Synthesis of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5- substituted-benzene thymidine mimics, some related α-anomers, and their evaluation as antiviral and anticancer agents

Article abstract of DOI:10.1081/NCN-100001435

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)-2.4-difluorobenzenes having a variety of C-5 substituents (H, Me, F, Cl, Br, I, CF₃, CN, NO₂, NH₂), designed as thymidine mimics, were synthesized for evaluation as anticancer and antiviral agents. The coupling reaction of 3,5-bis-O-(p-chlorobenzoyl) 2-deoxy-α-D-ribofuranosyl chloride with an organocadmium reagent [(2,4-difluorophenyl)₂Cd] afforded a mixture of the α- and β-anomeric products (α:β = 3:1 to 10:1 ratio). Treatment of the α-anomer with BF₃·Et₂O in nitroethane at 110-120°C for 30 min was developed as an efficient method for epimerization of the major α-anomer to the desired β-anomer. The 5-substituted (H, Me, Cl, I, NH₂) β-anomers exhibited negligible cytotoxicity in a MTT assay (CC₅₀ = 10^-3-10^-4 M range), relative to thymidine (CC₅₀ = 10^-3-10^-5 M range), against a variety of cancer cell lines. In contrast, the 5-NO₂ derivative was more cytotoxic (CC₅₀ = 10^-5-10^-6 M range). A number of 5-substituted β-anomers, and some related α-anomers, that were evaluated using 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 antiviral agents.

Full text of DOI:10.1081/NCN-100001435

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SYNTHESIS OF 1-(2-DEOXY-β-D-
RIBOFURANOSYL)-2,4-DIFLUORO-5-SUBSTITUTED-
BENZENE THYMIDINE MIMICS,* SOME RELATED α-
ANOMERS, AND THEIR EVALUATION AS ANTIVIRAL
AND ANTICANCER 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,
Alberta, 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,
Alberta, 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-BENZENE THYMIDINE MIMICS,* SOME
RELATED α-ANOMERS, AND THEIR EVALUATION AS ANTIVIRAL AND ANTICANCER AGENTS, Nucleosides, Nucleotides and
Nucleic Acids, 20:1-2, 11-40, DOI: 10.1081/NCN-100001435
To link to this article: http://dx.doi.org/10.1081/NCN-100001435
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(1&2), 11–40 (2001)
SYNTHESIS OF 1-(2-DEOXY-β-D-
RIBOFURANOSYL)-2,4-DIFLUORO-5-
SUBSTITUTED-BENZENE THYMIDINE MIMICS,∗
SOME RELATED α-ANOMERS, AND THEIR
EVALUATION AS ANTIVIRAL AND
ANTICANCER AGENTS
Zhi-Xian Wang,1 Weili Duan,1 Leonard I. Wiebe,1 Jan Balzarini,2
Erik De Clercq,2 and Edward E. Knaus1,†
1Faculty of Pharmacy and Pharmaceutical Sciences, University of
Alberta, Edmonton, Alberta, 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-difluoroben-
zenes having a variety of C-5 substituents (H, Me, F, Cl, Br, I, CF3, CN,
NO2, NH2), designed as thymidine mimics, were synthesized for evaluation
as anticancer and antiviral agents. The coupling reaction of 3,5-bis-O-(p-
chlorobenzoyl)-2-deoxy-α-D-ribofuranosyl chloride with an organocadmium
reagent [(2,4-difluorophenyl)2Cd] afforded a mixture of the α- and β-anomeric
products (α:β = 3:1 to 10:1 ratio). Treatment of the α-anomer with BF3·Et2O in
nitroethane at 110–120◦C for 30 min was developed as an efficient method for
epimerization of the major α-anomer to the desired β-anomer. The 5-substituted
(H, Me, Cl, I, NH2) β-anomers exhibited negligible cytotoxicity in a MTT as-
say (CC50 = 10−3–10−4 M range), relative to thymidine (CC50 = 10−3–10−5 M
range), against a variety of cancer cell lines. In contrast, the 5-NO2 derivative
was more cytotoxic (CC50 = 10−5–10−6 M range). A number of 5-substituted
∗Nucleoside-like numbering is used by analogy to nucleoside nomenclature.
†Address correspondence to E.E. Knaus. E-mail: eknaus@pharmacy.ualberta.ca
11
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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12
WANG ET AL.
β-anomers, and some related α-anomers, that were evaluated using a wide va-
riety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV),
vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV) and human im-
munodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural
C-aryl nucleoside mimics are inactive antiviral agents.
INTRODUCTION
Specific H-bonding patterns of the Watson–Crick A−T and G−C base pairs
plays an important role in the storage and replication of biological information (1).
The design of enzymatically replicable “unnatural base pairs” provides a method to
expand the biological and chemical genetic alphabet potential of DNA beyond the
natural A−T and G−C pairs. Accordingly, a substantial number of synthetic and
natural C-nucleosides have been prepared (2). In many cases, the target molecules
were designed to mimic the biological activity of the natural nucleoside analogs,
particularly as potential antiviral or anticancer agents (3,4).
Recently, Schweitzer and Kool (5) designed nonpolar hydrophobic isosteres
of pyrimidine nucleosides that retain the closest possible structural, steric and iso-
electronic relationship to the natural base, but that were not likely to form hydrogen
bonds. Compounds of this type were envisaged as valuable model compounds to
determine the importance of hydrogen bonding and base stacking in the formation
of stable DNA duplex structures. In this respect, the 2,4-difluoro-5-methylphenyl
isostere(1b)wasdesignedasananalogofthymidinewhereFistheisostericreplace-
ment for O, CH replaces NH, and the thymine N1 is replaced by a sp2 hybridized C
(see Fig. 1). F was considered to be the best possible substitution for the carbonyl-
type O from a structural and electronic viewpoint. Although aromatic-substituted
F-atoms can participate in weak intermolecular (6) or intramolecular (7) H-bonds,
NMR titration experiments with 2,4-difluorotoluene (the aryl group of 1b) did not
Figure 1. Structures of thymidine and 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzenes (1a,b).

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SYNTHESIS OF THYMIDINE MIMICS
13
show any evidence of H-bonding with potentially complementary partners. In ad-
dition, the aryl C−F bond has similar polar properties as a C=O bond, although it
is considerably smaller in magnitude.
The calculated bond lengths obtained from AM1 semiempirical calculations
for the natural thymine and unnatural 2,4-difluorotoluene moieties indicate that
the 2,4-difluoro-5-methylphenyl isostere (1b) is a nearly perfect steric mimic for
the natural thymidine structure (5). For example, the C−F bond lengths are within
◦
0.1 A of the C=O bonds, which they replace. Furthermore, data on atomic charges
indicates that the 2,4-difluoro-5-methylphenyl isostere has the same absolute signs
for the charges (although they are smaller in magnitude) on every atom when
compared to thymine indicating that the two moieties are isoelectronic. Although
the 2,4-difluoro-5-methylphenyl moiety is not a nucleobase, this aromatic group is
referred to as a “base” in analogy to the natural thymine base. The 5ꢁ-triphosphate of
1b (1b-TP) is inserted into replicating DNA strands by the Klenow fragment (KF,
-
exo mutant)ofEscherichiacoliDNApolymerase1(8). Steady-statemeasurements
have indicated that 1b-TP was inserted into a template opposite adenine (A) with an
efficacy (Vmax/Km) only 40-fold lower than the triphosphate of thymidine (dTTP).
Furthermore, it was inserted opposite A (relative to C, G, or T) with a selectivity
nearly as high as that for dTTP. Therefore, the 2,4-difluorotoluene moiety of 1b is
a shape mimic handled by the KF polymerase almost as if it were thymine (9–11).
It was therefore anticipated that 5-substituted nucleoside mimic derivatives of 1a
may be cytotoxic to rapidly multiplying cancer cells (inhibit tumor growth) and/or
act as antiviral agents due to their selective phosphorylation by virus-infected cells
(12), and as radiopharmaceutical agents to image (13), or chemotherapeutic agents
to treat (14), herpes simplex virus type-1 thymidine kinase positive (HSV-1 TK+)
gene-transfected tumors (gene therapy of cancer) (15). 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 (1) designed as 5-substituted-2ꢁ-deoxyuridine
(thymidine) mimics.
CHEMISTRY
A number of methodologies have been described for the synthesis of 2ꢁ-deoxy-
ribo-C- nucleosides (16). Synthesis of the thymidine mimic (1b), and some related
analogs, have been effected by reaction of an arylcadmium reagent with the well-
known Hoffer (17) α-chloro sugar synthon by Kool et al. (18–20). The resulting
α-nucleoside mimic 3ꢁ,5ꢁ-bis-O-toluoyl ester was then epimerized (α-epimer →
β-epimer) using benzenesulfonic acid in xylene at reflux temperature to afford the
desired β-anomeric product.
The iodophenyl derivatives (4c, d, h) were prepared by iodination of the cor-
responding precursor (3) with N-iodosuccinimide (NIS) in CF3SO3H (Method B)
using a method reported by Olah et al. (21). Although the use of CF3CO2H, in place
of CF3SO3H, as solvent was effective for the preparation of certain iodo-compounds

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14
WANG ET AL.
Scheme 1. Reagents and conditions: a) N-iodosuccinimide (NIS), CF3CO2H, 25◦C, 12 h (Method
A); b) N-iodosuccinimide, CF3SO3H, 25◦C, 2 h (Method B); c) Br2, Fe, 60◦C, 5 h (Method C).
such as 4b and 4f (Method A), CF3CO2H was an inferior solvent for iodination
of benzene derivatives (3) possessing more electron-withdrawing substituents such
as those in 3c and 3d. The bromo-compounds (4e, g) were prepared by treatment
of 3e and 3g with bromine in the presence of an iron catalyst (5) (Method C) as
illustrated in Scheme 1.
Hoffer’s original procedure (17) for the preparation of the α-chloro sugar syn-
thon [3,5-bis-O-(p-toluoyl)-2-deoxy-α-D-ribofuranosyl chloride] requires delicate
and tedious handling of anhydrous HCl gas. We, and others (22), have experienced
difficulties in obtaining acceptable yields using this method. Accordingly, 3,5-
bis-O-(p-chlorobenzoyl)-2-deoxy-α-D-ribofuranosyl chloride (6) (23) was used in
place of the Hoffer α-chloro sugar synthon since it is more stable and readily crys-
tallizes from the solvent. A synthetic route based on a reported procedure (23) is
illustrated in Scheme 2. The intermediates in this synthesis were not purified. Addi-
tion of a small amount of acetyl chloride to the saturated acetic acid–HCl solution
before adding the protected ribose methyl ether maintains the reaction mixture in
a dry state and affords higher product yield. The α-chloro sugar (6) prepared via
this method (73–82% yield) is sufficiently pure for immediate use without further
purification.
Scheme 2. Reagents and conditions: a) MeOH, acetyl chloride, 45 min at 25◦C and then pyridine;
b) pyridine, 4-dimethylaminopyridine, 4-chlorobenzoyl chloride, 1 h at 0◦C and then 12 h at 25◦C;
c) HOAc, dry HCl gas, 0◦C, 7–10 min.

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SYNTHESIS OF THYMIDINE MIMICS
15
Figure 2. Four-center transition state leading to the formation of the α-7 anomer.
Coupling of the α-chloro sugar (6) to organocadmium derivatives of 4a–h,
whichweregeneratedfromreactionoftheGrignardreagentwithcadmiumchloride,
wasperformedinTHFat25◦C(Scheme3). Asreportedintheliterature(18–20), this
coupling reaction yielded a mixture of α-7 and β-8 anomers. The major isomer in
all eight cases is formed with retention of configuration to afford the corresponding
α-anomeric C-nucleoside (7a–h) as the major product. The predominant formation
of the α-7 anomer may be the result of a four-center transition state at C-1 of the
chloro sugar (Fig. 2), as proposed by Hurd and Miles (24) for reactions involving
related pyranose systems with Grignard reagents, or by a dissociative mechanism
involvingpredominantalpha-faceattack(19). Theratioofthetwoisomers(α-7:β-8)
1
ranged from 3:1 to 10:1, as determined from the H NMR spectrum of the crude
reaction mixture. The two isomers could be separated via silica gel flash column
chromatography. The configuration at the C-1ꢁ carbon of the nucleosides 7b, 8b,
1
1b, and 2b possessing a 2,4-difluorotoluene moiety was determined by H NOE
studies (Fig. 3), and for other compounds it was determined by analysis of the
Figure 3. NOE stereochemical analysis of 7b, 8b, 2b, and 1b.

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16
WANG ET AL.
ꢁ
ꢁ
H1 –H2 coupling constants for the protected and deprotected nucleosides and by
comparison to 1H NMR spectral data reported by Kool (18–20).
Although the desired β-8 anomers could be isolated from the reaction, the
yields were less than ideal. Attempts to improve the yield of β-8 anomers via
modification of the coupling reaction conditions were unsuccessful. Kool reported
that the α-anomers of substituted benzene nucleoside mimics could be epimerized
to the desired β-anomers in the presence of benzenesulfonic acid in xylene at
reflux temperature (18–20). However, we observed considerable decomposition
under these epimerization conditions, and the ratio of β- to α-isomers was still in
the range of 1:2 and 1:1.5 for compounds 7a and 7b, respectively.
The α-7 → β-8 epimerization reaction may involve an acid-catalyzed re-
versible ring opening at the C-1ꢁ position. Therefore, different acids (BF3, BCl3,
CF3COOH, p-TsOH, PhSO3H)andconditions(solvent, temperature, reactiontime)
were evaluated. It was found that α-7a was epimerized in the presence of BF3·Et2O
in nitroethane at 110–120◦C for 45 min to afford β-8a (56–58% isolated yield) and
recovered α-7a (36–39%). The recovered α-7a was epimerized by repeating the
procedure to afford β-8a (77–79% total yield) and recovered α-7a (12–14% total
yield). This procedure was suitable for epimerization of all eight compounds 7a–h.
Compounds 7c–e and 7h with more electron-withdrawing substituents required
longer reaction times (1.5–2 h) and gave slightly lower ratios (1.2:1 to 1:1.2) of
β- to α-anomers. The p-chlorobenzoyl protecting groups in compounds 7 and 8
were removed quantitatively using NaOMe in MeOH at 25◦C. The β-1a–g and
α-2a–g anomers were all prepared in this way (see Scheme 3).
Scheme 3. Reagents and conditions: a) THF, Mg, I2 catalyst, 40◦C, 1 h; b) CdCl2, 1 h, reflux; c) 3,5-
bis-O-(p-chlorobenzoyl)-2-deoxy-α-D-ribofuranosyl chloride (6), 25◦C, 12 h; d) EtNO2, BF3·Et2O,
110–120◦C, 45 min; e) MeOH, NaOMe, 25◦C, 30 min.

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SYNTHESIS OF THYMIDINE MIMICS
17
Scheme 4. Reagents and conditions: a) N-iodosuccinimide, CF3CO2H, 20 min at 0◦C and 20 h
at 25◦C; b) NaOMe, MeOH, 25◦C, 10–30 min; c) ClCF2CO2Me, CuI, KF, DMF, 120◦C, 20 h;
d) Zn(CN)2, Pd(PPh3)4, DMF, 80–90◦C, 4 h; e) NH4NO3, (CF3CO)2O, CH2Cl2, 25◦C, 1.5 h; f) 1N
NaOH, THF, 25◦C, 6 h; g) 10% Pd-on-C, EtOH, 60–70◦C, H2 gas at 15 psi, 2 h.
The 5-iodo derivatives β-1i and α-2i (R = I, X = Y = F) were readily obtained
by treatment of the protected nucleosides β-8a and α-7a with N-iodosuccinimide in
the presence of trifluoroacetic acid, followed by treatment with NaOMe (Scheme 4).
CF3SO3H has been reported to be the best solvent for iodination of weakly reactive
aromatic compounds using NIS (21). In our case, the use of CF3SO3H as solvent
was not suitable due to compound decomposition. Compounds 1i and 2i could also
be obtained directly by treatment of nucleosides 1a and 2a with NIS, but separation
of the by-product succinimide from the target product was very difficult. Synthesis
of the 5-trifluoromethyl derivative β-1j was carried out using a reaction reported
by Chen (25). Thus, the protected 5-iodo compound 9a was treated with methyl
chlorodifluoroacetate in the presence of CuI and KF in DMF at 120◦C, followed
by deprotection with NaOMe in MeOH to afford β-1j in 97% yield. The 5-cyano
derivative 1k was also prepared from the protected 5-iodo derivative 9a by cyanation
with zinc cyanate in the presence of (PPh3)4Pd(0) (26), and then deprotection
using NaOMe in MeOH. Treatment of the 2,4-difluorophenyl compound 8a with
ammonium nitrate in trifluoroacetic anhydride afforded the 5-nitro derivative (27),
which on deprotection with NaOMe in MeOH at 25◦C gave the 2,4-difluoro-5-nitro
(1l, 59%) and 2-fluoro-4-methoxy-5-nitro (1m, 32%) derivatives. The formation of
1m is due to the fact that the 4-fluoro substituent is activated by the ortho-nitro
group, such that it is readily displaced by the nucleophilic methoxide anion (28). In
contrast, deprotection with 1N NaOH in THF afforded only the desired product 1l
in 93% yield. Hydrogenation of 1l using 10% Pd-on-C in the presence of hydrogen
gas in ethanol gave the 5-amino product 1n in 92% yield.

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WANG ET AL.
BIOLOGICAL RESULTS AND DISCUSSION
Replacement of the natural thymine base in thymidine by an unnatural aryl
isostere has attracted our attention since it could bestow new properties, which
the natural nucleosides lack, that may be useful in the design of a new class of
third generation C-aryl nucleoside mimics for use as antiviral and/or anticancer
agents. C-aryl nucleoside mimics offer a number of potential advantages relative to
classical 5-substituted-2ꢁ-deoxyuridines. Increased stability to pyrimidine phospho-
rylases (no glycosidic bond) would not require the presence of a 2ꢁ-ribo-F (29), or
2ꢁ-arabino-OH (30), substituent to provide in vivo stability. Compounds of this type
may elicit decreased host cell toxicity due to their inability to inhibit thymidylate
synthase (TS). Finally, their greater lipophilicity may alter tissue biodistribution,
particularly to treat brain viral infections and brain tumors due to an enhanced abil-
ity to cross the blood-brain-barrier (BBB). This latter advantage offers an attractive
alternative for preparing lipophilic prodrugs of nucleosides (31).
A number of 5-substituted β-anomers (1), and the reference compounds
5-iodo-2ꢁ-dioxyuridine (IUDR), 5-fluoro-2ꢁ-deoxyuridine (FUDR), and thymidine,
wereevaluatedusingtheMTTcytotoxicityassay(32)(seeTab. 1). The5-substituted
(H, Me, F, Cl, I, NH2) β-1 anomers exhibited negligible cytotoxicity (CC50 = 10−3–
10−4 M range), even when compared to thymidine (CC50 = 10−3–10−5 M range),
against KBALB, KBALB-STK, 143B, 143B-LTK, EMT-6, and R-970-5 cancer
cell lines. In contrast, the 5-NO2 derivative (1l) was more cytotoxic (CC50 = 10−5–
10−6 M range). These β-1 anomers (1) exhibited equipotent cytotoxicity against
nontransfected (KBALB, 143B) and the corresponding transfected (KBALB-STK,
143B-LTK) cancer cell lines expressing HSV-1 TK+.
The β-anomers (1c, R = F; 1n, R = NH2) and α-anomer (2c, R = F) were
also evaluated by the Developmental Therapeutics Program, Division of Cancer
Treatment, United States National Cancer Institute, using a 3-cell line primary
anticancer screen consisting of a panel of MCF7 (breast), NCI-H460 (lung), and
SF-268 (CNS) tumor cell lines (33). None of the three compounds evaluated in
this screen inhibited cell growth, relative to untreated control cells, at the highest
compound concentration used (1 × 10−4 M).
The 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes (1)
and related β-anomers (2) were evaluated for their antiviral activity in a wide va-
riety of assay systems (34). 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 de-
termined. These β-1 and α-2 compounds were generally inactive, or exhibited neg-
ligible activity in these antiviral assay systems (at concentrations up to 400 µg/mL).
The α-anomer 2g having a 4-chloro-2-fluoro-5-methylphenyl moiety, showed some
selectivity against HSV-1 (KOS strain, IC50 = 9.6 µg/mL or 36.8 µM) whereas,
the related α-anomer 2f having a 2-chloro-4-fluoro-5-methylphenyl moiety was
inactive (IC50 > 400 µg/mL or >1534 µM). In addition, the β- and α-anomers

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SYNTHESIS OF THYMIDINE MIMICS
19
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
KBALB-
STKc
143B-
LTKc
Compd (R)
KBALBb
143Bd
EMT-6e
R-970-5f
1a R = H
1b, R = Me
1c, R = F
1d, R = Cl
1i, R = I
3.1 × 10−3
1.4 × 10−3
2.9 × 10−3
2.0 × 10−3
5.4 × 10−4
8.3 × 10−6
3.2 × 10−3 3.7 × 10−3 5.7 × 10−3 4.4 × 10−3 5.4 × 10−3
1.7 × 10−3 1.3 × 10−3 1.5 × 10−3 2.1 × 10−3 1.5 × 10−3
2.2 × 10−3 2.0 × 10−3 1.8 × 10−3 3.9 × 10−3 2.7 × 10−3
2.0 × 10−3 1.5 × 10−3 2.5 × 10−3
–
–
5.4 × 10−4 5.4 × 10−4 8.1 × 10−4 6.1 × 10−4 7.7 × 10−4
6.0 × 10−6 6.1 × 10−6 7.1 × 10−5 9.0 × 10−6 7.0 × 10−6
5.7 × 10−3 6.3 × 10−3 8.0 × 10−3 8.3 × 10−3 5.1 × 10−3
1.0 × 10−5 7.0 × 10−3 7.4 × 10−3 3.8 × 10−4 6.2 × 10−4
1l, R = NO2
1m, R = NH2 5.5 × 10−3
IUDRg
9.7 × 10−5
FUdRh
6.0 × 10−11 8.8 × 10−11 9.0 × 10−5 1.0 × 10−4 9.0 × 10−12 5.5 × 10−5
Thymidine
9.5 × 10−5
1.0 × 10−4
–
–
1.3 × 10−4 2.5 × 10−3
a
The molar concentration of the test compound that killed 50% of the cells (or 50% cell survival)
upon incubation for 3–5 days at 37◦C in a humidified atmosphere of 95% air and 5% CO2 (Mean
value, n = 6).
b
Transformed fibroblast sarcoma cell line.
c These cells were transfected by and express the herpes simplex virus type 1 thymidine kinase (HSV-1
TK) gene.
d
Human osteosarcoma cell line.
Mouse mammary carcinoma cell line.
e
f
Human osteosarcoma cell line.
g
IUdR = 5-iodo-2ꢁ-deoxyuridine.
h
FUdR = 5-fluoro-2ꢁ-deoxyuridine.
(1d and 2d, R = Cl) did not reduce cytopathogenicity induced by parainfluenza-3
virus, reovirus-1, Sindbis virus, Coxsackie B4 virus or Punta Toro virus in Vero cell
cultures (IC50 > 80 µg/mL or >306 µM), or respiratory syncytial virus in HeLa
cell cultures (IC50 = >400 µg/mL or >1511 µM).
Antiviral studies using varicella-zoster virus (VZV) infected human embry-
onic lung cells [TK+ VZV (YS and OKA strains), and TK deficient VZV/TK−
(07/1 and YS/R strains)] 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 β-1 compounds (1a–c, e–g, i–k, and m, n) had IC50 values >2 × 10−4
M, whereas the 5-NO2 derivative (1l) exhibited IC50 values of 7.8 × 10−5 and
8.7 × 10−5 M against TK+ VZV (YS and OKA strains), and 7.2 × 10−5 and
>5 × 10−5 M against TK− VZV (07/1 and YS/R strains). The α-2 anomers (2a–c,
e–g, i)allshowedIC50 values>2 × 10−4 M againstbothTK+ andTK− VZV, except
for 2e (5-Br) which showed an IC50 value of 1.49 × 10−4 M against the YS strain
of TK+ VZV. The reference drug (E)-5-(2-bromovinyl)-2ꢁ-deoxyuridine (BVDU)
showed IC50 values of 8 × 10−9 and 9 × 10−9 M against VZV TK+ YS and OKA
strains, and >6 × 10−5 and 1.5 × 10−4 M against VZV TK− 07/1 and YS/R strains,

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WANG ET AL.
respectively. These results indicate that expression of the viral TK enzyme, which
would be expected to enhance phosphorylation to the 5ꢁ-monophosphate, does not
increase antiviral activity against VZV.
Antiviral activity against cytomegalovirus (CMV) strains, AD-169 and Davis,
was determined using a cytopathogenicty (CPE) reduction assay (35) in confluent
human embryonic lung (HEL) cells. All β-anomers tested (1a–c, e–g, i–k, m, n)
were inactive (IC50 > 200 µM) except for 1l [R = NO2, IC50 = 118 µM (AD-169);
IC50 = 100 µM (Davis)]. Related α-anomers (2a–c, e, f) were also inactive
(IC50 > 200 µM), although 2i (R = I) exhibited very weak activity against AD-169
(IC50 = 111 µM) and Davis (IC50 = 125 µM) strains of CMV. The α-anomer 2g,
having a 4-chloro-2-fluoro-5-methylphenyl moiety, also exhibited weak anti-CMV
activity against AD-169 (IC50 = 92 µM) and Davis (IC50 = 158µM) strains.
The β-1d and α-2d anomers (R = Cl) were also evaluated for their activity
against HIV-1 (strain IIIB) and HIV-2 (strain ROD) in human T-lymphocytes (CEM
cells). Neither 1d nor 2d inhibited replication of HIV-1 or HIV-2 (IC50 > 250µM)
at subtoxic concentrations (CC50 > 250µM). Furthermore, 1d and 2d did not pre-
vent Moloney murine sarcoma virus (MSV)-induced transformation of C3H/3T3
embryo murine fibroblasts in vitro (EC50 > 100 µM).
There are a number of plausible reasons why this novel class of 1-(2-deoxy-β-
D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes(1)areineffectiveanticancer/
antiviral agents. For example, the β-anomers 1 may not undergo phosphorylation
by thymidine kinase (TK) to the 5ꢁ-monophosphate (5ꢁ-MP). Support for this ex-
planation is based on the observations that there are generally negligible differ-
ences 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. This explanation does not unambiguously eliminate the
possibility that phosphorylation of the β-anomers 1 to the 5ꢁ-MP by non-transfected
and transfected cells, and their subsequent conversion to the active 5ꢁ-triphosphate
(5ꢁ-TP) are equally effective. In this regard, it has been shown that the 5ꢁ-TP of
β-1b is inserted into replicating DNA stands by the Klenow fragment of E. coli
DNA polymerase 1, and that continued chain elongation occurs even after insertion
of 1b (8). Thus, it is possible that these unnatural β-1 mimics may be incorporated
into DNA without producing a cytotoxic or antiviral effect. The potential validity
of this latter explanation could be determined by measuring incorporation of the
parent β-1 anomer into DNA. Alternatively, the β-1 anomers evaluated may be
devoid of anticancer/antiviral activity due to the fact that they are not inhibitors
of thymidylate synthase (TS). In this context, it is believed that the anticancer
activity exhibited by 5-fluoro-2ꢁ-deoxyuridine is primarily due to inhibition of
DNA biosynthesis by blocking TS, the enzyme which catalyzes the methylation of
2ꢁ-deoxyuridine-5ꢁ-monophosphate to thymidine-5ꢁ-monophosphate (36,37). In ad-
dition, it is known that the molecular target for the cytostatic effect exhibited by cer-
tain compounds such as (E)-5-(2-iodovinyl)-2ꢁ-deoxyuridine and 5-(1-azidovinyl)-
2ꢁ-deoxyuridine is due to inhibition of TS (38).

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SYNTHESIS OF THYMIDINE MIMICS
21
CONCLUSIONS
The novel class of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-substituted-
benzenes (1) reported here lacks anticancer/antiviral activity. The results obtained
in this study suggest that β-1 compounds are not suitable thymidine mimics. In
the event that the failure of β-1 compounds to undergo phosphorylation to the
5ꢁ-monophosphate is responsible for the lack of anticancer/antiviral activities ob-
served, it is possible that 5ꢁ-cycloSal-pronucleotide derivatives of β-1 designed
to adhere to the kinase-bypass concept (39,40) could result in the transforma-
tion of these inactive unnatural nucleoside mimics into a class of effective anti-
cancer/antiviral agents.
EXPERIMENTAL SECTION
General Methods. Melting points were determined with a Thomas Hoover
capillary apparatus and are uncorrected. 1H NMR, 13C NMR, and 19F NMR spectra
were measured on a Bruker AM-300 spectrometer in CDCl3 or DMSO-d6. 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 resonances 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, are within 0.4% of calculated
values for C, H, and N unless otherwise stated. Silica gel 60 (E. Merck Co.) was
employed for all silica gel column flash chromatography separations. The halo ben-
zene compounds (3b–h), 1-bromo-2,4-difluorobenzene (4a), and 2-deoxy-D-ribose
(5) were purchased from the Aldrich Chemical Co.
2,4-Difluoro-5-iodotoluene (4b). Method A. N-iodosuccinimide (3.9 g,
17 mmol) was added in aliquots to a cold (ice bath) solution of 3b (1.92 g, 15 mmol)
in CF3CO2H (5 mL). The resulting solution was stirred at 25◦C overnight, poured
onto ice-water (50 mL), and extracted with hexane (2 × 30 mL). The combined
organic extracts were washed with saturated aqueous sodium hydrosulfite, wa-
ter, and brine prior to drying (Na2SO4). Removal of the solvent in vacuo gave
a residue that was purified via a short silica gel column with hexane as eluant
1
to afford 4b (3.24 g, 85%) as a colorless liquid: H NMR (CDCl3) 7.56 (dd,
J = 7.9, 7.3 Hz, 1H), 6.97 (dd, J = 8.8, 8.2 Hz, 1H); 19F NMR (CDCl3) 67.50 (m),
48.76 (m).
1-Iodo-2,4,5-trifluorobenzene (4c). Method B. N-iodosuccinimide (5.4 g,
24mmol)wasaddedinaliquotstoacold(icebath)solutionof3c(2.98g, 22.5mmol)
in CF3SO3H (15 mL). The resulting solution was stirred at 25◦C for 2 h, poured onto
ice-water (50 mL), and extracted with hexane (3 × 30 mL). The combined organic

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WANG ET AL.
extracts were washed with saturated aqueous sodium hydrosulfite, water, and brine
prior to drying (Na2SO4). Removal of the solvent in vacuo afforded a residue, that
was purified via a short silica gel column with hexane as eluant to afford 4c (4.7 g,
80%) as a colorless liquid: 1H NMR (CDCl3) 7.57 (td, J = 8.8, 5.8 Hz, 1H), 6.97
(dt, J = 10.1, 7.0 Hz, 1H); 19F NMR (CDCl3) 66.22 (m), 29.47 (m), 21.25 (m).
5-Chloro-2,4-difluoro-1-iodobenzene (4d). Method B. Obtained as a col-
1
orless liquid, 87% yield, H NMR (CDCl3) 7.79 (dd, J = 7.9, 6.4 Hz, 1H), 6.95
(dd, J = 8.8, 7.3 Hz, 1H); 19F NMR (CDCl3) 70.55 (br s), 51.54 (br s).
1,5-Dibromo-2,4-difluorobenzene (4e). Method C. Iron (0.15 g) was
added to hot (60◦C) 3e (5 mL, 39 mmol). Bromine (6.2 g, 39 mmol) was then
added dropwise at 60◦C for 3 h with stirring, and the resulting red solution was
stirred at 60◦C for an additional 2 h. After cooling to 25◦C, the reaction mixture
was poured onto cold aqueous NaOH, and extracted with hexane (3 × 30 mL).
The combined organic extracts were washed with water and brine prior to drying
(Na2SO4). Removal of the solvent in vacuo afforded a residue that was purified via
silica gel column flash chromatography, using hexane as eluant to afford 4e (8.81 g,
84%) as colorless low melting point crystals: 1H NMR (CDCl3) 7.77 (t, J = 7.0 Hz,
1H), 6.99 (t, J = 8.1 Hz, 1H); 19F NMR (CDCl3) 58.26 (m, 2F).
4-Chloro-2-fluoro-5-iodotoluene (4f). Method A. Obtained as a colorless
liquid, 94% yield, 1H NMR (CDCl3) 7.66 (d, J = 8.2 Hz, 1H), 7.14 (dt, J = 9.1 Hz,
1H); 19F NMR (CDCl3) 46.28 (br s).
5-Bromo-2-chloro-4-fluorotoluene (4g). Method C. Obtained as a col-
1
orless liquid, 70% yield, H NMR (CDCl3) 7.42 (d, J = 7.3 Hz, 1H), 7.15 (d,
J = 7.9 Hz, 1H), 2.33 (s, 3H).
2,5-Difluoro-1-iodobenzene (4h). Method C. Obtained as a colorless liq-
uid, 48% yield, 1H NMR (CDCl3) 7.29 (ddd, J = 7.6, 5.5, 3.0 Hz, 1H), 7.08 (ddd,
J = 9.1, 7.9, 4.6 Hz, 1H), 6.99 (dddd, J = 9.1, 7.0, 3.7, 3.0 Hz, 1H); 19F NMR
(CDCl3) 48.08 (m), 44.61 (m).
3,5-Bis-O-( p-chlorobenzoyl)-2-deoxy-α-D-ribofuranosyl chloride (6).
Acetyl chloride (0.3 mL) was added to a solution of 5 (6.7 g, 0.05 mol) in MeOH
(100 mL), that generates HCl in situ, and the resulting solution was stirred at 25◦C
for 45 min. Pyridine (10 mL) was added to this solution and the solvents were
removed in vacuo. The residue was dissolved in dry pyridine (30 mL), and the
pyridine was removed in vacuo. The resulting syrup was dissolved in dry pyridine
(30 mL), and the solution was cooled in an ice-water bath. A catalytic amount of
4-dimethylaminopyridine was added, and then 4-chlorobenzoyl chloride (14 mL,
0.11 mol) was added drop wise for 20 min. After stirring at 0◦C for 1 h, the reac-
tion was allowed to proceed at 25◦C overnight. The reaction was quenched with
water (100 mL), and CH2Cl2 (120 mL) was added. The organic phase was sepa-
rated, and the aqueous fraction was extracted with CH2Cl2 (50 mL). The combined
organic extracts were washed with saturated aqueous NaHCO3 (2 × 100 mL), 15%
aqueous H2SO4 (2 × 100 mL), water and then brine, prior to drying (Na2SO4).
Removal of the solvent in vacuo gave a syrup that was dissolved in ether (100 mL),
and the white crystalline solid that formed was removed by suction filtration. The

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SYNTHESIS OF THYMIDINE MIMICS
23
filtrate was evaporated to dryness, and toluene (50 mL) was added and evaporated
repeatedly to remove moisture. The resulting syrup was dissolved in ether (20 mL),
cooled to 0◦C, and this solution was added to a cold solution of acetic acid (50 mL)
saturated with dry HCl gas. Dry HCl gas was bubbled through the resulting solu-
tion during which white crystals precipitated from solution after 2–5 min. Passage
of HCl gas was continued for another 5 min, and the white crystals were filtered
and washed with cold ether-hexane (1:1, v/v). This solid was dried in vacuum◦in
the presence of P2O5 to afford 6 (17.6 g, 82%) as white crystals: m.p. 130–131 C
(lit. (23) 131–132◦C); 1H NMR (CDCl3) 8.04, 7.96, 7.46, 7.43 (d, J = 8.5 Hz, 2H
each), 6.48 (d, J = 4.9 Hz, 1H, H-1), 5.54 (dd, J = 7.2, 3.5 Hz, 1H, H-3), 4.85
(dt, J = 4.6, 3.5 Hz, 1H, H-4), 4.70 (dd, J = 12.2, 3.5 Hz, 1H, H-5a), 4.61 (dd,
J = 12.2, 4.6 Hz, 1H, H-5b), 2.86 (ddd, J = 14.9, 7.2, 4.9 Hz, 1H, H-2α), 2.76
(d, J = 14.9 Hz, 1H, H-2β); 13C NMR (CDCl3) 165.26, 164.96, 139.97, 139.72,
131.11, 130.89, 128.79, 128.75, 127.83, 127.67, 94.96, 84.33, 73.79, 63.68, 44.32.
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-α-D-ribofuranosyl)-2,4-difluor-
obenzene (7a) and 1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-β-D-ribofuran-
osyl)-2,4-difluorobenzene (8a). Compound 4a (10.26 mL, 0.08 mol) was added
drop wise to a mixture of Mg (1.94 g, 0.08 mol) and one crystal of iodine in dry
THF (150 mL) with stirring under argon. The mixture was stirred at 40◦C for 1 h,
at which time formation of the Grignard reagent was complete, and a light brown
solution was produced. To this solution was added ground CdCl2 (7.33 g, 0.04 mol)
(dried under vacuum at 130◦C for 2 h), and the reaction mixture was stirred for
1 h at reflux temperature. After cooling to 25◦C, 6 (21.4 g, 0.05 mol) was added
in aliquots for 1.5 h, and the reaction was allowed to proceed at 25◦C with stir-
ring overnight. The reaction was quenched with saturated aqueous NH4Cl prior to
extraction with ether (3 × 50 mL). The combined organic extracts were washed
with water and brine, and the organic fraction was dried (Na2SO4). Removal of
the solvent in vacuo gave a residue, which was purified via silica gel column flash
chromatography (hexane-EtOAc, 17.5:1, v/v) to yield the β-anomer 8a (1.8 g,
7%) as a white solid, and the α-anomer 7a (16.4 g, 64%) as a light yellow syrup,
respectively.
BF3·Et2O (16 mL) was added to a solution of the α-anomer 7a (16.4 g,
32 mmol)in nitroethane(200mL)underargon, andtheresultingsolutionwas stirred
at 110–120◦C under argon for 45 min. After cooling to 25◦C, the reaction mixture
was poured onto cold water (100 mL), and neutralized with saturated aqueous
NaHCO3 priortoextractionwithether(3×100mL). Thecombinedorganicextracts
were washed with water and brine prior to drying (Na2SO4). Filtration, and then
removal of the solvent in vacuo, gave a residue that was purified via silica gel
column flash chromatography (hexane-EtOAc, 17.5:1, v/v) to give the β-anomer
8a (9.2 g, 56%) as white needles, and the α-anomer 7a (6.0 g, 37%) as a light
yellow syrup, respectively. The recovered α-anomer 7a (6.0 g) was treated again
with BF3· Et2O (6 mL) in nitroethane (80 mL) to give additional β-anomer 8a (3.5
g, 58%) and α-anomer 7a (2.2 g, 37%) affording collectively β-anomer (12.7 g,
77%) and recovered α-anomer 7a (2.2 g, 13%).

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WANG ET AL.
α-Anomer (7a): Colorless syrup; 1H NMR (CDCl3) 8.02, 7.69, 7.43, 7.35
(d, J = 8.5 Hz, 2H each), 7.55 (td, J = 8.6, 6.4 Hz, 1H), 6.90 (dd, J = 8.8, 7.9 Hz,
1H), 6.82 (ddd, J = 11.0, 8.8, 2.6 Hz, 1H), 5.60 (ddd, J = 6.4, 3.4, 3.0 Hz, 1H,
H-3ꢁ), 5.54 (dd, J = 7.0, 4.9 Hz, 1H, H-1ꢁ), 4.70 (td, J = 5.2, 3.0 Hz, 1H, H-4ꢁ),
4.61 (dd, J = 11.6, 5.2 Hz, 1H, H-5ꢁa), 4.55 (dd, J = 11.6, 5.2 Hz, 1H, H-5ꢁb),
2.96 (ddd, J = 14.0, 7.0, 6.4 Hz, 1H, H-2ꢁα), 2.31 (ddd, J = 14.0, 4.9, 3.4 Hz,
1H, H-2ꢁβ); 13C NMR (CDCl3) 165.29, 164.89, 162.45 (dd, J = 214.2, 12.1 Hz),
159.17 (dd, J = 215.3, 12.1 Hz), 139.84, 139.66, 131.02, 130.84, 128.77, 128.67,
128.89, 128.09, 127.74 (dd, J = 9.9, 5.3 Hz), 125.70 (dd, J = 13.2, 3.3 Hz), 111.0
(dd, J = 22.0, 3.8 Hz), 103.76 (t, J = 25.3 Hz), 82.15, 76.51, 74.89, 64.84, 39.22;
19F NMR (CDCl3) 50.16 (m), 47.88 (m).
β-Anomer (8a): White needles (recrystallized from hexane); m.p. 70–71◦C;
1H NMR(CDCl3) 7.95, 7.89, 7.39, 7.33 (d, J = 8.5 Hz, 2H each), 7.40 (m, 1H),
6.8–6.7 (m, 2H), 5.53 (br d, J = 6.4 Hz, 1H, H-3ꢁ), 5.37 (dd, J = 10.7, 5.2 Hz, 1H,
H-1ꢁ), 4.62 (dd, J = 11.9, 4.4 Hz, 1H, H-5ꢁa), 4.56 (dd, J = 11.9, 4.3 Hz, 1H, H-5ꢁb),
4.44 (m, 1H, H-4ꢁ), 2.58 (br dd, J = 13.7, 5.2 Hz, 1H, H-2ꢁα), 2.09 (ddd, J = 13.7,
10.7, 6.4 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.18, 165.02, 162.32 (dd, J = 248.3,
12.1 Hz), 159.70 (dd, J = 249.4, 12.1 Hz), 139.86, 139.57, 130.96, 130.88, 128.91,
128.69, 128.09, 127.92, 127.74 (dd, J = 8.8, 6.1 Hz), 123.75 (dd, J = 13.2, 3.3 Hz),
111.31 (dd, J = 20.9, 3.3 Hz), 103.59 (dd, J = 26.3, 25.3 Hz), 82.54, 77.24, 74.66
(d, J = 2.2 Hz), 64.72, 39.97; 19F NMR (CDCl3) 50.74 (m), 46.87 (m). Anal. calcd.
for C25H18Cl2F2O5: C, 59.19; H, 3.58. Found: C, 59.20; H, 3.45.
Compounds 7b–h and 8b–h were prepared, and α-7b–h were epimerized to
β-8b–h, using similar procedures to those described above for 7a and 8a.
5-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-α-D-ribofuranosyl)-2,4-difluor-
otoluene (7b) and 5-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢁ-deoxy-β-D-ribofuranosyl)-
2,4-difluorotoluene (8b). The coupling reaction of 4b with 6 afforded 7b (51%)
and 8b (11%). Epimerization of α-7b (repeated once after the initial separation of
the α- and β-anomers) yielded β-8b (75%) and recovered 7b (14%).
α-Anomer (7b): Colorless syrup; 1H NMR (CDCl3) 8.02, 7.72, 7.43, 7.36
(d, J = 8.5 Hz, 2H each), 7.38 (t, J = 8.8 Hz, 1H), 6.76 (t, J = 9.8 Hz, 1H), 5.60
(ddd, J = 6.1, 3.0, 2.5 Hz, 1H, H-3ꢁ), 5.53 (dd, J = 7.0, 4.6 Hz, 1H, H-1ꢁ), 4.72 (ddd,
J = 5.2, 4.9, 2.5 Hz, 1H, H-4ꢁ), 4.62 (dd, J = 11.7, 4.9 Hz, 1H, H-5ꢁa), 4.54 (dd,
J = 11.7, 5.2 Hz, 1H), H-5ꢁb), 2.94 (ddd, J = 13.8, 7.0, 6.1 Hz, 1H, H-2ꢁα), 2.29
(ddd, J = 13.8, 4.6, 3.0 Hz, 1H, H-2ꢁβ), 2.23 (s, 3H, CH3); 13C NMR (CDCl3)
165.30, 164.87, 161.2 (dd), 157.4 (dd), 139.84, 139.66, 131.03, 130.84, 128.89 (m),
128.77, 128.65, 128.13, 127.86, 125.04 (dd), 120.45 (dd), 103.25 (dd, J = 26.4,
25.3 Hz), 82.15, 76.58, 74.79 (d, J = 2.2 Hz), 64.67, 39.25, 14.03.
β-Anomer (8b): White needles (recrystallized from hexane); m.p. 70–
71.5◦C; 1H NMR (CDCl3) 8.03, 7.99, 7.46, 7.41 (d, J = 8.5 Hz, 2H each), 7.29 (t,
J = 8.4 Hz, 1H), 6.75 (t, J = 9.8 Hz, 1H), 5.61 (br d, J = 5.5 Hz, 1H, H-3ꢁ), 5.43
(dd, J = 10.7, 5.2 Hz, 1H, H-1ꢁ), 4.73 (dd, J = 11.7, 4.3 Hz, 1H, H-5ꢁa), 4.64 (dd,
J = 11.7, 4.0 Hz, 1H, H-5ꢁb), 4.50 (m, 1H, H-4ꢁ), 2.62 (dd, J = 13.7, 5.2 Hz, 1H,

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SYNTHESIS OF THYMIDINE MIMICS
25
H-2ꢁα), 2.29 (ddd, J = 13.7, 10.7, 5.5 Hz, 1H, H-2ꢁβ), 2.14 (s, 3H, CH3); 13C NMR
(CDCl3) 165.26, 165.14, 160.12 (dd), 157.1 (dd), 139.93, 139.68, 131.03, 130.94,
128.82, 128.77, 128.60 (dd), 128.18, 128.0, 123.01 (dd), 120.81 (dd), 103.16 (dd),
82.61, 77.38, 74.64, 64.76, 40.18, 13.90. Anal. calcd. for C26H20Cl2F2O5: C, 59.90;
H, 3.87. Found: C, 59.83; H, 3.57.
1-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢀ-deoxy-α-D-ribofuranosyl)-2,4,5-triflu-
orobenzene (7c) and 1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢀ-deoxy-β-D-ribofuran-
osyl)-2,4,5-trifluorobenzene (8c). The coupling reaction of 4c with 6 afforded
7c (32%) and 8c (4%). Epimerization of α-7c once (120◦C, 2 h) yielded β-8c (38%
yield) and recovered 7c (41%).
α-Anomer (7c): White crystals (recrystallized from hexane); m.p. 121–
1
122◦C; H NMR (CDCl3) 8.01, 7.73, 7.43, 7.37 (d, J = 8.5 Hz, 2H each), 7.40
(m, 1H), 6.92 (td, J = 6.9, 6.4 Hz, 1H), 5.60 (ddd, J = 6.1, 3.7, 2.4 Hz, 1H, H-3ꢁ),
5.51 (dd, J = 7.3, 4.6 Hz, 1H, H-1ꢁ), 4.73 (ddd, J = 5.2, 4.9, 2.4 Hz, 1H, H-4ꢁ),
4.60 (dd, J = 11.7, 5.2 Hz, 1H, H-5ꢁa), 4.53 (dd, J = 11.7, 4.9 Hz, 1H), H-5ꢁb),
2.98 (ddd, J = 14.0, 7.3, 6.1 Hz, 1H, H-2ꢁα), 2.26 (ddd, J = 14.0, 4.6, 3.7 Hz,
1H, H-2ꢁβ); 13C NMR (CDCl3) 165.21, 164.77, 153.90 (ddd), 148.9 (ddd), 146.8
(ddd), 139.92, 139.68, 130.95, 130.72, 128.75, 128.71, 127.96, 127.57, 126.50 (m),
114.87 (dd, J = 20.9, 5.5 Hz), 105.4 (dd, J = 27.5, 21.4 Hz), 82.29, 76.32, 74.20,
64.44, 39.14.
β-Anomer (8c): Colorless syrup; 1H NMR (CDCl3) 8.01, 7.96, 7.45, 7.40 (d,
J = 8.5 Hz each 2H), 7.35 (m, 1H), 6.92 (t, J = 9.8 Hz, 1H), 5.59 (br d, J = 6.1 Hz,
1H, H-3ꢁ), 5.40 (dd, J = 10.7, 5.2 Hz, 1H, H-1ꢁ), 4.72 (dd, J = 11.7, 4.3 Hz, 1H,
H-5ꢁa), 4.64 (dd, J = 11.7, 4.3 Hz, 1H), H-5ꢁb), 4.52 (m, 1H, H-4ꢁ), 2.67 (br dd,
J = 14.0, 5.2 Hz, 1H, H-2ꢁα), 2.10 (ddd, J = 14.0, 10.7, 6.1 Hz, 1H, H-2ꢁβ); 13
C
NMR (CDCl3) 165.18, 165.0, 154.25 (ddd), 149.98 (ddd), 146.53 (ddd), 139.92,
139.65, 130.96, 130.85, 128.78, 128.73, 127.98, 127.80, 124.57 (dt, J = 16.5,
4.4 Hz), 114.77 (dd, J = 20.9, 5.5 Hz), 105.35 (dd, J = 27.5, 21.6 Hz), 82.71,
77.09, 74.09, 64.51, 40.0.
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢀ-deoxy-α-D-ribofuranosyl)-5-chloro-2,
4-difluorobenzene (7d) and 1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢀ-deoxy-β-D-
ribofuranosyl)-5-chloro-2,4-difluorobenzene (8d). The coupling reaction of 4d
with 6 afforded 7d (33% yield) and 8d (3% yield). Two successive epimerizations of
7d (120◦C, 1.5 h), and separation of the α- and β-anomers after each epimerization,
yielded β-8d (44% yield) and recovered α-7d (35%).
α-Anomer (7d): White crystals (recrystallized from hexane); m.p. 72–73◦C;
1HNMR(CDCl3)8.01, 7.73, 7.44, 7.38(d, J = 8.5Hz, 2Heach), 7.67(t, J = 7.9Hz,
1H), 6.90 (dd, J = 9.8, 8.8 Hz, 1H), 5.62 (ddd, J = 6.4, 3.4,
2.1 Hz, 1H, H-3ꢁ), 5.53 (dd, J = 7.9, 4.6 Hz, 1H, H-1ꢁ), 4.76 (ddd, J = 5.2, 4.9,
2.1 Hz, 1H, H-4ꢁ), 4.59 (dd, J = 11.9, 5.2 Hz, 1H, H-5ꢁa), 4.52 (dd, J = 11.9, 4.9 Hz,
1H, H-5ꢁb), 2.97 (ddd, J = 14.3, 7.9, 6.4 Hz, 1H, H-2ꢁα), 2.26 (ddd, J = 14.3, 4.6,
3.4 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.27, 162.80, 157.44 (dd, J = 248.3,
9.9 Hz), 157.14 (dd, J = 250.5, 12.1 Hz), 139.98, 139.77, 131.04, 130.83, 128.83

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WANG ET AL.
(4C), 128.21 (d, J = 5.5 Hz), 128.05, 127.68, 127.44 (dd, J = 14.3, 4.4 Hz), 116.51
(dd, J = 17.6, 4.4 Hz), 105.04 (t, J = 25.3 Hz), 82.56, 76.41, 74.34, 64.50, 39.18.
Anal. calcd. for C25H17Cl3F2O5: C, 55.63; H, 3.17. Found: C, 55.18; H, 2.95.
β-Anomer (8d): Colorless syrup; 1H NMR (CDCl3) 8.01, 7.97, 7.44, 7.40
(d, J = 8.8 Hz, 2H each), 7.57 (dd, J = 7.9, 7.6 Hz, 1H), 6.89 (dd, J = 9.5,
8.8 Hz, 1H), 5.59 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.40 (dd, J = 10.7, 5.2 Hz, 1H,
H-1ꢁ), 4.73 (dd, J = 11.7, 4.0 Hz, 1H, H-5ꢁa), 4.65 (dd, J = 11.7, 4.0 Hz, 1H,
H-5ꢁb), 4.52 (td, J = 4.0, 2.1 Hz, 1H, H-4ꢁ), 2.65 (br dd, J = 13.7, 5.2 Hz, 1H,
H-2ꢁα), 2.10 (ddd, J = 13.7, 10.7, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.15,
164.97, 157.72 (dd, J = 249.4, 9.9 Hz), 157.22 (dd, J = 251.6, 13.2 Hz), 139.92,
139.64, 130.93, 130.86, 128.76, 128.72, 128.07 (d, J = 5.5 Hz), 128.01, 127.85,
125.28 (dd, J = 15.4, 4.4 Hz), 116.72 (dd, J = 17.6, 4.4 Hz), 104.84 (dd, J =
26.6, 25.3 Hz), 82.76, 77.11, 74.11, 64.46, 40.05.
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢀ-deoxy-α-D-ribofuranosyl)-5-bromo-2,
4-difluorobenzene (7e) and 1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢀ-deoxy-β-D-
ribofuranosyl)-5-bromo-2,4-difluorobenzene (8e). The coupling reaction of 4e
with 6 afforded 7e (36.4% yield) and 8e (5.45% yield). Two consecutive epimer-
izations of 7e (120◦C, 2 h), and separation of the α- and β-anomers after each
epimerization, yielded β-8e (52% yield) and recovered α-7e (14%).
α-Anomer (7e): White crystals (recrystallized from hexane); m.p. 77–80◦C;
1H NMR (CDCl3) 8.01, 7.72, 7.43, 7.34 (d, J = 8.5 Hz, 2H each), 7.81 (t, J = 7.6
Hz, 1H), 6.88 (dd, J = 9.8, 8.2 Hz, 1H), 5.63 (ddd, J = 6.1, 2.4, 2.1 Hz, 1H, H-3ꢁ),
5.53 (dd, J = 7.9, 4.6 Hz, 1H, H-1ꢁ), 4.76 (td, J = 5.2, 2.1 Hz, 1H, H-4ꢁ), 4.58 (dd,
J = 11.7, 5.2 Hz, 1H, H-5ꢁa), 4.51 (dd, J = 11.7, 5.2 Hz, 1H, H-5ꢁb), 2.96 (ddd,
J = 14.0, 7.9, 6.1 Hz, 1H, H-2ꢁα), 2.27 (ddd, J = 14.0, 4.6, 2.4 Hz, 1H, H-2ꢁβ); 13C
NMR (CDCl3) 165.16, 164.70, 158.20 (dd), 158.12 (dd), 139.86, 139.67, 130.95,
130.77, 128.73 (4C), 128.54 (d, J = 8.8 Hz), 127.98, 127.80 (dd, J = 14.3, 3.3 Hz),
127.58, 104.86 (t, J = 26.4 Hz), 103.70 (dd, J = 20.9, 4.4), 82.52, 76.34, 74.22 (d,
J = 2.2 Hz), 64.43, 39.09.
β-Anomer (8e): Colorless syrup; 1H NMR (CDCl3) 8.01, 7.98, 7.45, 7.41
(d, J = 8.5 Hz, 2H each), 7.73 (t, J = 7.7 Hz, 1H), 6.87 (dd, J = 9.8, 8.2 Hz,
1H), 5.59 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.40 (dd, J = 10.7, 5.2 Hz, 1H, H-1ꢁ), 4.73
(dd, J = 11.7, 4.3 Hz, 1H, H-5ꢁa), 4.64 (dd, J = 11.7, 4.0 Hz, 1H, H-5ꢁb), 4.52 (m,
1H, H-4ꢁ), 2.65 (br dd, J = 13.8, 5.2 Hz, 1H, H-2ꢁα), 2.09 (ddd, J = 13.7, 10.7, 6.1
Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.18, 164.99, 158.53 (dd, J = 248.3, 11.0
Hz), 158.30 (dd, J = 249.4, 12.1 Hz), 139.93, 139.65, 130.95, 130.89, 128.76,
128.69, 127.99, 127.82, 127.66 (dd, J = 9.9, 3.3 Hz), 125.70 (dd, J = 14.3, 3.3
Hz), 104.76 (t, J = 26.4 Hz), 104.11 (dd, J = 20.9, 4.4 Hz), 82.79, 77.14, 74.06,
64.46, 40.11.
5-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢀ-deoxy-α-D-ribofuranosyl)-4-chloro-2-
fluorotoluene (7f) and 5-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢀ-deoxy-β-D-ribofuran-
osyl)-4-chloro-2-fluorotoluene (8f). The coupling reaction of 4f with 6 afforded
a mixture of 7f and 8f (49% yield). Epimerization of this mixture (120◦C, 1 h)
yielded β-8f (49% yield) and recovered α-7f (31%).

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SYNTHESIS OF THYMIDINE MIMICS
27
1
α-Anomer (7f): Light yellow syrup; H NMR (CDCl3) 8.03, 7.74, 7.44,
7.36 (d, J = 8.5 Hz, 2H each), 7.47 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 8.8 Hz,
1H), 5.62–5.54 (m, 2H, H-1ꢁand H-3ꢁ), 4.77 (td, J = 5.2, 2.1 Hz, 1H, H-4ꢁ), 4.61
(dd, J = 11.7, 5.2 Hz, 1H, H-5ꢁa), 4.53 (dd, J = 11.7, 5.2 Hz, 1H, H-5ꢁb), 3.04
(ddd, J = 13.7, 7.6, 6.1 Hz, 1H, H-2ꢁα), 2.24 (d, J = 1.5 Hz, 3H, CH3), 2.17 (m,
1H, H-2ꢁβ).
β-Anomer (8f): White crystals (recrystallized from hexane); m.p. 114–
1
115◦C; H NMR (CDCl3) 8.03, 7.99, 7.46, 7.41 (d, J = 8.5 Hz, 2H each), 7.39
(d, J = 7.9 Hz, 1H), 7.03 (d, J = 9.1 Hz, 1H), 5.60 (br d, J = 6.4 Hz, 1H, H-3ꢁ),
5.50 (dd, J = 10.4, 5.2 Hz, 1H, H-1ꢁ), 4.78 (dd, J = 11.8, 4.0 Hz, 1H, H-5ꢁa), 4.67
(dd, J = 11.8, 4.0 Hz, 1H, H-5ꢁb), 4.52 (m, 1H, H-4ꢁ), 2.76 (dd, J = 13.7, 5.2 Hz,
1H, H-2ꢁα), 2.14 (s, 3H, CH3), 1.99 (ddd, J = 13.7, 10.4, 6.4 Hz, 1H, H-2ꢁβ); 13
C
NMR (CDCl3) 165.19, 165.09, 160.04 (d, J = 248.3 Hz), 139.89, 139.67, 133.88
(d, J = 4.4 Hz), 130.96, 130.86, 128.78, 128.74, 128.63 (d), 128.2 (d), 128.09,
127.92, 124.0 (d, J = 17.6 Hz), 116.01 (d, J = 26.3 Hz), 82.58, 77.22 (d, J = 4.4
Hz), 64.67, 39.97, 14.10 (d, J = 3.3 Hz). Anal. calcd. for C26H20Cl3FO5: C, 58.07;
H, 3.75. Found: C, 57.94; H, 3.63.
5-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢀ-deoxy-α-D-ribofuranosyl)-2-chloro-4-
fluorotoluene (7g) and 5-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢀ-deoxy-β-D-ribofuran-
osyl)-2-chloro-4-fluorotoluene (8g). The coupling reaction of 4g with 6 afforded
7g (30% yield) and 8g (5% yield). Epimerization of α-7g once yielded β-8g (51%
yield) and recovered α-7g (34%).
1
α-Anomer (7g): Light yellow syrup; H NMR (CDCl3) 8.02, 7.64, 7.43,
7.34 (d, J = 8.5 Hz, 2H each), 7.42 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 9.8 Hz, 1H),
5.60 (ddd, J = 5.8, 3.0, 2.4 Hz, 1H, H-3ꢁ), 5.54 (dd, J = 7.6, 4.9 Hz, 1H, H-1ꢁ),
4.72 (td, J = 4.9, 2.4 Hz, 1H, H-4ꢁ), 4.60 (dd, J = 11.7, 4.9 Hz, 1H, H-5ꢁa), 4.53
(dd, J = 11.7, 4.9 Hz, 1H, H-5ꢁb), 2.93 (ddd, J = 13.7, 7.6, 5.8 Hz, 1H, H-2ꢁα),
2.31 (s, 3H, CH3), 2.32 (m, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.26, 164.77, 157.20
(d, J = 246.1 Hz), 139.79, 139.64, 133.25 (d, J = 11.0 Hz), 131.68 (d, J = 3.3
Hz), 130.96, 130.76, 128.72, 128.59, 128.41 (d, J = 4.4 Hz), 128.18 (d), 128.02,
127.72, 116.14 (d, J = 25.3 Hz), 82.26, 76.45, 74.84, 64.57, 39.05, 19.31.
β-Anomer (8g): Colorless syrup; 1H NMR (CDCl3) 8.03, 7.97, 7.46, 7.41
(d, J = 8.5 Hz, 2H each), 7.34 (d, J = 7.9 Hz, 1H), 7.07 (d, J = 10.1 Hz, 1H),
5.60 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.42 (dd, J = 10.7, 5.2 Hz, 1H, H-1ꢁ), 4.74
(dd, J = 11.9, 4.0 Hz, 1H, H-5ꢁa), 4.64 (dd, J = 11.9, 4.0 Hz, 1H, H-5ꢁb), 4.52 (td,
J = 4.0, 2.1 Hz, 1H, H-4ꢁ), 2.65 (br dd, J = 14.0, 5.0 Hz, 1H, H-2ꢁα), 2.23 (s, 3H,
CH3), 2.16 (ddd, J = 14.0, 10.7, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.19,
165.08, 157.50 (d, J = 246.1 Hz), 139.89, 139.64, 133.65 (d, J = 9.9 Hz), 132.03
(d, J = 3.3 Hz), 130.96, 130.86, 128.76, 128.72, 128.31 (d, J = 4.4 Hz), 128.05,
127.86, 126.21 (d, J = 13.2 Hz), 116.00 (d, J = 24.2 Hz), 82.63, 77.28, 74.59,
64.64, 40.07, 19.19.
1-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢀ-deoxy-α-D-ribofuranosyl)-2,5-difluor-
obenzene (7h) and 1-(3,5-Bis-O-(p-chlorobenzoyl)-2ꢀ-deoxy-β-D-ribofura-
nosyl)-2,5-difluorobenzene (8h). The coupling reaction of 4h with 6 afforded

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WANG ET AL.
α-7h (49% yield) and β-8h (7% yield). Epimerization of α-7h once (120◦C, 2 h)
yielded β-8h (19% yield) and recovered α-7h (62%).
α-Anomer (7h): White crystals (recrystallized from hexane); m.p. 119–
1
120◦C; H NMR (CDCl3) 8.06, 7.73, 7.48, 7.39 (d, J = 8.5 Hz, 2H each), 7.37
(m, 1H), 7.1–6.9 (m, 2H), 5.65 (ddd, J = 6.4, 3.4, 2.1 Hz, 1H, H-3ꢁ), 5.60 (dd,
J = 7.9, 4.9 Hz, 1H, H-1ꢁ), 4.78 (td, J = 5.2, 2.1 Hz, 1H, H-4ꢁ), 4.64 (dd, J = 11.7,
5.2 Hz, 1H, H-5ꢁa), 4.58 (dd, J = 11.7, 5.2 Hz, 1H, H-5ꢁb), 3.02 (ddd, J = 14.0, 7.9,
6.4 Hz, 1H, H-2ꢁα), 2.35 (br d, J = 14.0 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.72,
165.27, 159.32 (d, J = 241.7 Hz), 155.62 (dd, J = 241.7, 2.2 Hz), 140.30, 140.16,
132.42 (dd, J = 15.4, 7.7 Hz), 131.41, 131.28, 129.24, 129.14, 128.55, 128.19,
116.76 (dd, J = 24.2, 8.8 Hz), 115.37 (dd, J = 25.3, 8.8 Hz), 113.97 (dd, J = 25.3,
4.4 Hz), 82.85, 76.87, 75.30, 65.06, 39.58. 19F NMR (CDCl3) 43.21 (m), 37.70 (m).
β-Anomer (8h): Colorless syrup; 1H NMR (CDCl3) 8.06, 8.01, 7.50, 7.43
(d, J = 8.5 Hz, 2H each), 7.31 (m, 1H), 7.1–6.9 (m, 2H), 5.64 (br d, J = 6.1 Hz,
1H, H-3ꢁ), 5.49 (dd, J = 10.7, 5.5 Hz, 1H, H-1ꢁ), 4.75 (dd, J = 11.7, 4.3 Hz, 1H,
H-5ꢁa), 4.69 (dd, J = 11.7, 3.7 Hz, 1H, H-5ꢁb), 4.58 (ddd, J = 4.3, 3.7, 2.1 Hz,
1H, H-4ꢁ), 2.73 (br d, J = 13.7, 5.5 Hz, 1H, H-2ꢁα), 2.17 (ddd, J = 13.7, 10.7, 6.1
Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.26, 165.07, 158.90 (dd, J = 241.7, 2.2
Hz), 155.44 (dd, J = 241.7, 2.2 Hz), 139.94, 139.34, 131.03, 130.94, 129.97 (dd,
J = 16.5, 7.7 Hz), 128.83, 128.76, 128.13, 127.96, 116.24 (dd, J = 24.2, 8.8 Hz),
115.27 (dd, J = 24.2, 8.8 Hz), 113.36 (dd, J = 26.4, 4.4 Hz), 82.72, 77.24, 74.67,
64.65, 39.98; 19F NMR (CDCl3) 43.89 (m), 36.96 (m).
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluorobenzene (1a). To a suspen-
sion of 8a (0.51 g, 1 mmol) in MeOH (10 mL) was added NaOMe (0.16 g, 3 mmol),
and the resulting solution was stirred at 25◦C for 30 min. The reaction was quenched
with solid NH4Cl (0.2 g), and ether (50 mL) was added. The mixture was filtered,
the filtered solid was washed with EtOAc, and the solvent from the filtrate was
removed in vacuo. Purification of the residue obtained by silica gel column flash
chromatography (hexaneEtOAc, 1:2, v/v) afforded 1a (0.227 g, 99%) as a white
solid, which was recrystallized from hexane-ether to give white crystals (0.22 g,
96%); m.p. 131–132◦C; 1H NMR (CDCl3) 7.41 (td, J = 8.5, 6.7 Hz, 1H), 6.82 (td,
J = 8.5, 2.4 Hz, 1H), 6.75 (ddd, J = 11.0, 8.8, 2.4 Hz, 1H), 5.30 (dd, J = 10.1, 5.8
Hz, 1H, H-1ꢁ), 4.33 (dt, J = 6.4, 3.0 Hz, 1H, H-3ꢁ), 3.94 (td, J = 5.0, 3.0 Hz, 1H,
H-4ꢁ), 3.70 (d, J = 5.0 Hz, 2H, H-5ꢁ), 2.72 (br s, 2H, OH), 2.27 (br dd, J = 13.1, 5.8
Hz, 1H, H-2ꢁα), 1.96 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 13C NMR (DMSO-
d6) 161.43 (dd, J = 246.1, 12.1 Hz), 159.26 (dd, J = 247.2, 12.1 Hz), 128.73 (dd,
J = 8.8, 6.0 Hz), 125.98 (dd, J = 13.2, 4.4 Hz), 111.38 (dd, J = 20.9, 3.3 Hz),
103.46 (dd, J = 26.4, 25.3 Hz), 87.68, 72.72 (d, J = 2.2 Hz), 72.38, 62.39, 42.22;
19F NMR (CDCl3) 50.19 (m), 47.07 (m). Anal. calcd. for C11H12F2O3: C, 57.39;
H, 5.25. Found: C, 57.15; H, 5.02.
Compounds 1b–g and 2a–g listed below were prepared using a similar pro-
cedure to that described above for the preparation of 1a.
1-(2-Deoxy-α-D-ribofuranosyl)-2,4-difluorobenzene
syrup; 91% yield; H NMR (CDCl3) 7.53 (td, J = 8.5, 6.4 Hz, 1H), 6.89 (ddd,
(2a). Colorless
1

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SYNTHESIS OF THYMIDINE MIMICS
29
J = 8.5, 8.2, 2.1 Hz, 1H), 6.79 (ddd, J = 11.0, 8.8, 2.1 Hz, 1H), 5.30 (dd, J = 7.6,
6.4 Hz, 1H, H-1ꢁ), 4.47 (ddd, J = 7.0, 6.7, 5.5 Hz, 1H, H-3ꢁ), 4.10 (ddd, J = 5.5, 4.9,
4.0 Hz, 1H, H-4ꢁ), 3.83 (dd, J = 11.7, 4.0 Hz, 1H, H-5ꢁa), 3.74 (dd, J = 11.7, 4.9
Hz, H-5ꢁb), 2.74 (ddd, J = 12.8, 6.7, 6.4 Hz, 1H, H-2ꢁα), 2.02 (br s, 2H, OH), 2.00
(ddd, J = 12.8, 7.6, 7.0 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 162.16 (dd, J = 248.3,
12.1 Hz), 159.62 (dd, J = 248.3, 12.1 Hz), 127.74 (dd, J = 9.9, 5.5 Hz), 125.70
(dd, J = 13.2, 3.3 Hz), 111.12 (dd, J = 22.0, 4.4 Hz), 103.56 (t, J = 25.3 Hz),
85.20, 73.66 (d, J = 2.2 Hz), 72.68, 62.39, 42.15. Anal. calcd. for C11H12F2O3: C,
57.39; H, 5.25. Found: C, 57.40; H, 5.47.
5-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluorotoluene (1b). White crystals
(recrystallized from hexane); 98% yield; m.p. 84.5–86◦C; 1H NMR (CDCl3) 7.19
(t, J = 8.4 Hz, 1H), 6.70 (t, J = 9.9 Hz, 1H), 5.30 (dd, J = 10.1, 5.6 Hz, 1H, H-1ꢁ),
4.44 (m, 1H, H-3ꢁ), 4.0 (m, 1H, H-4ꢁ), 3.84 (dd, J = 11.4, 4.1 Hz, 1H, H-5ꢁa), 3.77
(dd, J = 11.4, 4.9 Hz, 1H, H-5ꢁb), 2.24 (dd, J = 13.4, 5.6 Hz, 1H, H-2ꢁα), 2.19 (s,
3H, CH3), 1.97 (ddd, J = 13.4, 10.1, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 160.28
(dd, J = 247.2, 12.1 Hz), 158.12 (dd, J = 247.2, 11.0 Hz), 129.27 (dd, J = 6.6,
5.5 Hz), 123.46 (dd, J = 13.2, 4.4 Hz), 120.52 (dd, J = 17.6, 4.4 Hz), 103.27 (dd,
J = 26.4, 25.3 Hz), 87.06, 74.45, 73.43, 63.18, 42.16, 13.89 (d, J = 3.3 Hz); 19
F
NMR (CDCl3) 47.20 (m), 42.92 (m). Anal. calcd. for C12H14F2O3: C, 59.01; H,
5.78. Found: C, 58.72; H, 5.57.
5-(2-Deoxy-α-D-ribofuranosyl)-2,4-difluorotoluene
(2b). Colorless
syrup; 92% yield; 1H NMR (CDCl3) 7.33 (t, J = 8.5 Hz, 1H), 6.72 (dd, J = 10.1,
9.8 Hz, 1H), 5.23 (dd, J = 8.2, 6.4 Hz, 1H, H-1ꢁ), 4.43 (ddd, J = 7.2, 6.7, 5.8 Hz,
1H, H-3ꢁ), 4.06 (ddd, J = 5.8, 4.9, 4.0 Hz, 1H, H-4ꢁ), 3.81 (dd, J = 11.7, 4.0 Hz,
1H, H-5ꢁa), 3.72 (dd, J = 11.7, 4.9 Hz, 1H, H-5ꢁb), 2.69 (ddd, J = 12.8, 6.7, 6.4
Hz, 1H, H-2ꢁα), 2.5 (br s, 2H, OH), 2.23 (s, 3H, CH3), 1.97 (ddd, J = 12.8, 8.2, 7.2
Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 160.15 (dd, J = 246.1, 12.1 Hz), 157.71 (dd,
J = 246.1, 12.1 Hz), 128.79 (dd, J = 6.6, 5.5 Hz), 125.0 (dd, J = 13.2, 4.4 Hz),
120.55 (dd, J = 16.5, 4.3 Hz), 103.15 (dd, J = 26.4, 25.3 Hz), 85.17, 73.68 (d,
J = 2.2 Hz), 72.76, 62.46, 42.33, 13.97 (d, J = 2.2 Hz); 19F NMR (CDCl3) 46.78
(m), 42.34 (m). Anal. calcd. for C12H14F2O3: C, 59.01; H, 5.78. Found: C, 58.68;
H, 5.90.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4,5-trifluorobenzene (1c). White crys-
1
tals (recrystallized from hexane-CH2Cl2); 95% yield; m.p. 72–73◦C; H NMR
(CDCl3) 7.29 (ddd, J = 11.0, 8.8, 6.7 Hz, 1H), 6.81 (td, J = 9.8, 6.4 Hz, 1H),
5.22 (dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.24 (ddd, J = 6.1, 2.7, 1.5 Hz, 1H, H-3ꢁ),
3.87 (td, J = 4.9, 2.7 Hz, 1H, H-4ꢁ), 3.78 (br s, 2H, OH), 3.63 (dd, J = 11.7, 4.9
Hz, 1H, H-5ꢁa), 3.58 (dd, J = 11.7, 4.9 Hz, 1H, H-5ꢁb), 2.23 (ddt, J = 13.1, 5.8,
1.5 Hz, 1H, H-2ꢁα), 1.79 (ddd, J = 13.1, 10.1, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR
(CDCl3) 154.42 (ddd, J = 245.0, 8.8, 2.2 Hz), 149.05 (ddd, J = 249.4, 14.3, 12.1
Hz), 146.78 (ddd, J = 242.8, 12.1, 3.3 Hz), 125.63 (ddd, J = 15.38, 5.5, 4.4 Hz),
114.91 (dd, J = 20.9, 5.5 Hz), 105.16 (dd, J = 27.5, 20.9 Hz), 87.15, 73.23, 72.70,
62.61, 42.09; 19F NMR (CDCl3) 40.72 (m), 25.77 (m), 18.67 (m). Anal. calcd. for
C11H11F3O3: C, 53.23; H, 4.47. Found: C, 53.05, H, 4.24.

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WANG ET AL.
1-(2-Deoxy-α-D-ribofuranosyl)-2,4,5-trifluorobenzene (2c). Colorless
1
syrup; 97% yield; H NMR (CDCl3) 7.36 (ddd, J = 10.7, 8.8, 7.0 Hz, 1H), 6.87
(td, J = 9.8, 6.4 Hz, 1H), 5.21 (dd, J = 7.6, 7.0 Hz, 1H, H-1ꢁ), 4.40 (ddd, J = 6.4,
5.2, 4.6 Hz, 1H, H-3ꢁ), 4.02 (ddd, J = 4.6, 4.6, 3.4 Hz, 1H, H-4ꢁ), 3.80 (dd, J =
12.2, 3.4 Hz, 1H, H-5ꢁa), 3.67 (dd, J = 12.2, 4.6 Hz, 1H, H-5ꢁb), 3.01 (br s, 2H,
OH), 2.69 (ddd, J = 12.6, 7.0, 5.2 Hz, 1H, H-2ꢁα), 1.90 (ddd, J = 12.6, 7.6, 6.4 Hz,
1H, H-2ꢁβ); 13C NMR (CDCl3) 154.17 (ddd, J = 245.0, 9.9, 2.2 Hz), 148.98 (ddd,
J = 243.9, 12.1, 3.3 Hz), 126.57 (dt, J = 15.4, 4.4 Hz), 114.71 (dd, J = 20.9, 5.5
Hz), 105.27 (dd, J = 27.5, 21.4 Hz), 85.38, 73.19, 72.46, 62.26, 41.98; 19F NMR
(CDCl3) 41.11 (m), 26.41 (m), 19.39 (m). Anal. calcd. for C11H11F3O3: C, 53.23;
H, 4.47. Found: C, 52.96; H, 4.29.
1-(2-Deoxy-β-D-ribofuranosyl)-5-chloro-2,4-difluorobenzene
(1d).
White crystals (recrystallized from hexane-CH2Cl2); 92% yield; m.p. 106–108◦C;
1H NMR (CDCl3) 7.43 (dd, J = 8.2, 7.6 Hz, 1H), 6.77 (dd, J = 9.8, 8.8 Hz, 1H),
5.18 (dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.23 (ddd, J = 6.4, 3.0, 1.8 Hz, 1H, H-3ꢁ),
3.85 (ddd, J = 5.2, 4.9, 3.0 Hz, 1H, H-4ꢁ), 3.63 (dd, J = 11.6, 4.9 Hz, 1H, H-5ꢁa),
3.58 (dd, J = 11.6, 5.2 Hz, 1H, H-5ꢁb), 3.28 (br s, 2H, OH), 2.20 (dddd, J = 13.1,
5.8, 1.8, 1.5 Hz, 1H, H-2ꢁα), 1.79 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 13C
NMR (CDCl3) 157.86 (dd, J = 248.3, 11.0 Hz), 156.99 (dd, J = 251.6, 12.1 Hz),
128.04 (d, J = 20.9 Hz), 126.11 (dd, J = 14.3, 3.3 Hz), 116.30 (dd, J = 17.6, 4.4
Hz), 104.74 (dd, J = 26.4, 25.3 Hz), 87.14, 73.50, 72.90, 62.82, 42.19; 19F NMR
(CDCl3) 48.94 (m), 45.31 (m). Anal. calcd. for C11H11ClF2O3: C, 49.92; H, 4.19.
Found: C, 49.57; H, 3.86.
1-(2-Deoxy-α-D-ribofuranosyl)-5-chloro-2,4-difluorobenzene
(2d).
White crystals (recrystallized from hexane-CH2Cl2); m.p. 89–90.5◦C; H NMR
(CDCl3) 7.58 (t, J = 7.9 Hz, 1H), 6.86 (t, J = 9.2 Hz, 1H), 5.21 (dd, J = 7.6, 6.4
Hz, 1H, H-1ꢁ), 4.40 (ddd, J = 7.6, 6.7, 5.8 Hz, 1H, H-3ꢁ), 4.02 (ddd, J = 5.8, 4.9,
4.3 Hz, 1H, H-4ꢁ), 3.78 (dd, J = 11.9, 4.3 Hz, 1H, H-5ꢁa), 3.69 (dd, J = 11.9, 4.9
Hz, 1H, H-5ꢁb), 2.87 (br s, 2H, OH), 2.70 (ddd, J = 12.8, 6.7, 6.4 Hz, 1H, H-2ꢁα),
1.91 (dt, J = 12.8, 7.6 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 157.58 (dd, J = 248.3,
9.9 Hz), 156.96 (dd, J = 250.5, 12.1 Hz), 128.01 (d, J = 5.5 Hz), 127.28 (dd,
J = 14.3, 4.4 Hz), 116.42 (dd, J = 17.6, 4.4 Hz), 104.80 (dd, J = 26.4, 25.3 Hz),
85.33, 73.22, 72.39, 62.24, 41.99. Anal. calcd. for C11H11ClF2O3: C, 49.92; H,
4.19. Found: C, 49.86; H, 4.12.
1
1-(2-Deoxy-β-D-ribofuranosyl)-5-bromo-2,4-difluorobenzene
(1e).
White crystals (recrystallized from hexane-CH2Cl2); 95% yield; m.p. 87–89◦C;
1H NMR (CDCl3) 7.65 (t, J = 7.6 Hz, 1H), 6.87 (dd, J = 9.8, 8.5 Hz, 1H), 5.29
(dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.41 (br s, 1H, H-3ꢁ), 4.0 (m, 1H, H-4ꢁ), 3.78
(br s, 2H, H-5ꢁ), 2.33 (br dd, J = 13.1, 5.8 Hz, 1H, H-2ꢁα), 1.95 (ddd, J = 13.1,
10.1, 6.4 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 158.77 (dd, J = 249.4, 10.4 Hz),
158.20 (dd, J = 249.4, 13.2 Hz), 131.08 (d, J = 5.5 Hz), 126.30 (dd, J = 14.3, 3.8
Hz), 104.84 (t, J = 26.4 Hz), 103.72 (dd, J = 20.9, 3.8 Hz), 87.05, 73.73, 73.37,
63.13, 42.57. Anal. calcd. for C11H11BrF2O3: C, 42.74; H, 3.59. Found: C, 42.64;
H, 3.44.

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31
1-(2-Deoxy-α-D-ribofuranosyl)-5-bromo-2,4-difluorobenzene
(2e).
White crystals (recrystallized from hexane-CH2Cl2); 95% yield; m.p. 94–96◦C;
1H NMR (CDCl3) 7.72 (t, J = 7.6 Hz, 1H), 6.83 (dd, J = 10.1, 8.2 Hz, 1H), 5.20
(dd, J = 7.6, 6.4 Hz, 1H, H-1ꢁ), 4.37 (ddd, J = 7.6, 7.0, 5.5 Hz, 1H, H-3ꢁ), 4.01
(ddd, J = 5.5, 4.9, 4.3 Hz, 1H, H-4ꢁ), 3.75 (dd, J = 11.9, 4.3 Hz, 1H, H-5ꢁa), 3.67
(dd, J = 11.9, 4.9 Hz, 1H, H-5ꢁb), 2.70 (br s, 2H, OH), 2.68 (ddd, J = 12.8, 7.0, 6.4
Hz, 1H, H-2ꢁα), 1.89 (dt, J = 12.8, 7.6 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 158.38
(dd, J = 249.4, 11.0 Hz), 158.07 (dd, J = 249.4, 12.1 Hz), 130.84 (d, J = 5.5
Hz), 127.77 (dd, J = 14.3, 3.3 Hz), 104.68 (dd, J = 27.5, 26.4 Hz), 103.71 (dd,
J = 25.3, 3.3 Hz), 85.35, 73.16 (d, J = 2.2 Hz), 72.27, 62.20, 41.98. Anal. calcd.
for C11H11BrF2O3: C, 42.74; H, 3.59. Found: C, 43.03; H, 3.45.
5-(2-Deoxy-β-D-ribofuranosyl)-4-chloro-2-fluorotoluene (1f).
White
crystals (recrystallized from hexane-ether); 97% yield; m.p. 100.5–102◦C; 1H NMR
(CDCl3) 7.33 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 9.2 Hz, 1H), 5.40 (dd, J = 10.1, 5.8
Hz, 1H, H-1ꢁ), 4.41 (m, 1H, H-3ꢁ), 4.01 (td, J = 4.6, 4.0 Hz, 1H, H-4ꢁ), 3.85 (m,
2H, H-5ꢁ), 2.44 (ddd, J = 13.3, 5.8, 2.2 Hz, 1H, H-2ꢁα), 2.24 (d, J = 1.8 Hz, 3H,
CH3), 2.15 (br s, 2H, OH), 1.85 (ddd, J = 13.3, 10.1, 6.6 Hz, 1H, H-2ꢁβ); 13C NMR
(CDCl3) 159.83 (d, J = 248.3 Hz), 134.33 (d, J = 3.3 Hz), 129.02 (d, J = 9.9 Hz),
128.76 (d, J = 5.5 Hz), 123.71 (d, J = 17.6 Hz), 115.89 (d, J = 25.3 Hz), 86.93,
76.38, 72.89, 62.87, 41.99, 14.08 (d, J = 2.2 Hz); 19F NMR (CDCl3) 44.73 (dd,
J = 9.2, 6.1 Hz). Anal. calcd. for C12H14ClFO3: C, 55.29; H, 5.41. Found: C, 54.92;
H, 5.17.
5-(2-Deoxy-α-D-ribofuranosyl)-4-chloro-2-fluorotoluene (2f). Colorless
syrup; 95% yield; 1H NMR (CDCl3) 7.40 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.8 Hz,
1H), 5.25 (dd, J = 8.2, 6.4 Hz, 1H, H-1ꢁ), 4.39 (ddd, J = 7.0, 6.7, 5.8 Hz, 1H, H-3ꢁ),
4.07 (ddd, J = 5.8, 4.9, 4.0 Hz, 1H, H-4ꢁ), 3.78 (dd, J = 11.6, 4.0 Hz, 1H, H-5ꢁa),
3.69 (dd, J = 11.6, 4.9 Hz, 1H, H-5ꢁb), 2.77 (ddd, J = 12.8, 6.7, 6.4 Hz, 1H, H-2ꢁα),
2.23 (d, J = 1.8 Hz, 3H, CH3), 3.07 (br s, 1H, OH), 3.05 (br s, 1H, OH), 1.79 (ddd,
J = 12.8, 8.2, 7.0 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 159.86 (d, J = 247.2 Hz),
135.76 (d, J = 3.3 Hz), 128.60 (d, J = 5.5 Hz), 128.60, 123.84 (d, J = 16.5 Hz),
115.99 (d, J = 25.3 Hz), 85.55, 76.38, 72.68, 62.45, 42.18, 14.17 (d, J = 2.2 Hz).
Anal. calcd. for C12H14ClFO3: C, 55.29; H, 5.41. Found: C, 55.11; H, 5.62.
2-Chloro-4-fluoro-5-[1ꢁ-[2ꢁ-deoxy-β-D-ribofuranosyl]]toluene
(1g).
White crystals (recrystallized from hexane-CH2Cl2); 89% yield; m.p. 103–105◦C;
1H NMR (CDCl3) δ 7.27 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 10.1 Hz, 1H), 5.27 (dd,
J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.33 (ddd, J = 6.1, 3.0, 2.4 Hz, 1H, H-3ꢁ), 3.94 (m,
1H, H-4ꢁ), 3.70 (m, 2H), 2.9 (br s, 2H, OH), 2.29 (s, 3H, CH3), 2.25 (m, 1H, H-2ꢁα),
1.96 (m, 1H, H-2ꢁβ); 13C NMR (CDCl3) δ 157.73 (d, J = 247.2 Hz), 133.35 (d,
J = 9.9 Hz), 131.75 (d, J = 4.4 Hz), 128.73 (d, J = 5.5 Hz), 126.90 (d, J = 13.2
Hz), 115.97 (d, J = 25.3 Hz), 87.10, 74.17, 73.0, 62.93, 42.12, 19.12; 19F NMR
(CDCl3) δ 40.90 (t, J = 9.1 Hz). Anal. calcd. for C12H14ClFO3: C, 55.29; H, 5.41.
Found: C, 55.09; H, 5.34.
5-(2-Deoxy-α-D-ribofuranosyl)-2-chloro-4-fluorotoluene (2g). Colorless
syrup; 91% yield; 1H NMR (CDCl3) 7.35 (d, J = 7.9 Hz, 1H), 7.02 (d, J = 10.1 Hz,

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1H), 5.19 (dd, J = 7.8, 7.0 Hz, 1H, H-1ꢁ), 4.39 (ddd, J = 7.2, 6.4, 5.2 Hz, 1H, H-3ꢁ),
4.02 (m, 1H, H-4ꢁ), 3.76 (dd, J = 11.7, 4.0 Hz, 1H, H-5ꢁa), 3.67 (dd, J = 11.7, 4.3
Hz, 1H, H-5ꢁb), 3.05 (br s, 2H, OH), 2.67 (ddd, J = 12.8, 7.0, 6.4 Hz, 1H, H-2ꢁα),
2.31 (s, 3H, CH3), 1.97 (ddd, J = 12.8, 7.8, 7.2 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3)
157.46 (d, J = 247.2 Hz), 133.34 (d, J = 11.0 Hz), 131.93 (d, J = 3.3 Hz), 128.34
(d, J = 5.5 Hz), 128.15 (d, J = 13.2 Hz), 116.03 (d, J = 24.2 Hz), 85.15, 73.68,
73.65, 62.28, 42.13, 19.33. Anal. calcd. for C12H14ClFO3: C, 55.29; H, 5.41. Found:
C, 55.54; H, 5.11.
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-β-D-ribofuranosyl)-2,4-difluoro-
5-iodobenzene(9a). Aliquotsof N-iodosuccinimide(4.5g, 20mmol)wereadded
over a period of 20 min to a solution of 8a (5.07 g, 10 mmol) in CF3CO2H (20 mL)
cooled in an ice-water bath. The resulting black-red solution was stirred at 25◦C
for 20 h. The reaction mixture was poured onto cold water (100 mL), and ex-
tracted with ether (3 × 100 mL). The combined organic extracts were washed
with saturated aqueous NaHCO3 (2 × 100 mL), saturated aqueous sodium hy-
drosulfite (100 mL) and brine, and the organic fraction was dried (Na2SO4). Re-
moval of the solvent in vacuo gave a residue that was purified via silica gel col-
umn flash chromatography (hexane-ether, 5:1, v/v) to give 9a (5.8 g, 91%) as a
colorless syrup, which was recrystallized from hexane-ether to give white nee-
1
dles (5.23 g, 82%); m.p. 104–105◦C; H NMR (CDCl3) 8.02, 7.99, 7.47, 7.43
(d, J = 8.5 Hz, 2H each), 7.90 (t, J = 7.5 Hz, 1H), 6.80 (dd, J = 9.8, 7.8 Hz,
1H), 5.59 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.40 (dd, J = 10.7, 5.2 Hz, 1H, H-1ꢁ),
4.74 (dd, J = 11.9, 4.3 Hz, 1H, H-5ꢁa), 4.66 (dd, J = 11.9, 4.0 Hz, 1H, H-5ꢁb),
4.52 (ddd, J = 4.3, 4.0, 2.1 Hz, 1H, H-4ꢁ), 2.64 (br dd, J = 14.0, 5.2 Hz, 1H, H-
2ꢁα), 2.10 (ddd, J = 14.0, 10.7, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 165.16,
165.0, 161.07 (dd, J = 247.2, 12.1 Hz), 159.82 (dd, J = 249.4, 11.0 Hz), 139.92,
139.64, 136.60 (dd, J = 5.5, 3.3 Hz), 130.96 (4C), 128.79 (4C), 127.98, 127.82,
126.16 (dd, J = 14.3, 3.3 Hz), 104.03 (dd, J = 27.5, 26.4 Hz), 82.80, 77.19, 75.02
(dd, J = 25.3, 4.4 Hz), 74.02, 64.50, 40.16; 19F NMR (CDCl3) 70.91 (m), 46.60
(m). Anal. calcd. for C25H17Cl2F2IO5: C, 47.42; H, 2.71. Found: C, 47.36; H,
2.54.
1-(3,5-Bis-O-( p-chlorobenzoyl)-2ꢁ-deoxy-α-D-ribofuranosyl)-2,4-diflu-
oro-5-iodobenzene (9b). It was prepared using a procedure similar to that used
for the synthesis of 9a from 7a in 85% yield; White crystals (recrystallized from
1
hexane); m.p. 72–74◦C; H NMR (CDCl3) 8.02, 7.73, 7.45, 7.40 (d, J = 8.5 Hz,
2H each), 7.99 (dd, J = 7.6, 7.3 Hz, 1H), 6.83 (dd, J = 10.1, 7.6 Hz, 1H), 5.62
(dd, J = 6.4, 3.7, 1.8 Hz, 1H, H-3ꢁ), 5.52 (dd, J = 7.9, 4.3 Hz, 1H, H-1ꢁ), 4.76
(td, J = 5.2, 1.8 Hz, 1H, H-4ꢁ), 4.57 (dd, J = 11.9, 5.2 Hz, 1H, H-5ꢁa), 4.51 (dd,
J = 11.9, 5.2 Hz, 1H, H-5ꢁb), 2.95 (ddd, J = 12.5, 7.9, 6.4 Hz, 1H, H-2ꢁa), 2.25
(br d, J = 12.5 Hz, 1H, H-2ꢁb); 13C NMR (CDCl3) 165.16, 164.71, 160.93 (dd,
J = 248.3, 12.1 Hz), 159.48 (dd, J = 249.4, 11.0 Hz), 139.93, 139.68, 136.59 (dd,
J = 5.5, 2.2 Hz), 130.95, 130.80, 128.79, 128.73, 128.27 (dd, J = 14.3, 3.3 Hz),
128.01, 127.63, 104.10 (dd, J = 27.5, 26.4 Hz), 74.65 (dd, J = 25.3, 4.3 Hz), 82.49,
76.50, 74.17, 64.41, 39.09; 19F NMR (CDCl3) 70.36 (m), 47.49 (m).

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33
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene
(1i).
Compound 9a (3.17 g, 5 mmol) was treated with NaOMe (0.81 g, 15 mmol) in
MeOH (100 mL) for 20 min, and then the reaction was quenched with solid NH4Cl
(1.5 g). Removal of the solvent in vacuo gave a residue that was dissolved in EtOAc
(100 mL), and the mixture was filtered and washed with EtOAc. Removal of the
solvent from the filtrate gave a residue that was purified via silica gel column flash
chromatography (hexane-EtOAc, 1:1 to 1:1.5 v/v) to give 1i (1.75 g, 98%) as a
white solid, which was recrystallized from hexane-CH2Cl2 to give white needles
(1.6 g, 90%); m.p. 70–72◦C; 1H NMR (CDCl3) 7.83 (t, J = 7.4 Hz, 1H), 6.83 (dd,
J = 10.1, 7.6 Hz, 1H), 5.29 (dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.47 (ddd, J = 6.4, 3.0,
1.8 Hz, 1H , H-3ꢁ), 4.02 (ddd, J = 4.9, 4.3, 3.0 Hz, 1H, H-4ꢁ), 3.86 (dd, J = 11.6,
4.3 Hz, 1H, H-5ꢁa), 3.79 (dd, J = 11.6, 4.9 Hz, 1H, H-5ꢁb), 2.33 (ddt, J = 13.4, 5.8,
1.8 Hz, 1H, H-2ꢁα), 1.99 (ddd, J = 13.4, 10.1, 6.4 Hz, 1H, H-2ꢁβ), 1.89 (br s, 2H,
OH); 13C NMR (CDCl3) 161.02 (dd, J = 247.2, 12.1 Hz), 160.14 (dd, J = 249.4,
11.0 Hz), 136.77 (dd, J = 5.5, 3.3 Hz), 126.71 (dd, J = 14.3, 4.4 Hz), 104.21 (dd,
J = 27.4, 26.4 Hz), 87.10, 74.89 (dd, J = 25.3, 4.4 Hz), 73.74, 73.30, 63.13, 42.33;
19F NMR (CDCl3) 70.52 (m), 47.42(m). Anal. calcd. for C11H11F2IO3: C, 37.10;
H, 3.11. Found: C, 37.30; H, 2.94.
1-(2-Deoxy-α-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (2i). It was
prepared similarly from 9b in 91% yield; white solid (recrystallized from hexane–
1
CH2Cl2); m.p. 86–88◦C; H NMR (CDCl3) 7.90 (t, J = 7.3 Hz, 1H), 6.79 (dd,
J = 9.8, 7.9 Hz, 1H), 5.19 (dd, J = 7.6, 6.4 Hz, 1H, H-1ꢁ), 4.40 (ddd, J = 7.3, 5.8,
4.9 Hz, 1H, H-3ꢁ), 4.03 (ddd, J = 4.9, 4.58, 4.0 Hz, 1H, H-4ꢁ), 3.78 (dd, J = 11.7,
4.0 Hz, 1H, H-5ꢁa), 3.69 (dd, J = 11.7, 4.9 Hz, 1H, H-5ꢁb), 3.02 (br s, 2H, OH),
2.69 (ddd, J = 12.5, 7.3, 6.4 Hz, 1H, H-2ꢁα), 1.91 (ddd, J = 12.5, 7.6, 5.8 Hz, 1H,
H-2ꢁβ); 13C NMR (CDCl3) 160.88 (dd, J = 247.2, 12.1 Hz), 159.73 (dd, J = 249.4,
11.0 Hz), 136.50 (dd, J = 5.5, 3.3 Hz), 128.10 (dd, J = 14.3, 4.4 Hz), 104.12 (dd,
J = 27.5, 26.4 Hz), 85.27, 74.89 (dd, J = 25.3, 3.3 Hz), 73.23 (d, J = 2.2 Hz),
72.47, 62.31, 42.10; 19F NMR (CDCl3) 70.22 (s), 47.01 (d, J = 9.2 Hz). Anal.
calcd. for C11H11F2IO3: C, 37.10; H, 3.11. Found: C, 37.45; H, 2.83.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-trifluoromethylbenzene
(1j). A mixture of 9a (0.32 g, 0.5 mmol), CuI (0.14 g, 0.75 mmol), and ground
anhydrous KF (0.044 g, 0.75 mmol) in DMF (1.5 mL) was stirred with heating.
When the reaction temperature reached 60◦C, ClCF2COOMe (0.5 mL) was added,
the reaction flask was sealed with a septum, and the temperature was slowly in-
creased to 120◦C. The reaction was allowed to proceed at 120◦C for 20 h with
stirring, and then the reaction mixture was cooled to 25◦C before pouring onto
water (50 mL). This mixture was filtered, washed with ether, the filtrate was ex-
tracted with ether (3 × 20 mL), and washed with water and brine prior to drying
(Na2SO4). Removal of the solvent in vacuo gave a residue which was purified via
silica gel column flash chromatography (hexane-EtOAc, 20:1, v/v) to give the bis-
p-chlorobenzoyl ester of 1j (0.28 g, 97%) as a colorless syrup; 1H NMR (CDCl3)
7.87, 7.79, 7.31, 7.24 (d, J = 8.5 Hz, each 2H), 7.67 (t, J = 7.9 Hz, 1H), 6.79 (dd,
J = 10.1, 9.8 Hz, 1H), 5.45 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.28 (dd, J = 10.7, 5.2

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34
WANG ET AL.
Hz, 1H, H-1ꢁ), 4.60 (dd, J = 11.8, 4.0 Hz, 1H, H-5ꢁa), 4.51 (dd, J = 11.8, 4.0 Hz,
1H, H-5ꢁb), 4.38 (td, J = 4.0, 2.1 Hz, 1H, H-4ꢁ), 2.52 (br dd, J = 14.0, 5.2 Hz,
1H, H-2ꢁα), 1.94 (ddd, J = 14.0, 10.7, 6.1 Hz, 1H, H-2ꢁβ). The product obtained
above (0.28 g, 0.48 mmol) was treated with NaOMe (0.080 g, 1.5 mmol) in MeOH
(5 mL) at 25◦C for 10 min. The reaction was quenched with solid NH4Cl (0.15 g),
and ether (50 mL) was added. Filtration, removal of the solvent from the filtrate
in vacuo, and purification of the residue obtained via silica gel column flash chro-
matography (hexane-EtOAc, 1:1 to 1:1.5 v/v) gave 1j (0.14 g, 967%) as a colorless
syrup; 1H NMR (CDCl3) 7.75 (t, J = 7.9 Hz, 1H), 6.92 (t, J = 9.8 Hz, 1H), 5.32
(dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.42 (ddd, J = 6.1, 3.0, 1.8 Hz, 1H, H-3ꢁ), 4.01
(td, J = 4.9, 3.0 Hz, 1H, H-4ꢁ), 3.78 (d, J = 4.9 Hz, 2H, H-5ꢁ), 3.25 (br s, 1H,
OH), 2.91 (br s, 1H, OH), 2.35 (ddt, J = 13.3, 5.8, 1.8 Hz, 1H, H-2ꢁα), 1.95 (ddd,
J = 13.3, 10.1, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 161.71 (dd, J = 256.0,
12.1 Hz), 159.48 (dd, J = 257.1, 13.2 Hz), 125.91 (d, J = 2.2 Hz), 122.12 (q,
J = 272.5 Hz), 114.60 (m), 105.22 (dd, J = 24.2, 20.9 Hz), 87.07, 73.66, 73.29,
63.01, 42.32; 19F NMR (CDCl3) 100.94 (d, J = 12.2 Hz, 3F), 53.91 (q, J = 9.2 Hz),
50.22 (m). Anal. calcd. for C12H11F5O3: C, 48.33; H, 3.72. Found: C, 48.44; H,
3.61.
1-(2-Deoxy-β-D-ribofuranosyl)-5-cyano-2,4-difluorobenzene (1k).
A
mixture of compound 9a (0.25 g, 0.4 mmol), Zn(CN)2 (0.026 g, 0.22 mmol),
and Pd(PPh3)4 (0.045 g, 0.04 mmol) in dry DMF (1 mL) was stirred at 80–90◦C
under argon for 4 h. After cooling to 25◦C, the reaction mixture was diluted with
toluene (30 mL), and washed with 2N NH4OH twice, and then brine prior to dry-
ing (Na2SO4). Removal of the solvent in vacuo and purification of the residue
obtained via silica gel column flash chromatography (hexane-EtOAc, 10:1 v/v) to
give the bis-(p-Cl-benzoyl) ester of 1k (0.2 g, 94%) as light yellow crystals; m.p.
94–95◦C; 1H NMR (CDCl3) 7.98, 7.93, 7.41, 7.37 (d, J = 8.5 Hz, 2H each), 7.83
(dd, J = 7.6, 7.3 Hz, 1H), 6.93 (t, J = 9.2 Hz, 1H), 5.59 (br d, J = 6.1 Hz, 1H,
H-3ꢁ), 5.35 (dd, J = 10.7, 5.2 Hz, 1H, H-1ꢁ), 4.74 (dd, J = 11.9, 4.0 Hz, 1H, H-5ꢁa),
4.63 (dd, J = 11.9, 4.3 Hz, 1H, H-5ꢁb), 4.53 (ddd, J = 4.3, 4.0, 1.8 Hz, 1H, H-4ꢁ),
2.69 (br dd, J = 13.7, 5.2 Hz, 1H, H-2ꢁα), 2.05 (ddd, J = 13.7, 10.7, 6.1 Hz, 1H, H-
2ꢁβ); 13C NMR (CDCl3) 165.10, 164.94, 163.02 (dd, J = 261.5, 13.2 Hz), 162.08
(dd, J = 259.3, 12.1 Hz), 139.90, 139.68, 131.88 (d, J = 5.5 Hz), 130.96, 130.83,
128.81, 128.76, 127.88, 127.72, 126.36 (dd, J = 13.2, 4.4 Hz), 112.87, 105.02 (dd,
J = 26.4, 24.2 Hz), 98.04 (dd, J = 15.4, 4.4 Hz), 82.95, 77.00, 73.92, 64.32, 39.89;
IR (NaCl): 2236 (CN), 1724 (C=O) cm−1.
The above compound (0.15 g, 0.28 mmol) was treated with NaOMe (0.05 g,
0.9 mmol) in MeOH (5 mL) at 25◦C with stirring for 15 min. The reaction was
quenched with solid NH4Cl, and ether (50 mL) was added. After filtration, removal
of the solvent in vacuo gave a residue that was purified via silica gel column flash
chromatography (hexane-EtOAc, 1:2 v/v) to afford 1k (0.06 g, 84%) as white
1
needles after recrystallization from hexane-EtOAc; m.p. 149.5–151◦C; H NMR
(CDCl3) 7.86 (t, J = 7.4 Hz, 1H), 6.88 (t, J = 9.3 Hz, 1H), 5.22 (dd, J = 10.1,

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SYNTHESIS OF THYMIDINE MIMICS
35
5.8 Hz, 1H, H-1ꢁ), 4.25 (ddd, J = 6.1, 2.7, 1.8 Hz, 1H, H-3ꢁ), 3.88 (td, J = 4.9, 2.7
Hz, 1H, H-4ꢁ), 3.62 (d, J = 4.9 Hz, 2H, H-5ꢁ), 2.26 (ddt, J = 13.1, 5.8, 1.8 Hz, 1H,
H-2ꢁα), 1.76 (ddd, J = 13.1, 10.1, 6.1 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3) 162.81
(dd, J = 260.4, 13.2 Hz), 162.23 (dd, J = 259.3, 12.1 Hz), 132.18 (d, J = 6.6 Hz),
127.68 (dd, J = 15.4, 4.4 Hz), 113.16, 104.69 (dd, J = 26.4, 23.5 Hz), 97.44 (d,
J = 3.3 Hz), 87.33, 72.94 (d, J = 2.2 Hz), 72.61, 62.46, 42.14; 19F NMR (CDCl3)
58.07 (q, J = 9.2 Hz), 56.78 (q, J = 9.2 Hz). Anal. calcd. for C12H11F2NO3: C,
56.47; H, 4.34; N, 5.49. Found: C, 56.57; H, 4.20; N, 4.27.
1-(2-Deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-nitrobenzene(1l)and1-(2-
Deoxy-β-D-ribofuranosyl)-2-fluoro-4-methoxy-5-nitrobenzene(1m). NH4NO3
(0.16 g, 2 mmol) was added to a solution of 8a (0.507 g, 1 mmol) in (CF3CO)2
(5 mL) and CH2Cl2 (1 mL), and the resulting solution was stirred at 25◦C for 1 h. A
second aliquot of NH4NO3 (0.08 g, 1 mmol) was added and the reaction was allowed
to proceed for another 30 min with stirring. The reaction mixture was poured onto
water (100 mL), and extracted with CH2Cl2 (3 × 20 mL). The combined organic
extracts were washed with saturated aqueous NaHCO3 and brine prior to drying
(Na2SO4). Removal of the solvent in vacuo gave a residue which was purified via
silica gel column flash chromatography (hexane-EtOAc, 10:1 to 5:1, v/v) to give
the 5-nitro derivative of 8a (0.43 g, 79%) as a pale yellow syrup; 1H NMR (CDCl3)
8.02, 7.99, 7.47, 7.42 (d, J = 8.8 Hz, 2H each), 7.04 (t, J = 9.8 Hz, 1H), 6.35 (t,
J = 7.7 Hz, 1H), 5.61 (br d, J = 6.1 Hz, 1H, H-3ꢁ), 5.43 (dd, J = 10.7, 5.2 Hz,
1H, H-1ꢁ), 4.75 (dd, J = 11.9, 4.0 Hz, 1H, H-5ꢁa), 4.68 (dd, J = 11.9, 4.0 Hz, 1H,
H-5ꢁb), 4.57 (td, J = 4.0, 2.1 Hz, 1H, H-4ꢁ), 2.71 (br dd, J = 14.0, 5.2 Hz, 1H,
H-2ꢁα), 2.12 (ddd, J = 14.0, 10.7, 6.1 Hz, H-2ꢁβ); 19F NMR (CDCl3) 58.05 (m),
48.87 (dd, J = 11.4, 9.2 Hz).
Method A: The above compound (0.4 g, 0.72 mmol) was treated with a
solution of NaOMe (0.12 g, 2.2 mmol) in MeOH (10 mL) for 10 min. Completion
of the reaction as described previously, and purification of the product via silica gel
column flash chromatography (hexane-EtOAc, 1:2 to 0:1, v/v) afforded 1l (0.11 g,
59%) as light yellow needles (recrystallized from ether-hexane) and 1m (0.06 g,
32%) as a light yellow solid (recrystallized from ether-hexane).
Method B: Aqueous NaOH (2 mL of 1N, 4 mmol) was added to a solution of
above compound (0.28 g, 0.5 mmol) in THF (10 mL). The reaction was allowed to
proceed at 25◦C for 6 h with stirring, the reaction was quenched with solid NH4Cl,
themixturewas filtered, andthesolventwasremovedinvacuo. Theresidueobtained
was extracted with acetone (10 mL), filtered, and the solid was washed with acetone.
Removal of the solvent from the filtrate in vacuo gave a residue which was purified
via silica gel column flash chromatography (hexane-EtOAc, 1:2 to 1:5 v/v) to give
a yellow solid. Recrystallization from hexane-ether afforded 1l (0.128 g, 93%) as
pale yellow crystals.
1l: Yellow needles; m.p. 100–101◦C; 1H NMR (CDCl3) 8.32 (t, J = 7.8 Hz,
1H), 6.99 (t, J = 9.9 Hz, 1H), 5.31 (dd, J = 9.8, 5.8 Hz, 1H, H-1ꢁ), 4.36 (ddd,
J = 6.4, 3.0, 2.1 Hz, 1H, H-3ꢁ), 3.98 (ddd, J = 4.9, 4.6, 3.0 Hz, 1H, H-4ꢁ), 3.76 (dd,

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36
WANG ET AL.
J = 11.6, 4.6 Hz, 1H, H-5ꢁa), 3.71 (dd, J = 11.6, 4.9 Hz, 1H, H-5ꢁb), 2.36 (dddd,
J = 13.1, 5.8, 2.1, 1.8 Hz, 1H, H-2ꢁα), 1.89 (ddd, J = 13.1, 9.8, 6.4 Hz, 1H, H-2ꢁβ);
13C NMR (CDCl3) 163.1 (dd, J = 259.3, 11.0 Hz), 155.4 (dd, J = 265.9, 12.1 Hz),
133.83 (m), 127.12 (dd, J = 15.4, 4.4 Hz), 125.22 (dd, J = 5.5, 2.2 Hz), 106.18
(dd, J = 27.5, 25.3 Hz), 87.37, 73.01, 72.76, 62.62, 42.10; 19F NMR (CDCl3) 58.43
(m), 47.79 (dd, J = 11.4, 9.1 Hz). Anal. calcd. for C11H11F2NO5: C, 48.01; H, 4.03;
N, 5.09. Found: C, 48.00; H, 5.93; N, 4.99.
1m: Pale yellow crystals; m.p. 127–128◦C; 1H NMR (CDCl3) 8.09
(d, J = 7.6 Hz, 1H), 6.76 (d, J = 11.6 Hz, 1H), 5.28 (dd, J = 10.7, 5.8 Hz, 1H,
H-1ꢁ), 4.35 (ddd, J = 6.4, 3.0, 1.8 Hz, 1H, H-3ꢁ), 3.94 (m, 1H, H-4ꢁ), 3.92 (s, 3H,
OCH3), 3.72 (d, J = 4.9 Hz, 2H, H-5ꢁ), 2.57 (s, 2H, OH), 2.30 (dddd, J = 13.1, 5.8,
1.8, 1.5 Hz, 1H, H-2ꢁα), 1.92 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 19F NMR
(CDCl3) 55.77 (dd, J = 11.6, 7.6 Hz). Anal. calcd. for C12H14FNO6: C, 50.18; H,
4.91; N, 4.88. Found: C, 49.91; H, 4.67; N, 5.27.
1-(2-Deoxy-β-D-ribofuranosyl)-5-amino-2,4-difluorobenzene (1n).
A
mixture of compound 1l (0.072 g, 0.26 mmol) and 10% Pd-on-C (0.015 g) in
EtOH (3 mL) was stirred at 60–70◦C under 1 atm of hydrogen gas for 2 h. After
cooling to 25◦C, the reaction mixture was filtered and washed with EtOH. Re-
moval of the solvent from the filtrate in vacuo gave pale brown crystals, which
were recrystallized from ether to afford 1n (0.059 g, 92%) as light yellow crys-
1
tals; m.p. 126–128◦C; H NMR (CDCl3) 6.88 (dd, J = 9.8, 7.3 Hz, 1H), 6.63
(t, J = 10.3 Hz, 1H), 5.20 (dd, J = 10.1, 5.8 Hz, 1H, H-1ꢁ), 4.21 (ddd, J = 6.4,
3.0, 2.1 Hz, 1H, H-3ꢁ), 3.84 (td, J = 4.9, 3.0 Hz, 1H, H-4ꢁ), 3.75 (br s, 4H,
NH2 and OH), 3.60 (d, J = 4.9 Hz, 2H, H-5ꢁ), 2.17 (br dd, J = 13.1, 5.8 Hz,
1H, H-2ꢁβ), 1.82 (ddd, J = 13.1, 10.1, 6.4 Hz, 1H, H-2ꢁβ); 13C NMR (CDCl3)
151.88 (dd, J = 239.5, 11.0 Hz), 150.11 (dd, J = 241.7, 12.1 Hz), 130.34 (dd,
J = 12.1, 3.3 Hz), 124.39 (dd, J = 14.3, 3.3 Hz), 114.61 (t, J = 4.4 Hz), 103.18
(dd, J = 27.5, 23.1 Hz), 87.05, 73.86 (d, J = 2.2 Hz), 72.71, 62.71, 42.15. Anal.
calcd. for C11H13F2NO3: C, 53.88; H, 5.34; N, 5.71. Found: C, 53.83; H, 5.26; N,
5.59.
In Vitro Cell Cytotoxicity (MTT assay). KBALB, KBALB-STK, human
143B, human 143B-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
at 8 × 102 cells/well, and incubated at 37◦C in a humidified 5% CO2 atmosphere
for 24 h.
The test compound was dissolved in DMEM medium, and 100 µL of this
solution was added to cells in 96-well plates to produce the preselected test 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 consist-
ing 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

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SYNTHESIS OF THYMIDINE MIMICS
37
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 incubated at 37◦C for 4 h. The medium was removed from the wells, dimethyl
sulfoxide (150 µL) was added to each well, and the plates were placed on a shaker
for 15 min to dissolve the formazan crystals. The absorbance at 540 nm (A540) was
measured immediately in each well, using a scanning multi-well spectrophotome-
ter (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 concentration 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 drug.
In Vitro Anticancer Screen. A 3-cell line panel consisting of MCF7 (breast),
HNCI-460 (lung), and SF-268 (CNS) is currently used as a primary screen by the
Developmental Therapeutics Program, Division of Cancer Treatment and Diagno-
sis, U.S. National Cancer Institute. In this protocol, each cell line is inoculated and
preincubated on a microtiter plate. Test compounds are then added at a single con-
centration, and the culture is incubated for 48 h. End-point determinations are made
with sulforhodamine B, a protein-binding dye. Results for each test compound are
reported as the percent of growth of the treated cells when compared to the untreated
control cells. Compounds that reduce the growth of any one of the cell lines to 32%
or less (negative numbers indicate cell kill) are passed on for evaluation in the full
panel of 60 cell lines over a 5-log dose range (33).
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
(34,41–43). 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, Sindbis
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 varying 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.
InhibitionofHIV-InducedGiantCellFormation. CEMcellcultureswere
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 sus-
pension was transferred to 200 µL microtitre plate wells containing 100 µL of

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WANG ET AL.
serial dilutions of the test compound solutions. After 4 days of incubation at 37◦C,
cell cultures were examined for syncytium formation as previously described (44).
Cytostatic Activity Assays. The cytostatic assays were performed as pre-
viously described (45). Briefly, 100-µL aliquots of the cell suspensions (7.5 ×
105 human T-lymphocyte CEM cells/mL) were added to the wells of a microtitre
plate containing 100 µL of varying concentrations of the test compounds. After
a 3-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 expressed as the compound concentration that reduced the number of viable cells
by 50% (CC50). The cytotoxicity measurements were based on microscopically
visible alteration of normal cell morphology (E6SM, HeLa, Vero) or inhibition of
cell growth (HEL), as previously described (43).
ACKNOWLEDGMENTS
We are grateful to the Medical Research Council of Canada (Grant. No.
MT-14480) for financial support of this research, and to the Alberta Heritage Foun-
dation for Medical Research for a fellowship to one of us (Z.X.W.). We would
also like to thank the United States National Cancer Institute for providing some in
vitro anticancer data. 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 Gemeen-
schap 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.
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