iodo-2′-deoxyuridine (5, 1.0 g, 2.82 mmol)
was dissolved in MeCN/Et N (66 mL of 1:1, v/v) under an argon
atmosphere. Trimethylsilylacetylene (1.6 mL, 11.3 mmol), bis-
triphenylphosphine)palladium(II) chloride (42.2 mg, 0.60 mmol),
and CuI (28 mg, 0.15 mmol) were added, a condenser was fitted
to the flask, and the reaction flask was immersed into a preheated
oil bath (50 °C). The reaction was allowed to proceed for 3.5 h,
and the solvents were removed in vacuo to give a residue that was
purified by silica gel flash column chromatography. Elution with
3
(
CHCl
0.73 g, 79%), mp 63-64 °C; H NMR (MeOH-d
H-6), 6.21 (dd, J ) 4.5, 4.5 Hz, 1H, H-1′), 4.38 (ddd, J ) 5.5, 4.2,
.9 Hz, 1H, H-3′), 3.92 (dd, J ) 4.7, 3.1 Hz, 1H, H-4′), 3.81 (dd,
J ) 10.9, 3.1 Hz, 1H, H-5′), 3.72 (dd, J ) 12.8, 3.7 Hz, 1H, H-5′′),
.28 (m, 2H, H-2′), 0.19 [s, 1H, Si(CH ]; Exact mass (HRMS)
calcd for C14 Si, 324.1141; found, 324.1134. Anal.
Si) C, H, N.
-(2-Deoxy-â-D-ribofuranosyl)-5-ethynyluracil (7, EDU). A
3
/MeOH (9:1, v/v) afforded TMSEDU as a beige solid
1
(
4
) δ 8.52 (s, 1H,
27
recommended by Still et al. A Bruker AM-300 NMR spectrometer
3
1
was used to acquire H NMR spectra with TMS as internal standard.
Coupling constant (J) values are estimated in hertz (Hz), and spin
multiples are given as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), and br (broad). Microanalyses determined for C, H,
and N were within (0.4% of theoretical values.
2
3 3
)
20 2 5
H N O
14 20 2 5
(C H N O
1
1
-(â-D-Arabinofuranosyl)-5-[2-(trimethylsilyl)ethynyl]uracil
2, TMSEAU). In an argon purged flask, TMSEAU-TA (1,
.27 g, 0.58 mmol) was dissolved in anhydrous MeOH (5 mL). A
solution of NaOMe in MeOH (33 mL of 0.05 N) was added to a
solution of TMSEDU (6, 159 mg, 0.34 mmol) in anhydrous MeOH
(4 mL) under an argon atmosphere, and the reaction was allowed
to proceed with stirring at 25 °C for 2 h. The pH of the solution
was adjusted to pH 5-6 using prewashed Amberlite IR-120 acidic
resin, and the mixture was filtered through a Celite pad. Removal
of the solvent from the filtrate in vacuo furnished a residue that
was purified by silica gel column flash chromatography using
3
(
0
solution of 0.05 N NaOMe in MeOH (46 mL) was added, and the
reaction was allowed to proceed with stirring at 25 °C for 1 h. The
pH of the solution was adjusted to pH 5-6 using prewashed
Amberlite IR-120 acidic resin, and the mixture was filtered through
a Celite pad. Removal of the solvent from the filtrate in vacuo
furnished a residue that was purified by silica gel flash column
CHCl /MeOH (4:1, v/v) as eluent to yield EDU (7) as a white
9
chromatography using CHCl
3
/MeOH (4:1, v/v) as eluent to afford
crystalline solid (80 mg, 87%), mp 178-180 °C (dec) [lit
175 °C]; H NMR (DMSO-d ) δ 11.60 (s, 1H, NH), 8.30 (s, 1H,
H-6), 6.11 (dd, J ) 3.6, 3.6 Hz, 1H, H-1′), 5.28 (d, J ) 4.5 Hz,
1H, C-3′ OH), 5.16 (t, J ) 4.5 Hz, 1H, C-5′ OH), 4.24 (m, 1H,
H-3′), 4.12 (s, 1H, acetylene proton), 3.79 (m, 1H, H-4′), 3.58 (m,
2H, H-5′), 2.10 (m, 2H, H-2′).
1
TMSEAU (2; 114 mg, 81%) as a white crystalline solid, mp 115-
6
1
1
18 °C; H NMR (DMSO-d
6
) δ 11.65 (s, 1H, NH), 7.93 (s, 1H,
H-6), 5.95 (d, J ) 4.5 Hz, 1H, H-1′), 5.61 (d, J ) 5.0 Hz, 1H, C-2′
OH), 5.48 (d, J ) 4.5 Hz, 1H, C-3′ OH), 5.12 (t, J ) 5.3 Hz, 1H,
C-5′ OH), 4.00 (br q, J ) 4.4 Hz, 1H, H-2′), 3.90 (br q, J )
3
2
C
.9 Hz, 1H, H-3′), 3.73 (br q, J ) 3.4 Hz, 1H, H-4′), 3.62 (br m,
H, C-5′), 0.18 [s, 9H, -Si(CH ]; Exact mass (HRMS) calcd for
Si, 340.1091; found, 340.1078. Anal. (C14 Si)
1-(2-Deoxy-â-D-ribofuranosyl)-5-(2-iodoethynyl)uracil (8,
IEDU). TMSEDU (6, 1.2 g. 3.70 mmol) was added, under an argon
atmosphere, to the reaction flask containing anhydrous DMF
(24 mL), N-iodosuccinimide (1.17 g, 5.18 mmol), and AgNO3
(130 mg, 0.74 mmol) with stirring. The reaction flask was covered
with aluminum foil, and the reaction was allowed to proceed at
25 °C for 3 h with stirring. The solvent was coevaporated using
toluene (3 × 25 mL), and the crude product was purified by silica
3 3
)
14
H N O
20 2 6
20 2 6
H N O
C, H, N.
-(â-D-Arabinofuranosyl)-5-ethynyluracil (3, EAU). A solution
of TMSEAU (2, 0.27 g, 0.58 mmol) in benzene (6.0 mL) was added
to a solution of K CO (70 mg, 0.63 mmol) in MeOH (12.0 rnL)
with stirring at 25 °C, and the reaction was allowed to proceed for
h. Removal of the solvent in vacuo afforded a residue that was
purified by silica gel column flash chromatography using CHCl
MeOH (4:1, v/v) as eluent to yield EAU (3) as a white solid (0.14
1
2
3
6
3
gel flash column chromatography using CHCl /MeOH (9:1, v/v)
3
/
as eluent. Recrystallization of the eluted product from MeOH-
O afforded IEDU (8, 56 mg, 40%) as a pale yellow solid, mp
140 °C (dec) [lit mp 135-137 °C]; H NMR (MeOH-d ) δ 8.32
4
H
2
9
1
20
1
g, 90%), mp > 230 °C (dec) [lit mp > 250 °C]; H NMR (DMSO-
d
1
1
6
) δ 11.70 (s, 1H, NH), 7.98 (s, 1H, H-6), 5.98 (d, J ) 4.5 Hz,
(s, 1H, H-6), 6.21 (dd, J ) 3.2, 3.2 Hz, 1H, H-1′), 4.38 (ddd, J )
6.5, 5.9, 3.5 Hz, 1H, H-3′), 3.93 (q, J ) 3.5 Hz, 1H, H-4′), 3.81
(dd, J ) 12.0, 3.0 Hz, 1H, H-5′), 3.72 (dd, J ) 12.0, 3.5 Hz, 1H,
H, H-1′), 5.62 (d, J ) 5.3 Hz, 1H, C-2′ OH), 5.48 (d, J ) 4.5 Hz,
H C-3′ OH), 5.14 (t, J ) 5.1 Hz, 1H, C-5′ OH), 4.08 (s, 1H,
acetylene proton), 3.98 (dd, J ) 8.8, 4.5 Hz, 1H, H-2′), 3.90 (br q,
2 5
H-5′′), 2.25 (m, 2H, H-2′). Anal. (Cl1Hl1IN O ) C, H, N.

2
856 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Calculation of Bond Angles and Distances. The C-C5-C6
Cristofoli et al.
(5) Griengl, H.; Bodenteich, M.; Hayden, W.; Wanek, E.; Streicher, W.;
Stutz, P.; Backmayer, H.; Ghazzouli, I.; Rosenwirth, B. 5-(Haloalkyl)-
bond angles and the interspatial distances between iodine and the
C5 carbon atom in IEDU and IVDU (see structures in Figure 1)
were calculated following PM3 geometry optimization using the
Alchemy 2000 program (Version 2.0, Tripos Inc., St. Louis, MO).
Antiviral Activity Assays. The antiviral assays were based on
2
′-deoxyuridines: A novel type of potent antiviral nucleoside
analogue. J. Med. Chem. 1985, 28, 1679-1684.
(
6) De Clercq, E.; Rosenwirth, B. Selective in vitro and in vivo activities
of 5-(2-haloalkyl) pyrimidine nucleoside analogs, particularly 5-(2-
chloroethyl)-2′-deoxyuridine against herpes simplex virus. Antimi-
crob. Agents Chemother. 1985, 28, 246-251.
(7) For a review, see: P e´ rigaud, C.; Gosselin, G.; Imbach, J. L.
Nucleoside analogues as chemotherapeutic agents: A review.
Nucleosides Nucleotides 1992, 11, 903-945.
(8) Goodchild, J.; Porter, R. A.; Raper, R. H.; Sim, I. S.; Upton, R. M.;
Viney, J.; Wadsworth, H. J. Structural requirements of olefinic
6
an inhibition of virus-induced cytopathicity in E SM, HeLa, Vero,
or HEL cell cultures, following previously established procedures.2
8-31
HSV-1 (KOS), HSV-2 (G), vaccinia virus, vesicular stomatitis virus
-
(VSV), the thymidine kinase-deficient HSV-1 TK (B2005), and
-
+
6
HSV-1 TK /TK (VMW 1837) strains were exposed to E SM cell
cultures, parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie
B4 virus, and Punta Toro virus to Vero cell cultures, respiratory
syncytial virus to HeLa cell cultures, and CMV (AD-169, Davis),
and VZV (YS, OKA), and the thymidine kinase-deficient VZV (07/
5
-substituted deoxyuridines for antiherpes activity. J. Med. Chem.
1983, 26, 1252-1257.
(
9) Rahim, S. G.; Trevidi, N.; Selway, J.; Darby, G.; Collins, P.; Powell,
K. L.; Purifoy, D. J. M. 5-Alkynyl pyrimidine nucleosides as potential
selective inhibitors of varicella-zoster virus. AntiViral Chem. Chemoth-
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(10) De Clercq, E. Biochemical aspects of the selective antiherpes activity
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H. L.; Anderson, W. F. Gene therapy for the treatment of brain tumors
using intra-tumoral transduction with the thymidine kinase gene and
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1
, YS/R) strains to HEL cell cultures. Briefly, confluent cell cultures
in 96-well microtiter trays were incubated with 100 CCID50 of virus,
with one 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 of the test compounds. Viral cytopathicity
was recorded as soon as it reached completion in the control virus-
infected cell cultures.
Cytostatic and Cytotoxic Activity Assays. The cytostatic assays
for HEL cells were performed as follows. Aliquots (100 µL) of
the HEL cell suspensions were added to the wells of a 96-well
microtiter plate containing 100 µL of varying concentrations of the
test compounds. After a 3-day incubation period at 37 °C in a
(
(
(14) Balzarini, J.; Morin, K. W.; Knaus, E. E.; Wiebe, L. I.; De Clercq,
E. Novel (E)-5-(2-iodovinyl)-2′-deoxyuridine derivatives as potential
cytostatic agents against herpes simplex virus-thymidine kinase gene-
transfected tumors. Gene Ther. 1995, 2, 317-322.
2
humidified CO -controlled incubator, the number of viable cells
was determined using a Coulter counter. Cytostatic activity was
expressed as the compound concentration that reduced the number
of viable cells by 50% (CC50). The cytotoxicity measurements were
based on a microscopically visible alteration of normal cell
(
15) Balzarini, J.; Andrei, G.; Kumar, R.; Knaus, E. E.; Wiebe, L. I.; De
Clercq, E. The cytostatic activity of 5-(1-azidovinyl)-2′-deoxyuridine
AzVDU) against herpes simplex virus thymidine kinase gene-
(
morphology (E
HEL), as previously described.31
The cytostatic activity assays for the various FM3A cell lines
6
SM, HeLa, Vero, HEL) or inhibition of cell growth
transfected FM3A cells is due to inhibition of thymidylate synthase
and enhanced by UV light (λ ) 254 nm) exposure. FEBS Lett. 1995,
373, 41-44.
(
4
were as follows: 6 × 10 cells suspended in growth medium were
allowed to proliferate in 200-µL wells of microtiter plates in the
presence of 5-fold dilutions of the test compounds at 37 °C in a
(16) Morin, K. W.; Atrazheva, E. D.; Knaus, E. E.; Wiebe, L. I. Synthesis
1
25
and cellular uptake of 2′-substituted analogues of (E)-5-(2-[ I]-
iodovinyl)-2′-deoxyuridines in tumor cells transduced with the herpes
simplex type-1 thymidine kinase gene. Evaluation as probes for
monitoring gene therapy. J. Med. Chem. 1997, 40, 2184-2190.
17) Robins, M. J.; Manfredini, S.; Wood, S. G.; Wanklin, R. J.; Rennie,
B. A.; Sacks, S. L. Nucleic acid related compounds. 65. New
syntheses of 1-(â-D-arabinofuranosyl)-5-(E)-2-iodovinyl)uracil (IVAraU)
from vinylsilane precursors. Radioiodine uptake as a marker for
thymidine kinase postitive herpes viral infections. J. Med. Chem.
1991, 34, 2275-2280.
2
humidified CO -controlled atmosphere. After 48 h, the number of
cells was counted in a Coulter counter. The IC50 value was defined
as the compound concentration required to inhibit cell proliferation
by 50%.
(
To evaluate the cytostatic activity of the nucleoside analogues
4
against the osteosarcoma cell lines, 10 cells/well were plated in
9
subsequently incubated at 37 °C in a humidified CO -controlled
atmosphere in the presence of 5-fold dilutions (in normal cell growth
medium) of the compounds. After 3 days, the cells were detached
with trypsin solution (Gibo) and counted in a Coulter counter
6-well microtiter plates (Falcon) and allowed to adhere. Cells were
(18) Nishikawa, T.; Shibuya, S.; Hosokawa, S.; Isobe, M. One pot
2
synthesis of haloacetylenes from trimethylsilylacetylenes. Synlett
1
994, 485-486.
(
19) Takahashi, S.; Kuroyama, Y.; Sonogashira, K.; Hagihara, N. A
convenient synthesis of ethynylarenes and diethynylarenes. Synthesis
(Coulter Electronics Ltd., Harpenden Hertz, U.K.). The IC50 was
1
980, 627-630.
defined as the drug concentration required to inhibit cell prolifera-
tion by 50%.
(
20) Escuret, V.; Aucagne, V.; Joubert, N.; Durantel, D.; Rapp, K. L.;
Schinazi, R. F.; Zoulim, F.; Agrofoglio, L. A. Synthesis of 5-halo-
ethynyl- and 5-(1,2-dihalo)vinyluracil nucleosides: Antiviral activity
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Acknowledgment. We are grateful to the Canadian Institutes
of Health Research (Grant No. MOP-14480) for financial
support of this research. Work done at the Rega Institute was
supported by the IDO grant of the K.U. Leuven and the FWO
(21) Wang, Z.-X.; Duan, W.; Wiebe, L. I.; Balzarini, J.; De Clercq, E.;
Knaus, E. E. Synthesis of 1-(2-deoxy-â-D-ribofuranosyl)-2,4-difluoro-
-substituted-benzenes: “Thymine replacement” analogs of deox-
ythymidine for evaluation as anticancer and antiviral agents. Nucle-
osides, Nucleotides Nucleic Acids 2001, 20, 41-58.
5
(No. G.0267-04). We thank Anita Camps, Frieda De Meyer,
Lizette van Berckelaer, Lies Vandenheurck, Anita Van Lierde,
and Bart Degr e` ve for excellent technical assistance.
(
(
(
22) Andrei, G.; Snoeck, R.; Reymen, D.; Liesnard, C.; Goubau, P.;
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T. Thymidylate synthetase as a target enzyme for the inhibitory
activity of 5-substituted-2′-deoxyuridines on the growth of human
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