Synthesis of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes: "thymine replacement" analogs of thymidine for evaluation as anticancer and antiviral agents

Article abstract of DOI:10.1081/NCN-100001436

A group of unnatural 1-(2-deoxy-ss-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 two-carbon substituents [-C≡C - X, X = I, Br; -C≡CH; (E)-CH=CH-X, X = I, Br; -CH=CH₂; -CH₂CH₃; -CH(N₃) CH₂

Full text of DOI:10.1081/NCN-100001436

This article was downloaded by: [University of Chicago Library]
On: 20 December 2014, At: 03:02
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
SYNTHESIS OF 1-(2-DEOXY-β-D-RIBOFURANOSYL)-2,4-
DIFLUORO-5-SUBSTITUTED-BENZENES*: “THYMINE
REPLACEMENT” ANALOGS OF THYMIDINE FOR
EVALUATION AS ANTICANCER AND ANTIVIRAL AGENTS
Zhi-Xian Wang a , Weili Duan a , Leonard I. Wiebe a , Jan Balzarini b , Erik De Clercq b
Edward E. Knaus c
&
a Faculty of Pharmacy and Pharmaceutical Sciences , University of Alberta , Edmonton,
AB, T6G 2N8, Canada
b Rega Institute for Medical Research , Minderbroedersstraat 10, Leuven, B-3000,
Belgium
c Faculty of Pharmacy and Pharmaceutical Sciences , University of Alberta , Edmonton,
AB, T6G 2N8, Canada
Published online: 17 Aug 2006.
To cite this article: Zhi-Xian Wang , Weili Duan , Leonard I. Wiebe , Jan Balzarini , Erik De Clercq & Edward E.
Knaus (2001) SYNTHESIS OF 1-(2-DEOXY-β-D-RIBOFURANOSYL)-2,4-DIFLUORO-5-SUBSTITUTED-BENZENES*: “THYMINE
REPLACEMENT” ANALOGS OF THYMIDINE FOR EVALUATION AS ANTICANCER AND ANTIVIRAL AGENTS, Nucleosides,
Nucleotides and Nucleic Acids, 20:1-2, 41-58, DOI: 10.1081/NCN-100001436
To link to this article: http://dx.doi.org/10.1081/NCN-100001436
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(1&2), 41–58 (2001)
SYNTHESIS OF 1-(2-DEOXY-β-D-
RIBOFURANOSYL)-2,4-DIFLUORO-5-
SUBSTITUTED-BENZENES∗: “THYMINE
REPLACEMENT” ANALOGS OF THYMIDINE FOR
EVALUATION AS ANTICANCER AND
ANTIVIRAL AGENTS
Zhi-Xian Wang,1 Weili Duan,1 Leonard I. Wiebe,1 Jan Balzarini,2
Erik De Clercq,2 and Edward E. Knaus,1,†
1Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, AB, Canada T6G 2N8
2Rega Institute for Medical Research, Minderbroedersstraat 10,
Leuven B-3000, Belgium
ABSTRACT
A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-
benzenes having a variety of C-5 two-carbon substituents [−C≡C−X, X = I,
Br; −C≡CH; (E)−CH=CH−X, X = I, Br; −CH=CH2; −CH2CH3; −CH(N3)
CH2Br], designedasnucleosidemimics, weresynthesizedforevaluationasanti-
cancer and antiviral agents. The 5-substituted (E)−CH=CH−I and −CH2CH3
compounds exhibited negligible cytotoxicity in a MTT assay (CC50 = 10−3 to
10−4 M range), relative to thymidine (CC50 = 10−3 to 10−5 M range), against
a variety of cancer cell lines. In contrast, the C-5 substituted −C≡C−I and
−CH(N3)CH2Br compounds were more cytotoxic (CC50 = 10−5 to 10−6 M
range). The −C≡C−I and −CH2CH3 compounds exhibited similar cytotoxic-
ity against non-transfected (KBALB, 143B) and HSV-1 TK+ gene transfected
(KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex
virus type 1 (HSV-1) thymidine kinase gene (TK+). This observation indicates
∗ Nucleoside-like numbering is used by analogy to nucleoside nomenclature.
†Address correspondence to Edward E. Knaus. E-mail: eknaus@pharmacy.ualberta.ca
41
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
REPRINTS
42
WANG ET AL.
that expression of the viral TK enzyme did not provide a gene therapeutic
effect. The parent group of 5-substituted compounds, that were evaluated us-
ing a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster
virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV), and
human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of
unnatural C-aryl nucleoside mimics are inactive and/or weakly active antiviral
agents.
INTRODUCTION
The development of new methods for the synthesis of 2ꢁ-deoxyuridines pos-
sessing novel 2-carbon substituents at the C-5 position, which are potent and se-
lective antivirals, represented an important area of antiviral drug design. Great
progress in antiviral therapy was made with the discovery of (E)-5-(2-bromovinyl)-
2ꢁ-deoxyuridine (BVDU, Brivudin), (E)-5-(2-bromovinyl)arabinouridine (BVAU,
Brovavir), and 5-(prop-1-ynyl)arabinouridine (PAU, Zonavir). Earlier antiherpet-
ics, such as 5-iodo- and 5-trifluoromethyl-2ꢁ-deoxyuridine, exhibited little if any
selectivity in their antiviral action. Conversely, BVDU, BVAU, and PAU are highly
specific inhibitors of herpes simplex virus (HSV-1) and varicella-zoster virus (VZV)
replication. Their selectivity is due to specific phosphorylation by virus-encoded
thymidine kinase (TK) in virus-infected, but not in normal, host cells, (1–4).
Ofthemany5-substitutedpyrimidinenucleosidesinvestigated, (E)-5-[2-halo(iodo,
IVDU; bromo, BVDU; chloro, CVDU]-vinyl (1,2) and 5-(2-chloroethyl)-2ꢁ-deoxy-
uridine (CEDU) (5) are among the most potent and selective in their action against
HSV-1. CEDU is effective against systemic HSV-1 infection and HSV-1 encephali-
tis in mice at a 5–15-fold lower dose than BVDU (6). In contrast, the less po-
tent 5-ethyl-2ꢁ-deoxyuridine (EDU) is approximately equiactive against HSV-1 and
HSV-2 (1,2).
Modification of pyrimidine nucleoside bases can bestow new properties (oral
bioavailability, metabolic stability, and pharmacokinetic) (7), which the natural
bases lack, and, when incorporated into nucleic acids by enzymatic processes, can
further alter the structure and/or function of these biopolymers. In this respect,
2ꢁ-deoxyuridine derivatives, which possess a C-5 substituent two-carbon atoms in
length, generally exhibit antiviral, but not anticancer, activity. Structure–activity
relationship correlations (SARCs) for 5-olefinic 2ꢁ-deoxyuridine analogs showed
that optimum inhibition of HSV-1 in vitro occurred when the 5-substituent was
unsaturated 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)−CH=CH−I) (8). There has been considerable interest in
5-alkynyl pyrimidine nucleosides to treat VZV infections. Although 5-ethynyl-2ꢁ-
deoxyuridine(5−C≡CH)washighlyactiveagainstHSV-1, CMV, andVZVinvitro,
it was also cytotoxic. In contrast, 5-(−C≡C−CH3) derivatives of 2ꢁ-deoxyuridine
and arabinouridine (Zonavir) exhibited highly selective anti-VZV activity and
they were noncytotoxic. Thus, changing the C-5 substituent from −C≡CH to

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
43
−C≡C−CH3 greatly increased VZV selectivity and decreased cytotoxicity, ef-
fects attributed to an inability of the −C≡C−CH3 analogs to act as substrates
for cellular TK, or to decreased affinity of the monophosphate for thymidylate
synthase (TS) (9). A number of C-nucleosides exhibit anticancer and antiviral ac-
tivities, that is likely due to their structural resemblance to natural N-nucleosides
(10−12). Accordingly, nonpolar hydrophobic isosteres of pyrimidine nucleosides
that retain close structural, steric, and isoelectronic relationships to the natural base
were recently designed by Schweitzer and Kool (13). Thus, the 2,4-difluoro-5-
methylphenyl isostere 1 was designed as an unnatural mimic of thymidine (2). The
5ꢁ-triphosphate of 1 (1-TP) was inserted into replicating DNA strands by the Klenow
fragment (KF, exo− mutant) of E. coli DNA polymerase 1 (14). Steady-state mea-
surements indicated that 1-TP was inserted opposite adenine (A) with an efficacy
(Vmax/Km) only 40-fold lower than the triphosphate of thymidine (dTTP). In addi-
tion, it was inserted opposite A, relative to C, G, or T, with a selectivity nearly as
high as that for dTTP. Consequently, the 2,4-difluoro-5-methylphenyl moiety of 1
is a shape mimic utilized by KF polymerase similar to the natural thymine base that
it replaces (15–17). It was, therefore, anticipated that nucleoside mimic derivatives
of 1, possessing a C-5 two-carbon substituent, may be cytotoxic to rapidly multi-
plying cancer cells (inhibit tumor growth) and/or act as antiviral agents due to their
selective phosphorylation by virus-infected cells (18), and as radiopharmaceutical
agents for imaging (19), or as chemotherapeutic agents for treating (20), herpes
simplex virus type-1 thymidine kinase positive (HSV-1 TK+) gene-transfected tu-
mors (gene therapy of cancer) (21). We now report the synthesis, antiviral, and
anticancer activities for a group of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-
substituted-benzenes (5, 7) designed as 5-substituted-2ꢁ-deoxyuridine (thymidine)
mimics (Fig. 1).
Figure 1. Structures of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-methylbenzene (1) and
thymidine (2).

ORDER
REPRINTS
44
WANG ET AL.
CHEMISTRY
Reaction of the 5-iodo-2,4-difluorophenyl compound 3a with TMS−C≡C−H
in the presence of (Ph3P)2PdCl2 and CuI in Et3N (22) afforded the corresponding
5-(trimethylsilyl)ethynyl derivative 4 (see Scheme 1). The 5-iodoethynyl 5a, or
5-bromoethynyl 5b derivatives were prepared by reaction of the 5-(trimethylsilyl)
ethynyl compound 4 with either N-iodosuccinimide or N-bromosuccinimide, in
the presence of AgNO3 catalyst in acetone (23), and then removal of the p-
chlorobenzoyl protecting groups in the sugar moiety, using NaOMe in MeOH.
Treatment of the 5-(trimethylsilyl)ethynyl p-chlorobenzoyl protected compound 4
with NaOMe in MeOH simultaneously removed the trimethylsilyl and p-chloro-
benzoyl groups, to afford the 5-ethynyl nucleoside mimic 5c.
Synthesis of the (E)-5-(2-trimethylsilylvinyl) derivative 6b was performed
usingamethodologysimilartothatreportedpreviously(24)forthesynthesisof(E)-
5-(2-iodovinyl)-2ꢁ-fluoro-2ꢁ-deoxyuridine (IVFRU), as illustrated in Scheme 2. Ac-
cordingly, reaction of the protected 5-iodo compound 3a with (E)-1-trimethylsilyl-
2- tributylstannylethene (25) catalyzed by (Ph3P)2PdCl2 in MeCN with subsequent
removalofthe p-chlorobenzoylprotectinggroups, usingNaOMeinMeOHafforded
6b. The (E)-5-(2-trimethylsilylvinyl) group is stable under these reaction condi-
tions. The proton NMR spectrum for 6b showed a JCH=CH = 19.5 Hz coupling
constant that is indicative of the (E)-5-(−CH=CH−TMS) stereochemistry. Com-
pound 6b was also prepared directly from the unprotected 5-iodo compound 3b,
using the same procedure.
The (E)-5-(2-iodovinyl) compound 7a was synthesized by the reaction of 6b
with ICl in MeCN at 25◦C (24). Proton NMR spectral analysis of 7a indicated
that only the (E)-isomer was produced (JCH=CH = 15.0 Hz). In contrast, reaction
of the (E)-5-(2-trimethylsilylvinyl) compound 6b with Br2 or BrCl, under similar
Scheme 1. Reagents and conditions: (a) (Ph3P)2PdCl2, H–C≡C–TMS, CuI, Et3N, 25◦C, 12 h;
(b) N-iodosuccinimide, AgNO3, acetone, 25◦C, 2 h (5a); (c) N-iodosuccinimide, AgNO3, acetone,
25◦C, 1.5 h (5b); (d) NaOMe, MeOH, 25◦C, 15 min (5a) or 20 min (5b); and (e) NaOMe, MeOH,
25◦C, 30 min.

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
45
Figure 2. Mechanism for the iodination of silicon-stabilized cationic species.
reaction conditions gave a mixture of (E)- and (Z)-isomers in a ratio of about
2:1 with J values of 14.2 and 10.1 Hz, respectively. The production of both (E)-
and (Z)-isomers is attributed to the fact that the trans-double bond generates a
trans-dibromide adduct that converts to the (Z)-bromovinyl product via a trans-
elimination reaction (26). Miller and McGarvey (27) reported that halogenation
of trans-β-trimethylsilylstyrene in CH2Cl2 at low temperature proceeds via a
silicon-stabilized cationic species, which upon attack by a halide anion would
afford a dihalide adduct that is formally the product of cis-halogenation. Trans-
elimination of this dihalide would then give a trans-β-halostyrene, an effective
replacement of silicon by halide with retention of stereochemistry about the double
bond (see Fig. 2) (27,28). In this respect, addition of Br2 to 6b at −78◦C, followed by
treatment of the reaction mixture with a cold saturated solution of KF in DMSO at
0◦C, afforded the (E)-5-(2-bromovinyl) product 7b (JCH=CH = 14.2 Hz) as the sole
product (see Scheme 2). Exclusive formation of the (E)−CH=CH−Br product 7b
is likely due to stabilization of the silicon-bridged cationic species at −78◦C and the
effect of the phenyl moiety (27). Desilylation of the (E)-5-(2-trimethylsilylvinyl)
compound 6b upon treatment with CF3CO2H (29,30) for 5–10 min gave the 5-
vinyl product 7c. A similar desilylation using CF3CO2D, in place of CF3CO2H,
produced the (E)-5-(2-deuteriovinyl) product 7d (70% D-labelled as determined
by 1H NMR). Hydrogenation of the 5-vinyl compound 7c, using 10% Pd-on-C and
H2 gas at 15 psi in EtOH afforded the 5-ethyl derivative 7e. Reaction of the 5-vinyl
compound 7c with BrN3, generated in situ by the treatment of NaN3 with Br2 in
CH2Cl2 in the presence of concentrated HCl (31), in MeNO2 at 25◦C afforded the
5-(1-azido-2-bromoethyl) product 7f. Product 7f exists as a mixture of two insepa-
rable diastereomers due to the presence of an asymmetric center at the C-1 position
of the 5-(1-azido-2-bromoethyl) moiety.
BIOLOGICAL RESULTS AND DISCUSSION
Nucleoside mimics, in which the natural thymine base in thymidine is replaced
by an unnatural aryl isostere, could induce new properties that the natural nucleo-
sides lack thereby providing a strategy to design a new class of C-aryl nucleoside
mimics. C-aryl nucleoside mimics possess some potential advantages, relative to
classical 5-substituted-2ꢁ-deoxyuridines, that include i) in vivo resistance to gly-
cosyl bond cleavage by pyrimidine phosphorylases, ii) decreased host cell toxicity

ORDER
REPRINTS
46
WANG ET AL.
Scheme 2. Reagents and conditions: (a) (Ph3P)2PdCl2, (E)-TMS–CH=CHSnBu3, MeCN, 60◦C, 4 h
(3a → 6a, 3b → 6b), and then NaOMe, MeOH, 25◦C, 30 min (6a → 6b); (b) 6b, ICl, MeCN, 25◦C,
30 min (7a); (c) 6b, Br2, CH2Cl2, −78◦C, 2 h, and then KF/DMSO at 0◦C for 1 h and at 25◦C for 2 h
(7b); (d) 6b, CF3CO2H, 25◦C, 5 min (7c); (e) 6a, CF3CO2D, 25◦C, 5 min (7d); (f) 10% Pd-on-C, H2
gas at 15 psi, EtOH, 60◦C, 5 h; and (g) BrN3, MeNO2, CH2Cl2, 25◦C, 10 min.
due to an inability to serve as substrates for thymidylate synthase (TS), and iii) in-
creased lipophilicity that could enhance their ability to penetrate the blood-brain-
barrier (BBB) to improve their efficacy for the treatment of brain viral infections
and brain tumors. A group of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzenes
having a variety C-5 two-carbon substituents [−C≡C−X, X = I, Br; −C≡C−H;
(E)−CH=CH−X, X = I, Br; −CH=CH2; CH2CH3;−CH(N3)CH2Br] were in-
vestigated to probe the effect of size, electronic, hybridization [sp (−C≡C−), sp2
(−CH=CH−), sp3 (−CH−CH−)], and lipophilic parameters that are potential
determinants of antiviral efficacy.
5-Substituted β-anomers 5a, 7a, 7e, 7f, and the reference compounds 5-iodo-
2ꢁ-deoxyuridine (IUDR), 5-fluoro-2ꢁ-deoxyuridine (FUDR), and thymidine, were
evaluated using the MTT cytotoxicity assay (32) (see Tab. 1). The 5-substituted
[(E)−CH=CH−I 7a; −CH2CH3 7e] compounds exhibited negligible cytotoxic-
ity (CC50 = 10−3 to 10−4 M range), even when compared to thymidine (CC50
=
10−3 to 10−5 M range), against KBALB, KBALB-STK, 143B, 143B-LTK, EMT-
6, and R-970-5 cancer cell lines. In contrast, the C-5 substituted −C≡C−I 5a

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
47
Table 1. In Vitro Cell Cytotoxicity of 1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-substituted-
benzenes Determined Using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium Bromide
(MTT) Assay
Cellular Toxicity (CC50, M) Toward Various Cell Linesa
Compounds
KBALBb
KBALB-STKc
143Bd
143B-LTKc
EMT-6e
R-970-5f
5a
7a
7e
7f
6.8 × 10−6
1.7 × 10−4
2.3 × 10−3
1.3 × 10−6
9.7 × 10−5
6.0 × 10−11
9.5 × 10−5
4.0 × 10−5
–
4.0 × 10−5 6.0 × 10−6
2.6 × 10−6 3.0 × 10−5
1.7 × 10−4 1.8 × 10−4
1.5 × 10−4
–
2.6 × 10−3
2.3 × 10−3 8.9 × 10−4
–
–
–
–
–
–
–
IUDRg
FudRh
Thymidine
1.0 × 10−5
8.8 × 10−11
1.0 × 10−4
7.0 × 10−3 7.4 × 10−3
3.8 × 10−4 6.2 × 10−4
9.0 × 10−12 5.5 × 10−5
1.3 × 10−4 2.5 × 10−3
9.0 × 10−5 1.0 × 10−4
–
–
aThe 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).
bTransformed fibroblast sarcoma cell line.
cThese cells were transfected by, and expressed, the herpes simplex virus type 1 thymidine kinase
(HSV-1 TK) gene.
d Human osteosarcoma cell line.
eMouse mammary carcinoma cell line.
f Human osteosarcoma cell line.
gIUdR = 5-iodo-2ꢁ-deoxyuridine.
hFUdR = 5-fluoro-2ꢁ-deoxyuridine.
and −CH(N3)CH2Br 7f compounds were more cytotoxic (CC50 = 10−5 to 10−6 M
range). The C-5 −C≡C−I 5a and −CH2CH3 7e compounds exhibited similar
cytotoxicity against non-transfected (KBALB, 143B), and the corresponding trans-
fected (KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex
virus type 1 (HSV-1) thymidine kinase gene (TK+).
The 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes (5a
–c, 6b, 7a–c, 7f) were evaluated for their antiviral activity in a wide variety of
assay systems (33). Antiviral activities against herpes simplex virus type 1 (strains
KOS, F, McIntyre), herpes simplex virus type 2 (strains G, 196, Lyons), thymidine
kinase-deficient (TK−) herpes simplex virus type 1 (strains KOS ACVr, VMW-
1837), vaccinia virus, and vesicular stomatitis virus in E6SM cells were determined.
This group of compounds were generally inactive, or exhibited negligible activity,
as determined by reduction of virus-induced cytopathoicity, in these antiviral as-
say systems (at concentrations up to 400 µg/mL). In this regard, the −C≡C−Br
compound 5b exhibited distinct (IC50 = 9.6 µg/mL) and selective activity against
HSV-1 (KOS, F, McIntyre), the −C≡CH compound 5c showed weak activity
(IC50 = 48 µg/mL) against HSV-1 (F), the (E)−CH=CH−TMS compound 6b
exhibited weak activity (IC50 = 48 µg/mL) against HSV-1 (McIntyre), HSV-2
(Lyons) and vaccinia virus, and the (E)−CH=CH−I compound 7a showed weak
(IC50 = 48 µg/mL), but selective activity against HSV-1 (KOS, F, McIntyre). In

ORDER
REPRINTS
48
WANG ET AL.
addition, the (E)−CH=CH−Br 7b and −CH(N3)CH2Br 7f compounds did not re-
duce cytopathogenicity induced by parainfluenza-3 virus, reovirus-1, Sindbis virus,
Coxsackie B4 virus or Punta Toro virus in Vero cell cultures (IC50 > 16 µg/mL),
or respiratory syncytial virus in HeLa cell cultures [IC50 ≥ 400 µg/mL (7b) and
>80 µg/mL (7f)].
Antiviral studies using VZV infected human embryonic lung cells [TK+ VZV
(YSandOKAstrains), andTKdeficientVZV/TK− (07/1andYS/Rstrains)]showed
that the inhibitory concentration required to reduce virus plaque formation by 50%
(IC50) for a viral input of 20 plaque forming units (PFU) for the −C≡C−I 5a,
−C≡C−H 5c, (E)−CH=CH−TMS 6b, (E)−CH=CH−I 7a, and −CH=CH2 7c
compounds was >20, >50, >20, >50, and >200 µM, respectively, whereas the
−C≡C−Br compound 5b showed IC50 values >50 µM except for TK+ VZV (YS,
IC50 = 13 µM; OKA, IC50 = 29 µM). The reference drug (E)-5-(2-bromovinyl)-
2ꢁ-deoxyuridine (BVDU) showed IC50 values of 0.008 and 0.009 µM against VZV
TK+ YS and OKA strains, and >60 and >150 µM against VZV TK− 07/1 and YS/R
strains, respectively. These results indicate that expression of the viral TK enzyme,
which would be expected to enhance phosphorylation to the 5ꢁ-monophosphate and
5ꢁ-diphosphate (by the accompanying thymidylate kinase activity), did not increase
the activity against VZV.
Antiviral activity against cytomegalovirus (CMV), strains AD-169, and Davis
was determined using a cytopathicity (CPE) reduction assay (34) in confluent hu-
man embryonic lung (HEL) cells. All compounds tested were inactive at the high-
est concentration tested (−C≡C−I, IC50 > 20 µM; −C≡C−Br, IC50 > 50 µM;
−C≡C−H, IC50 > 200 µM; (E)−CH=CH−I, IC50 > 50 µM; −CH=CH2, IC50 >
200 µM) except for the (E)−CH=CH−TMS compound 6b, which showed an IC50
value of 9 µM against AD-169 strain.
The (E)−CH=CH−Br 7b and −CH(N3)CH2Br 7f compounds were also
evaluated for their activity against HIV-1 (strain IIIB) and HIV-2 (strain ROD)
in human T-lymphocytes (CEM cells). Neither 7b nor 7f inhibited replication of
HIV-1 or HIV-2 (IC50 > 50 µM) at subtoxic concentrations (CC50 ≥ 78 µM). Fur-
thermore, 7b (IC50 > 100 µM) and 7f (IC50 > 200 µM) did not prevent Moloney
murine sarcoma virus (MSV)-induced transformation of C3H/3T3 embryo murine
fibroblasts in vitro.
The weak, or absence of, anticancer/antiviral efficacy for this novel class of 1-
(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes could be due to
a number of factors. For example, it is possible that the sugar moiety does not
undergo phosphorylation by thymidine kinase (TK) to the 5ꢁ-monophosphate (5ꢁ-
MP). Support for this explanation is based on the observations that there are gener-
ally negligible differences in anticancer/antiviral activities between non-transfected
(KBALB, 143B) and viral TK-transfected (KBALB-STK, 143B-LTK) cell lines,
or between herpes simplex virus-1 (KOS), and either herpes simplex virus-1 TK−
KOS or VMW-1837 (TK−/TK+) viral strains. These results do not unambiguously
exclude the possibility that phosphorylation to the 5ꢁ-MP by non-transfected and
transfected cells, and their subsequent transformation to the active 5ꢁ-triphosphate

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
49
(5ꢁ-TP) are equally effective. Accordingly, it has been shown that the 5ꢁ-TP of the
thymidine mimic 1 is incorporated into replicating DNA stands by the Klenow frag-
ment of E. coli DNA polymerase 1, and that further chain elongation occurs even
after its incorporation (14). Therefore, it is possible that these unnatural mimics in-
vestigated may be incorporated into DNA without producing a cytotoxic or antiviral
effect. Validation of this latter explanation could be determined by measuring in-
corporation of the parent thymidine mimic 1 into DNA. Alternatively, the group of
nucleoside mimics evaluated may be devoid of anticancer/antiviral activity due to
the fact that they are not inhibitors of thymidylate synthase (TS). Credence for this
possibility is based on the belief that the anticancer activity exhibited by 5-fluoro-
2ꢁ-deoxyuridine is primarily due to inhibition of DNA biosynthesis by blocking TS,
the enzyme that catalyzes the methylation of 2ꢁ-deoxyuridine-5ꢁ-monophosphate to
thymidine-5ꢁ-monophosphate (35,36). Furthermore, inhibition of TS is the mech-
anism by which certain nucleosides, such as (E)-5-(2-iodovinyl)-2ꢁ-deoxyuridine
and 5-(1-azidovinyl)-2ꢁ-deoxyuridine, exhibit their cytostatic effect (37). These ex-
planations are consistent with previous biological data that showed that a related
class of thymidine mimics having a variety of C-5 substituents (H, Me, F, Cl, Br, I,
CF3, CN, NO2, and NH2) were also inactive anticancer and antiviral agents (38).
EXPERIMENTAL SECTION
General Methods. Melting points were determined with a Thomas Hoover
capillary apparatus and are uncorrected. 1H NMR, 13C NMR, and 19F NMR spec-
tra were measured on a Bruker AM-300 spectrometer. Proton chemical shifts (δ)
are given relative to internal TMS (δ = 0). 13C NMR spectra were acquired using
the J modulated spin echo technique where methyl and methine carbon reso-
nances appear as positive peaks and methylene and quaternary carbons appear as
negative peaks, and carbon chemical shifts (δ) are given relative to CDCl3 (δ = 77).
Fluorine chemical shifts (δ) are given relative to external C6F6 (δ = 0). Infrared
spectra were recorded on a Nicolet Magna 550 IR spectrometer, using air as ref-
erence. Elemental analyses, performed by the MicroAnalysis Service Laboratory,
Department of Chemistry, University of Alberta. Silica gel 60 (E. Merck) was em-
ployed for all silica gel column flash chromatography separations. 1-(3,5-Bis-O-(p-
chlorobenzoyl)-2ꢁ-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (3a) and
1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (3b) were prepared ac-
cording to a previously reported procedure (38).
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-β-D-ribofuranosyl)-2,4-difluoro-
5-(2-trimethylsilylethynyl)benzene (4). CuI (25 mg, 0.131 mmol), (Ph3P)2
PdCl2 (35 mg, 0.074 mmol) and (trimethylsilyl)acetylene (0.29 mL, 2 mmol) were
added to a solution of 3a (0.636 g, 1 mmol) in Et3N (25 mL) with stirring under
argon. The resulting pale yellow solution was stirred at 25◦C for 12 h, the reaction
mixture was diluted with Et2O (100 mL) and filtered to remove solid material.
Removal of the solvent from the filtrate gave a residue that was purified by silica

ORDER
REPRINTS
50
WANG ET AL.
gel column flash chromatography (hexane/EtOAc, 20:1, v/v) to afford 4 (0.59 g,
98%) as white crystals after recrystallization from hexane; m.p. 92−94◦C; 1H NMR
(CDCl3) 8.02, 7.98, 7.46, 7.41 (d, J = 8.5 Hz, 2H each), 7.65 (t, J = 7.9 Hz, 1H),
6.81 (dd, J = 10.1, 8.8 Hz, 1H), 5.58 (br d, J = 6.4 Hz, 1H, H-3ꢁ), 5.40 (dd, J =
10.7, 5.2 Hz, 1H, H-1ꢁ), 4.68 (d, J = 4.0 Hz, 2H, H-5ꢁ), 4.52 (td, J = 4.0, 2.1 Hz,
1H, H-4ꢁ), 2.62 (br d, J = 14.0, 5.2 Hz, 1H, H-2ꢁα), 2.12 (ddd, J = 14.0, 10.7,
6.4 Hz, 1H, H-2ꢁβ), 0.24 (s, 9H, Si(CH3)3); 13C NMR (CDCl3) 165.25, 165.0,
162.90 (dd), 159.39 (dd), 139.92, 139.60, 131.68 (dd, J = 6.6, 2.2 Hz), 130.96,
130.90, 128.78, 128.75, 128.02, 127.83, 124.13 (dd, J = 14.3, 4.4 Hz), 108.7 (dd),
103.93 (dd, J = 26.4, 25.3 Hz), 100.13, 96.53, 82.67, 77.19, 74.28, 64.62, 40.02,
−0.23(3C); 19F NMR (CDCl3) 55.17 (m), 49.38 (q, J = 9.1 Hz). Anal. Calcd. for
C30H26Cl2F2O5Si: C, 59.70; H, 4.34. Found: C, 59.50; H, 4.05.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-(2-iodoethynyl)benzene
(5a).
N-iodosuccinimide (0.09 g, 0.4 mmol) and ground AgNO3 (0.014 g,
0.08 mmol) were added to a solution of 4 (0.2 g, 0.33 mmol) in acetone (4 mL)
with stirring, and the reaction was allowed to proceed at 25◦C for 2 h in the dark.
Removal of the solvent in vacuo gave a residue that was purified via silica gel col-
umn flash chromatography (hexane/EtOAc, 10:1, v/v) to give white crystals (0.21 g,
97%), m.p. 161–162◦C. This product (0.2 g, 0.304 mmol) was suspended in MeOH
(10 mL), NaOMe (0.08 g, 1.48 mmol) was added, and the reaction was allowed
to proceed with stirring at 25◦C for 15 min at which time a clear solution was ob-
tained. The reaction was quenched with solid NH4Cl, and Et2O (15 mL) was added.
Filtration, and removal of the solvent from the filtrate in vacuo, gave a residue that
was purified via silica gel column flash chromatography (hexane/EtOAc, 1:1.5, v/v)
to afford 5a (0.11 g, 95%) as white crystals; m.p. 125–126◦C; 1H NMR (CDCl3)
7.54 (t, J = 7.9 Hz, 1H), 6.78 (dd, J = 9.8, 9.2 Hz, 1H), 5.26 (dd, J = 10.1,
5.8 Hz, 1H, H-1ꢁ), 4.36 (ddd, J = 6.4, 3.0, 2.1 Hz, 1H, H-3ꢁ), 3.96 (td, J = 5.2,
3.0 Hz, 1H, H-4ꢁ), 3.72 (d, J = 5.2 Hz, 2H, H-5ꢁ), 2.76 (s, 2H, OH), 2.29 (br dd,
J = 13.1, 5.8 Hz, 1H, H-2ꢁα), 1.90 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 13
C
NMR (CDCl3) 163.08 (dd, J = 253.8, 12.1 Hz), 159.61 (dd, J = 252.7, 11.0 Hz),
132.23 (dd, J = 6.6, 2.2 Hz), 125.06 (dd, J = 13.2, 3.3 Hz), 108.10 (dd, J = 15.2,
4.4 Hz), 103.95 (t, J = 25.3 Hz), 87.10, 86.22, 73.67 (d, J = 2.2 Hz), 73.07, 65.77,
62.97, 42.24. Anal. Calcd. for C13H11F2IO3: C, 41.08; H, 2.92. Found: C, 41.51;
H, 2.74.
1-(2-Deoxy-β-D-ribofuranosyl)-5-(2-bromoethynyl)-2,4-difluorobenzene
(5b). N-bromosuccinimide (36 mg, 0.2 mmol) and ground AgNO3 (0.01 g,
0.059 mmol) were added to a solution of 4 (0.11 g, 0.181 mmol) in acetone (2
mL) with stirring, and the reaction was allowed to proceed at 25◦C in the dark for
1.5 h. Removal of the solvent in vacuo gave a residue that was purified via silica
gel column flash chromatography (hexane/EtOAc, 10:1, v/v) to give white crystals
(0.095 g), m.p. 124–125.5◦C. This product was suspended in MeOH (5 mL), and
NaOMe (0.03 g, 0.55 mmol) was added. The reaction was allowed to proceed at
25◦C for 20 min during which time the solution turned clear. The reaction was
quenched with solid NH4Cl, and diluted with ether. Filtration, and removal of the

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
51
solvent from the filtrate in vacuo, gave a residue that was purified via silica gel col-
umn flash chromatography (hexane/EtOAc, 1:1.5, v/v) to afford 5b (0.048 g, 80%
from 4) as white crystals; m.p. 124–125◦C; 1H NMR (CDCl3) 7.56 (t, J = 7.9 Hz,
1H), 6.78 (dd, J = 10.1, 9.2 Hz, 1H), 5.26 (dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.32
(ddd, J = 6.4, 3.0, 1.8 Hz, 1H, H-3ꢁ), 3.93 (td, J = 4.9, 3.0 Hz, 1H, H-4ꢁ), 3.70
(d, J = 4.9 Hz, 2H, H-5ꢁ), 2.95 (br s, 2H, OH), 2.28 (br dd, J = 13.1, 5.8 Hz, 1H,
H-2ꢁα), 1.89 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 162.70
(dd, J = 253.8, 11.4 Hz), 159.53 (dd, J = 252.7, 11.0 Hz), 131.92 (dd, J = 6.6,
2.2 Hz), 125.25 (dd, J = 14.3, 3.3 Hz), 107.41 (dd, J = 15.4, 3.3 Hz), 103.97
(t, J = 25.3 Hz), 87.14, 73.60, 72.94, 72.62, 62.88, 54.76, 42.21. Anal. Calcd. for
C13H11BrF2O3: C, 46.87; H, 3.33. Found: C, 46.72; H, 3.13.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-ethynylbenzene(5c). Com-
pound 4 (0.30 g, 0.5 mmol) was added to a solution of NaOMe (0.11 g, 2 mmol)
in MeOH (10 mL) with stirring, and the reaction was allowed to proceed at 25◦C
for 30 min prior to quenching with solid NH4Cl. Removal of the solvent in vacuo
gave a residue that was dissolved in EtOAc (20 mL), the resulting mixture was
filtered, and washed with EtOAc (20 mL). Removal of the solvent from the fil-
trate gave a residue that was purified via silica gel column flash chromatography
(hexane/EtOAc, 1:1.5, v/v) to afford 5c (0.117 g, 92%) as white crystals after re-
crystallization from hexane–CH2Cl2; m.p. 143–144◦C; 1H NMR (CDCl3) 7.57 (t,
J = 8.0 Hz, 1H), 6.76 (dd, J = 9.9, 9.1 Hz, 1H), 5.23 (dd, J = 9.9, 5.9 Hz, 1H,
H-1ꢁ), 4.28 (ddd, J = 6.2, 2.9, 1.8 Hz, 1H, H-3ꢁ), 3.90 (td, J = 5.1, 2.9 Hz, 1H,
H-4ꢁ), 3.68 (dd, J = 11.7, 5.1 Hz, 1H, H-5ꢁα), 3.62 (dd, J = 11.7, 5.1 Hz, 1H,
H-5ꢁβ), 3.31 (br s, 2H, OH), 3.25 (s, 1H, –C≡CH), 2.25 (br dd, J = 13.2, 5.9
Hz, 1H, H-2’α), 1.86 (ddd, J = 13.2, 9.9, 6.2 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3)
162.59 (dd, J = 253.8, 12.1 Hz), 159.66 (dd, J = 252.7, 12.1 Hz), 132.08 (dd, J =
6.6, 2.2 Hz), 125.31 (dd, J = 14.3, 3.3 Hz), 106.77 (dd, J = 15.4, 3.3 Hz), 103.84
(dd, J = 26.4, 25.3 Hz), 87.17, 82.06, 73.52 (d, J = 2.2 Hz), 72.84, 62.80, 42.08;
19F NMR (CDCl3) 53.76 (q, J = 9.1 Hz), 50.20 (q, J = 9.1 Hz). Anal. Calcd. for
C13H12F2O3: C, 61.42; H, 4.76. Found: C, 61.30; H, 4.69.
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-β-D-ribofuranosyl)-2,4-difluoro-
(E)-5-(2-trimethylsilylvinyl)benzene(6a). (Ph3P)2PdCl2 (0.21g, 0.3mmol)was
added to a mixture of 3a (1.9 g, 3 mmol) and (E)-1-trimethylsilyl-2-tributylstannyl-
ethene (1.95 g, 5 mmol) in dry CH3CN (40 mL), and the mixture was stirred vig-
orously at 60◦C for 4 h under an argon atmosphere. Removal of the solvent in
vacuo gave a residue that was purified via silica gel column flash chromatography
(hexane/EtOAc, 20:1 to 15:1, v/v) to yield 6a (1.3 g, 72%) as white crystals after
recrystallization from hexane; m.p. 81–82◦C; 1H NMR (CDCl3) 8.04, 7.97, 7.47,
7.34 (d, J = 8.5 Hz, 2H each), 7.72 (t, J = 8.2 Hz, 1H), 6.94 (d, J = 19.5 Hz,
1H, CH=CHTMS), 6.78 (dd, J = 10.4, 10.1 Hz, 1H), 6.43 (d, J = 19.5 Hz, 1H,
CH=CHTMS), 5.61 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.44 (dd, J = 10.7, 5.2 Hz, 1H,
H-1ꢁ), 4.76 (dd, J = 11.9, 4.0 Hz, 1H, H-5ꢁα), 4.68 (dd, J = 11.9, 4.0 Hz, 1H,
H-5’β), 4.53 (td, J = 4.0, 2.1 Hz, 1H, H-4ꢁ), 2.65 (br dd, J = 13.7, 5.2 Hz, 1H,
H-2ꢁα), 2.16 (ddd, J = 13.7, 10.7, 6.1 Hz, 1H, H-2ꢁβ), 0.12 (s, 9H, Si(CH3)3); 13
C

ORDER
REPRINTS
52
WANG ET AL.
NMR (CDCl3) 165.24, 165.13, 159.44 (dd, J = 251.6, 12.1 Hz), 159.03 (dd, J =
248.3, 9.9 Hz), 139.97, 139.69, 134.0 (d, J = 3.3 Hz), 132.67, 131.04, 130.92,
128.85, 128.79, 128.20, 128.03, 124.74 (t, J = 5.5 Hz), 123.95 (dd, J = 13.2,
3.3 Hz), 122.87 (dd, J = 12.1, 4.4 Hz), 103.76 (dd, J = 26.4, 25.3 Hz), 82.80,
77.42, 74.85 (d, J = 2.2 Hz), 64.78, 40.24, −1.33.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-(E)-5-(2-trimethylsilylvinyl)
benzene (6b). (Ph3P)2PdCl2 (0.245 g, 0.35 mmol) was added to a mixture of
3b (1.25 g, 3.5 mmol) and (E)-1-trimethylsilyl-2-tributylstannylethene (2.75 g,
7 mmol) in dry MeCN (50 mL), and the mixture was stirred vigorously under argon
at 60◦C for 4 h. Removal of the solvent in vacuo gave a residue that was purified
via silica gel column flash chromatography (hexane/EtOAc, 1.5:1–1:1, v/v) to yield
6b (1.0 g, 86%) as light yellow crystals, which were recrystallized from hexane to
1
afford white crystals (0.88 g, 76%); m.p. 101–102◦C; H NMR (CDCl3) 7.58 (t,
J = 10.4 Hz, 1H), 6.97 (d, J = 19.5 Hz, 1H, CH=CHTMS), 6.76 (t, J = 10.4 Hz,
1H), 6.48 (d, J = 19.5 Hz, 1H, CH=CHTMS), 5.30 (dd, J = 10.1, 5.8 Hz, 1H,
H-1ꢁ), 4.42 (ddd, J = 6.4, 3.4, 2.1 Hz, 1H, H-3ꢁ), 4.00 (td, J = 4.9, 3.4 Hz, 1H,
H-4ꢁ), 3.79 (d, J = 4.9 Hz, 2H, H-5ꢁ), 2.90 (br s, 2H, OH), 2.30 (br dd, J = 13.1,
5.8 Hz, 1H, H-2ꢁα), 2.04 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ), 0.16 (s, 9H,
Si(CH3)3); 13C NMR (CDCl3) 159.33 (dd, J = 250.5, 12.1 Hz, 2C), 134.07 (d,
J = 3.3 Hz), 132.52, 125.21 (t, J = 5.5 Hz), 124.18 (dd, J = 13.2, 3.3 Hz), 122.52
(dd, J = 11.0, 3.3 Hz), 103.88 (t, J = 26.4 Hz), 87.04, 74.66, 73.45, 63.17, 42.35,
−1.37; 19F NMR (CDCl3) 46.44 (m), 44.88 (m). Anal. Calcd. for C16H22F2O3Si:
C, 58.51; H, 6.75. Found: C, 58.09; H, 6.75.
1-(2-Deoxy-β-D-ribofuranosyl)]-2,4-difluoro-(E)-5-(2-iodovinyl)benzene
(7a). ICl (0.065 g, 0.4 mmol) was added to a solution of 6b (0.132 g, 0.4 mmol)
in dry MeCN (5 mL) with stirring at 25◦C. The reaction was allowed to proceed at
25◦C for 30 min with stirring, the solvent was removed in vacuo, and the residue
obtained was purified via silica gel column flash chromatography (hexane/EtOAc,
1:1–1:1.5, v/v) to afford 7a (0.147 g, 96%) as a light yellow syrup, which was
recrystallized from hexane–CH2Cl2 to give slightly yellow crystals (0.130 g, 85%);
m.p. 85–87◦C; 1H NMR (CDCl3) 7.42 (d, J = 15.0 Hz, 1H, CH=CHI), 7.40 (t,
J = 8.2 Hz, 1H), 6.94 (d, J = 15.0 Hz, 1H, CH=CHI), 6.74 (t, J = 10.2 Hz, 1H),
5.27 (dd, J = 9.9, 5.9 Hz, 1H, H–1ꢁ), 4.35–4.42 (m, 1H, H-3ꢁ), 3.99 (td, J = 4.8,
3.0 Hz, 1H, H-4ꢁ), 3.76 (d, J = 4.8 Hz, 2H, H-5ꢁ), 3.14 (br s, 2H, OH), 2.30 (br
dd, J = 13.2, 5.9 Hz, 1H, H-2ꢁα), 1.92 (m, 1H, H-2ꢁβ); 13C NMR (CCl3) 159.22
(dd, J = 250.5, 12.1 Hz), 158.76 (dd, J = 252.7, 12.1 Hz), 136.67, 125.85 (dd,
J = 12.1, 5.5 Hz), 124.85 (dd, J = 14.3, 3.3 Hz), 121.77 (dd, J = 13.2, 3.3 Hz),
104.19 (dd, J = 26.4, 25.3 Hz), 87.02, 80.0 (d, J = 6.6 Hz), 74.18, 73.32, 63.12,
42.34. Anal. Calcd. for C13H13F2IO3: C, 40.86; H, 3.43. Found: C, 40.53; H, 3.15.
1-(2-Deoxy-β-D-ribofuranosyl)-(E)-5-(2-bromovinyl)-2,4-difluorobenzene
(7b). A solution of Br2 (0.085 g, 0.53 mmol) in CH2Cl2 (0.2 mL) was added drop
wise to a solution of 6b (0.164 g, 0.5 mmol) in CH2Cl2 (10 mL) with stirring at
−78◦C. The reaction was allowed to proceed at −78◦C for 2 h, a saturated solution
of dry KF in DMSO (5 mL) was added, and the reaction mixture was allowed to

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
53
warm to 0◦C with subsequent stirring at 0◦C for 1 h and then at 25◦C for 2 h.
The reaction mixture was diluted with water (10 mL) and extracted with EtOAc
(3 × 20 mL). The combined organic extracts were washed with water (10 mL)
and brine, and the organic fraction was dried (Na2SO4). After filtration, the solvent
was removed in vacuo, and the residue was purified via silica gel column flash
chromatography (hexane/EtOAc, 1:1.5, v/v) to afford 7b (0.14 g, 83%) as white
crystals after recrystallization from hexane–CH2Cl2; m.p. 116–117.5◦C; 1H NMR
(CDCl3) 7.44 (t, J = 8.2 Hz, 1H), 7.09 (d, J = 14.2 Hz, 1H, CH=CHBr), 6.89
(d, J = 14.2 Hz, 1H, CH=CHBr), 6.75 (t, J = 10.2 Hz, 1H), 5.28 (dd, J = 10.1,
5.8 Hz, 1H, H-1ꢁ), 4.33 (ddd, J = 6.4, 3.0, 2.1 Hz, 1H, H-3ꢁ), 3.94 (td, J = 4.9,
3.0 Hz, 1H, H-4ꢁ), 3.71 (d, J = 4.9 Hz, 2H, H-5ꢁ), 3.20 (br s, 2H, OH), 2.29 (br dd,
J = 13.1, 5.8 Hz, 1H, H-2ꢁα), 1.91 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 13
C
NMR (CDCl3) 159.06 (dd, J = 252.7, 13.2 Hz), 158.8 (dd, J = 252.7, 12.1 Hz),
129.24, 126.01 (t, J = 5.5 Hz), 125.19 (dd, J = 13.2, 4.4 Hz), 119.94 (dd, J =
13.2, 4.4 Hz), 109.38 (dd, J = 7.7, 2.2 Hz), 104.06 (dd, J = 26.4, 25.3 Hz), 87.10,
73.89, 72.89, 62.81, 42.21. Anal. Calcd. for C13H13BrF2O3: C, 46.59; H, 3.91.
Found: C, 46.62; H, 3.61.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-vinylbenzene (7c). A so-
lution of 6b (0.81 g, 2.47 mmol) in CF3CO2H (5 mL) was stirred at 25◦C for
5 min. Excess CF3CO2H was removed in vacuo, the residue was dissolved in
MeOH (10 mL), and this solution was treated with K2CO3 powder (0.5 g) for
10 min with stirring. Removal of the MeOH in vacuo gave a residue, which was
extracted with EtOAc (2 × 10 mL). Removal of the solvent from the extracts in
vacuo gave a residue, which was purified via silica gel column flash chromatog-
raphy (hexane/EtOAc, 1:1.5, v/v) to afford 7c (0.6 g, 95%) as white needles after
1
recrystallization from hexane–CH2Cl2; m.p. 116–118◦C; H NMR (CDCl3) 7.55
(t, J = 8.4 Hz, 1H), 6.79 (t, J = 10.4 Hz, 1H), 6.79 (dd, J = 18.0, 11.3 Hz, 1H,
CH=CH2), 5.77 [d, J = 18.0 Hz, 1H, (E)–CH=CHH], 5.36 [d, J = 11.3 Hz, 1H,
(Z)–CH=CHH], 5.32 (ddd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.47 (ddd, J = 6.4, 3.4,
1.8 Hz, 1H, H-3ꢁ), 4.03 (ddd, J = 4.9, 4.3, 3.4 Hz, 1H, H-4ꢁ), 3.86 (dd, J = 11.6,
4.3 Hz, 1H, H-5ꢁa), 3.78 (dd, J = 11.6, 4.9 Hz, 1H, H-5ꢁb), 2.33 (ddt, J = 13.1, 5.8,
1.8 Hz, 1H, H-2ꢁα), 2.07 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ), 1.99 (br s, 2H,
OH); 13C NMR (CDCl3) 159.28 (dd, J = 252.7, 13.2 Hz), 159.05 (dd, J = 250.5,
12.1 Hz), 128.27, 125.43 (d, J = 5.5 Hz), 124.56 (dd, J = 13.2, 3.3 Hz), 121.51
(dd, J = 12.1, 3.3 Hz), 116.19 (d, J = 3.3 Hz), 103.69 (t, J = 26.4 Hz), 87.10,
74.18, 72.90, 62.82, 42.0; 19F NMR (CDCl3) 46.31 (m), 45.87 (q, J = 9.2 Hz).
Anal. Calcd. for C13H14F2O3: C, 60.93; H, 5.51. Found: C, 60.83; H, 5.37.
Thedeuteroanalog7dwaspreparedbyasimilarprocedure, usingCF3CO2Din
place of CF3CO2H as described above. The atom percent deuterium was determined
from the 1H NMR integral for the (E)–CH=CHH resonance at δ = 5.77.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-ethylbenzene (7e). 10%
Pd-on-C (0.05 g) was added to a solution of 7c (0.128 g, 0.5 mmol) in EtOH
(5 mL), and the resultant mixture was stirred under 1 atm of hydrogen gas at 60◦C
for 5 h. After cooling to 25◦C, the reaction mixture was filtered and washed with

ORDER
REPRINTS
54
WANG ET AL.
EtOH (5 mL). Removal of the solvent from the filtrate in vacuo gave a residue,
which was recrystallized from hexane–Et2O to afford 7e (0.121 g, 94%) as white
crystals; m.p. 66–67.5◦C; 1H NMR (CDCl3) 7.24 (dd, J = 8.5, 8.2 Hz, 1H), 6.74
(t, J = 10.1 Hz, 1H), 5.30 (dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.44 (ddd, J = 6.4,
3.4, 2.1 Hz, 1H, H-3ꢁ), 4.0 (td, J = 4.6, 3.4 Hz, 1H, H-4ꢁ), 3.81 (dd, J = 11.6,
4.6 Hz, 1H, H-5ꢁα), 3.76 (dd, J = 11.6, 4.6 Hz, 1H, H-5ꢁβ), 2.62 (q, J = 7.6 Hz,
2H, CH2CH3), 2.29 (dd, J = 13.1, 5.8 Hz, 1H, H-2ꢁα), 2.06 (ddd, J = 13.1, 10.1,
6.4 Hz, 1H, H-2ꢁβ), 1.20 (t, J = 7.7, 3H, CH2CH3); 13C NMR (CDCl3) 160.14
(dd, J = 247.2, 12.1 Hz), 158.26 (dd, J = 247.2, 12.1 Hz), 128.02 (dd, J = 6.6,
4.6 Hz), 126.79 (dd, J = 16.5, 3.3 Hz), 123.69 (dd, J = 13.2, 4.4 Hz), 103.47
(dd, J = 26.4, 25.3 Hz), 87.10, 74.77, 73.55, 63.27, 42.38, 21.82 (d, J = 2.2 Hz),
14.41. Anal. Calcd. for C13H16F2O3: C, 60.46; H, 6.24. Found: C, 60.10; H, 6.62.
1-(2-Deoxy-β-D-ribofuranosyl)-5-(1-azido-2-bromoethyl)-2,4-difluoro-
benzene (7f). Concentrated HCl (2 mL) and water (0.5 mL) were added to a
suspension of NaN3 (3.25 g, 50 mmol) in CH2Cl2 (10 mL) cooled to 0◦C. This het-
erogeneous mixture was stirred for 10 min at 0◦C, then Br2 (0.8 g, 2.5 mmol) was
added dropwise, and the resulting mixture was stirred at 0◦C for 30 min. Separation
of the CH2Cl2 layer yielded a brown solution of BrN3, which was used directly
in the subsequent reaction. A solution of BrN3 (2.4 mL of 0.5 M, 1.2 mmol) ob-
tained above was added to a suspension of compound 7c (0.2 g, 0.78 mmol) in
MeNO2 (5 mL), and the resulting solution was stirred at 25◦C for 10 min. Removal
of the solvent in vacuo gave a residue, which was purified via silica gel column
flash chromatography (hexane/EtOAc, 1:1, v/v) to give 7f (0.24 g, 81%) as a col-
orless syrup. 19F NMR analysis showed this product consisted of a mixture of two
diastereomers (ratio A:B = 5:6) due to the chiral center at C-1 position of the 5-
(1-azido-2-bromoethyl) moiety. 1H NMR (CDCl3) 7.53 (dd, J = 8.2, 7.6 Hz, 1H),
6.87 (t, J = 9.9 Hz, 1H), 5.38 (dd, J = 9.8, 5.8 Hz, 1H, H-1ꢁ), 5.08 (dd, J = 6.7,
3.4 Hz, 1H, H-3ꢁ), 4.42–4.52 [m, 1H, –CH(N3)CH2Br], 4.04 (ddd, J = 4.6, 4.0, 3.4
Hz, 1H, H-4ꢁ), 3.88 (dd, J = 11.6, 4.0 Hz, 1H, H-5ꢁa), 3.81 and 3.80 (two dd, J =
11.6, 4.6 Hz, 1H total, H-5ꢁb), 3.48–3.70 [complex m, 2H total, –CH(N3)CH2Br],
2.39 (m, 1H, H-2ꢁα), 2.05 (m, 1H, H-2ꢁβ), 1.95 (br s, 2H, OH); 13C NMR (CDCl3)
159.83 (dm, J = 251.6 Hz), 159.21 (dm, J = 251.6 Hz), 126.74 (dd, J = 6.6,
5.5 Hz) and 126.42 (t, J = 5.5 Hz), 125.60 (m), 120.42 (dd, J = 4.4, 3.3 Hz) and
120.25 (t, J = 3.3 Hz), 104.26 (dd, J = 26.4, 25.3 Hz) and 104.22 (dd, J = 26.4,
25.3 Hz), 87.08, 74.22 and 74.09, 73.32, 63.09, 59.09, 59.96, 42.31, 33.71; 19F
NMR (CDCl3) 49.02 (m, A) and 48.90 (m, B), 46.32 (m, A) and 46.16 (m, B).
Anal. Calcd. for C13H14BrF2N3O3: C, 41.29; H, 3.73. Found: C, 41.18; H, 3.69.
In Vitro Cell Cytotoxicity (MTT Assay). KBALB, KBALB-STK, human
143B, human 14B-LTK, and R-970-5 cells were cultured in complete DMEM
medium supplemented with 10% fetal bovine serum (FBS), and EMT-6 cells were
cultured in complete MAYMOUTH medium in 10% FBS. Exponentially growing
cells were trypsinized, centrifuged, suspended in growth medium, and the cell
number was readjusted to 8 × 103 cells/mL. Cells were seeded into 96-well plates

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
55
at 8 × 102 cells/well, and incubated at 37◦C in a humidified 5% CO2 atmosphere
for 24 h.
The test compounds were dissolved in DMEM medium, and 100 µL of these
solutions was added to cells in 96-well plates to produce the preselected test com-
pound concentration. DMEM medium (100 µL) was added to control wells. The
plates were incubated for 3–5 days at 37◦C in a humidified atmosphere consisting
of 95% air and 5% CO2. At the end of the incubation, 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) was dissolved in phosphate-
buffered saline (PBS) to produce a concentration of 5 mg/mL, filtered through a
0.45 µm membrane filter, and diluted (1:5) with prewarmed DMEM medium. A
50 µL aliquot of this solution was added to each well, and the plates were incu-
bated 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 multi-well spectrophotometer (ELISA
reader). A540 values, corrected for the absorbance in medium blanks, reflected the
concentration of viable cells. The CC50 values reported are the test drug concen-
tration that reduced the A540 to 50% of the control value (mean value, n = 6). This
assay (32), which depends on the metabolic reduction of MTT to colored formazan,
measures cytostatic and cytotoxic effects of the test drugs.
Antiviral Activity Assays. The antiviral assays, other than the anti-HIV
assays, were based on an inhibition of virus-induced cytopathicity in either E6SM,
HeLa, Vero or HEL cell cultures, following previously established procedures
(33,39–41). Herpes simplex virus type 1 (HSV-1) (KOS, F, McIntyre), HSV-2
(G, 196. Lyons), vaccinia virus, vesicular stomatitis virus (VSV), the thymidine
kinase-deficient HSV-1 TK− KOS (ACVr), and HSV-1 TK−/TK+ (VMW 1837)
strains were exposed to E6SM cell cultures, parainfluenza-3 virus, reovirus-1, Sind-
bis virus, Coxsackie B4 virus and Punta Toro virus to Vero cell cultures, respiratory
syncytial virus to HeLa cell cultures, and cytomegalovirus (CMV) (AD-169, Davis)
and varicella-zoster virus (VZV) (YS, OKA), and the thymidine kinase-deficient
VZV (07/1, YS/R) strains to HEL cell cultures. Briefly, confluent cell cultures in
microtitre trays were incubated with 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 various concentrations (400, 100, .. µg/mL) of the test compounds.
Viral cytopathicity was recorded as soon as it reached completion in the control
virus-infected cell cultures.
Inhibition of HIV-Induced Giant Cell Formation. CEM cell cultures
were suspended at 250,000–300,000 cells/mL of culture medium, and infected with
HIV-1(IIIB) or HIV-2(ROD) at 100 CCID50/mL. Then, 100 µL of the infected cell
suspension were transferred to 200 µL microtiter plate wells containing 100 µL of
serialdilutionsofthetestcompoundsolutions. Afterfourdaysofincubationat37◦C,
cell cultures were examined for syncytium formation as previously described (42).

ORDER
REPRINTS
56
WANG ET AL.
Cytostatic Activity Assays. The cytostatic assays were performed as pre-
viously described (43). Briefly, 100 µL aliquots of the cell suspensions (7.5 × 105
human T-lymphocyte CEM cells/mL) were added to the wells of a microtiter plate
containing 100 µL of varying concentrations of the test compounds. After a three-
day incubation period at 37◦C in a humidified CO2-controlled incubator, the number
of viable cells was determined using a Coulter Counter. Cytostatic activity is ex-
pressed as the compound concentration that reduced the number of viable cells by
50% (CC50). The cytotoxicity measurements were based on microscopically visi-
ble alteration of normal cell morphology (E6SM, HeLa, Vero) or inhibition of cell
growth (HEL), as previously described (41).
ACKNOWLEDGMENTS
We are grateful to the Canadian Institutes of Health Research (Medical Re-
search Council of Canada) Grant No. MT-14480 for financial support of this re-
search, and to the Alberta Heritage Foundation for Medical Research for a fellow-
ship to one of us (Z.-X.W.). The experiments done at the Rega Institute were
supported by the Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen
Krediet No. 9.0104.98, and the Geconcerteerde Onderzoeksacties (GOA)-Vlaamse
Gemeenschap contract No. 2000/12. We thank Ann Absillis, Anita Camps, Frieda
De Meyer, Lizette van Berckelaer, Lies Vandenheurck, and Anita Van Lierde for
excellent technical assistance.
REFERENCES
1. De Clercq, E. Pure Appl. Chem. 1983, 55, 623–36.
2. De Clercq, E. Chemica Scripta 1986, 26, 41–47.
3. Sorivudine, B., Monograph in Drugs of the Future, 1995, 739–40.
4. Kinchington, D.; Goldthorpe, S. Current Antiviral, Agents. Part 1. Herpesviruses,
Hepatitis Viruses and Respiratory Viruses. Factfile 1995, 57–61; available in IAVN-
ONLINE on the Internet; developed by the International Society for Antiviral
Research.
5. Griengl, H.; Bodenteich, M.; Hayden, W.; Wanek, E.; Streicher, W.; Stutz, P.;
Backmayer, H.; Ghazzouli, I.; Rosenwirth, B. J. Med. Chem. 1985, 28, 1679–84.
6. De Clercq, E.; Rosenwirth, B. Antimicrob. Agents Chemother. 1985, 28, 246–51.
7. Perigaud, C.; Gosselin, G.; Imbach, J.L. Nucleosides & Nucleotides 1992, 11, 903–
19.
8. Goodchild, J.;Porter, R.A.;Raper, R.H.;Sim, I.S..;Upton, R.M.;Viney, J.;Wadsworth,
H.J. J. Med. Chem. 1983, 26, 1252–57.
9. Rahim, S.G.; Trevidi, N.; Selway, J.; Darby, G.; Collins, P.; Powell, K.L.; Purifoy, D.
J. M. Antiviral Chem. Chemother. 1992, 3, 293–97.
10. Hacksell, U.; Daves, G.D. Prog. Med. Chem. 1985, 22, 1–65.
11. Montgomery, J. A. Antiviral Res. 1989, 12, 113–31.

ORDER
REPRINTS
SYNTHESIS OF THYMIDINE ANALOGS
57
12. Mansuri, M.M.; Martin, J.C. Ann. Rept. Med. Chem., 1988, 23, 161–70.
13. Schweitzer, B.A.; Kool, E.T. J. Org. Chem. 1994, 59, 7238–42.
14. Moran, S.; Ren, R.R.-X.; Kool, E.T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10506–
11.
15. Moran, S.; Ren, R.R.-X.; Rumney, S.; Kool, E.T. J. Am. Chem. Soc. 1997, 119, 2056–
57.
16. Morales, J.; Kool, E.T. Nature Struct. Biol. 1998, 5, 950–54.
17. Guckian, K.M.; Krugh, T.R.; Kool, E.T. Nature Struct. Biol. 1998, 5, 954–59.
18. De Clercq, E. Biochem. Pharmacol. 1984, 33, 2159–69.
19. Morin, K.W.; Knaus, E.E.; Wiebe, L.I. Nucl. Med. Commun. 1997, 18, 557–66.
20. Oldfield, E.H.; Ram, Z.; Culver, K.W.; Blaese, R.M.; DeVroom, H.L.; Anderson, W. F.
Hum. Gene Ther. 1993, 4, 39–69.
21. Wiebe, L.I.; Morin, K.W.; Knaus, E.E. Q. J. Nucl. Med. 1997, 41, 79–89.
22. Takahashi, S.; Kuroyama, Y.; Sonogashira, K.; Hagihara, N. Synthesis 1980, 627–
30.
23. Nishikawa, T.; Shibuya, S.; Hosokawa, S.; Isobe, M. Synlett. 1994, 485–86.
24. Morin, K.W.; Atrazheva, E.D.; Knaus, E.E.; Wiebe, L.I. J. Med. Chem. 1997, 40,
2184–90.
25. Cunico, R.F.; Clayton, F.J. J. Org. Chem. 1976, 41, 1480–82.
26. Tamao, K.; Akita, M.; Maeda, K.; Kumada. J. Org. Chem. 1987, 52, 1100–06.
27. Miller, R.B.; McGarvey, G. J. Org. Chem. 1978, 43, 4424–31.
28. Hollinshead, D.M.; Howell, S.C.; Ley, S.V.; Mahon, M.; Ratcliffe, N.M.; Worthington,
P.A. J. Chem. Soc. Perkin Trans. 1983, 1, 1579–89.
29. Eaton, B.; King, J.A.; Vollhardt, K.P.C. J. Am. Chem. Soc. 1986, 108, 1359–60.
30. Yamaguchi, M.; Kido, Y.; Hayashi, A.; Hirama, M. Angew. Chem. Int. Ed. Engl. 1997,
36, 1313–15.
31. Hassner, A.; Boerwinkle, F.P.; Levy, A.B. J. Am. Chem. Soc. 1970, 92, 4879–83.
32. Alley, M.C.; Scudiero, D.A.; Monks, A.; Hursey, M.L.; Czerwinski, M.J.; Fine, D.L.;
Abbott, B.J.; Mayo, J.G.; Shoemaker, R.H.; Boyd, M.R. Cancer Res. 1988, 48, 589–
601.
33. De Clercq, E. In Vitro Detection of Antiviral Activity. In Vitro and Ex Vivo Test
Systems to Rationalize Drug Design and Delivery. In Minutes of an European Sympo-
sium; Crommelin, D., Couvreur, P., Duceˆne, D., Eds.; Editions de Sante´: Paris, 1994;
108–25.
34. Snoeck, R.; Schols, D.; Andrei, G.; Neyts, J.; De Clercq, E. Antiviral Res. 1991, 16,
1–9.
35. Langenbach, R.J.; Danenberg, P.V.; Heidelberger, C. Biochem. Biophys. Res. Com-
mun. 1972, 48, 1565–71.
36. Santi, D.V.; McHenry, C.S.; Sommer, H. Biochemistry 1974, 13, 471–81.
37. Balzarini, J.; Andrei, G.; Kumar, R.; Knaus, E.E.; Wiebe, L.I.; De Clercq, E. FEBS
Lett., 1995, 373, 41–44; and references cited therein.
38. Wang, Z.-X.; Duan, W.; Wiebe, L.I.; Balzarini, J.; De Clercq, E.; Knaus, E.E. Nucle-
osides, Nucleotides & Nucleic Acids, in press.
39. Schols, D.; De Clercq, E.; Balzarini, J.; Baba, M.; Witvrouw, M.; Hosoya, M.;
Andrei, G.; Snoeck, R.; Neyts, J.; Pauwels, R.; Nagy, M.; Gyo¨rgyi-Edele´nyi, J.;
Machovich, R.; Horva´th, I.; Low, M.; Go¨ru¨g, S. Antiviral Chem. Chemother. 1990, 1,
233–40.

ORDER
REPRINTS
58
WANG ET AL.
40. De Clercq, E.; Descamps, J.; Verhelst, G.; Walker, R.T.; Jones, A.S.; Torrence, P.F.;
Shugar, D. J. Infect. Dis. 1980, 141, 563–74.
41. De Clercq, E.; Holy´, A.; Rosenberg, L.; Sakuma, T.; Balzarini, J.; Maudgal, P.C.
Nature, 1986, 323, 464–67.
42. Balzarini, J.; Naesens, L.; Slachmuylders, J.; Niphuis, H.; Rosenberg, I.; Holy´, A.;
Schellekens, H.; De Clercq, E. AIDS 1991, 5, 21–28.
43. De Clercq, E.; Balzarini, J.; Torrence, P.F.; Mertes, M.P.; Schmidt, C.L.; Shugar, D.;
Barr, P.J.; Jones, A.S.; Verhelst, G.; Walker, R.T. Mol. Pharmacol. 1981, 19, 321–30.
Received July 27, 2000
Accepted September 25, 2000

Request Permission or Order Reprints Instantly!
Interested in copying and sharing this article? In most cases, U.S. Copyright
Law requires that you get permission from the article’s rightsholder before
using copyrighted content.
All information and materials found in this article, including but not limited
to text, trademarks, patents, logos, graphics and images (the "Materials"), are
the copyrighted works and other forms of intellectual property of Marcel
Dekker, Inc., or its licensors. All rights not expressly granted are reserved.
Get permission to lawfully reproduce and distribute the Materials or order
reprints quickly and painlessly. Simply click on the "Request
Permission/Reprints Here" link below and follow the instructions. Visit the
U.S. Copyright Office for information on Fair Use limitations of U.S.
copyright law. Please refer to The Association of American Publishers’
(AAP) website for guidelines on Fair Use in the Classroom.
The Materials are for your personal use only and cannot be reformatted,
reposted, resold or distributed by electronic means or otherwise without
permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the
limited right to display the Materials only on your personal computer or
personal wireless device, and to copy and download single copies of such
Materials provided that any copyright, trademark or other notice appearing
on such Materials is also retained by, displayed, copied or downloaded as
part of the Materials and is not removed or obscured, and provided you do
not edit, modify, alter or enhance the Materials. Please refer to our Website
User Agreement for more details.
Order now!
Reprints of this article can also be ordered at
http://www.dekker.com/servlet/product/DOI/101081NCN100001436