Synthesis and biological activity of 4′-thio-L-xylofuranosyl purine nucleosides

Article abstract of DOI:10.1080/15257770500269077

□ A series of some new 4′-thio-L-xylofuranosyl nucleosides were prepared and evaluated as potential anticancer agents. A versatile sugar intermediate for direct coupling with the purine moiety is also synthesized by an efficient and high-yielding route. Proof of structure and configuration at all chiral centers of the nucleosides was obtained by proton NMR. All target compounds were evaluated in a series of human cancer cell lines in vitro. The details of the synthesis of the carbohydrate precursor 1-O-acetyl-2,3,5-tri-O- benzyl-4-thio-L-xylofuranose (6) and corresponding purine nucleosides are presented in the manuscript. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770500269077

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Synthesis and Biological Activity of 4′-Thio-L-
xylofuranosyl Purine Nucleosides
Kamal N. Tiwari a , Lea Messini a , John A. Montgomery a & John A. Secrist III a
a Southern Research Institute , Birmingham, Alabama, USA
Published online: 16 Aug 2006.
To cite this article: Kamal N. Tiwari , Lea Messini , John A. Montgomery & John A. Secrist III (2005) Synthesis and Biological
Activity of 4′-Thio-L-xylofuranosyl Purine Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 1895-1906, DOI:
10.1080/15257770500269077
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1895–1906, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500269077
SYNTHESIS AND BIOLOGICAL ACTIVITY OF
4 -THIO-L-XYLOFURANOSYL PURINE NUCLEOSIDES
Kamal N. Tiwari, Lea Messini, John A. Montgomery, and John A. Secrist III
ᮀ
Southern Research Institute, Birmingham, Alabama, USA
ᮀ
A series of some new 4 -thio-L-xylofuranosyl nucleosides were prepared and evaluated as
potential anticancer agents. A versatile sugar intermediate for direct coupling with the purine
moiety is also synthesized by an efficient and high-yielding route. Proof of structure and
configuration at all chiral centers of the nucleosides was obtained by proton NMR. All target
compounds were evaluated in a series of human cancer cell lines in vitro. The details of the
synthesis of the carbohydrate precursor 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-L-xylofuranose (6) and
corresponding purine nucleosides are presented in the manuscript.
Keywords Nucleosides; 4 -Thionucleosides; Purines; Antiviral activity
INTRODUCTION
In our ongoing initiative to develop novel anticancer agents, we have
pursued the synthesis of many different types of nucleosides. These efforts
have resulted in the development of fludarabine,[1,2] which received FDA
approval over a decade ago, and clofarabine,[3–8] which received initial ap-
proval by the FDA at the end of 2004 for the treatment of pediatric acute
lymphoblastic leukemia (ALL). Other nucleosides used regularly in cancer
chemotherapy include gemcitabine, cladribine, pentostatin, capecitabine,
and cytarabine.[9] For gemcitabine, the utility extends beyond hematologic
malignancies to include some solid tumor indications. There continues to
be a need for new agents, and nucleosides continue to show promise;[10]
Dedicated to the memory of John A. Montgomery.
Received 8 February 2005; accepted 20 June 2005.
This investigation was supported by NIH Grant No. CA34200. We gratefully acknowledge the
data received from the following laboratories at Southern Research Institute: Dr. W. B. Parker and
Dr. W. R. Waud (cytotoxicity data), R.W. Buckheit (antiviral data), Sheila Campbell (HPLC analyses),
Jerry Frye (large-scale synthesis), Dr. J. M. Riordan, M. D. Richardson, J. C. Bearden, and J. W. Truss
of the Molecular Spectroscopy Laboratory (spectral data).
Address correspondence to John A. Secrist III, Southern Research Institute, P.O. Box 55305,
Birmingham, AL 35255-5305. E-mail: secrist@sri.org
1895

1896
K. N. Tiwari et al.
they also have the advantage of having the potential for multiple sites of
action as well as activity against leukemias and lymphomas as well as solid
tumors. For some years we have focused our attention on 4ꢁ-thionucleosides,
which have structural similarities to natural nucleosides such that many of
them function well as both substrates and inhibitors for enzymes on the nu-
cleic acid biosynthetic pathways.[11] In particular, we focused initially on 4ꢁ-
thio-ara-C,[12–17] which had been made previously[18] but had not been fully
explored because of the synthetic challenge it presented. Suitable routes
have been developed to prepare it efficiently, and it has been found to have
significant activity and selectivity in a wide range of human tumor xenograft
models.[12] In addition, we have studied its mechanism of action extensively
in our laboratories.[19–23] On the basis of all of this research, 4ꢁ-thio-ara-C
was moved into Phase Iclinical trials. [18] Based upon the activity of this com-
pound as well as 2ꢁ-deoxy[24] and other arabino 4ꢁ-thionucleosides,[25,26] we
have continued to explore other types of 4ꢁ-thionucleosides. Herein we de-
scribe the preparation of a series of new purine 4ꢁ-thio-L-xylonucleosides as
well as certain aspects of their biological activity. This series was prepared
as a part of our program to explore the scope and limitations of activation
of nucleosides by the enzyme deoxycytidine kinase as well as other kinases.
Deoxycytidine kinase serves as the initial metabolic enzyme for many of the
approved nucleoside anticancer agents, including both purine and pyrimi-
dine nucleosides, as well as for 4ꢁ-thio-ara-C. Based upon our previous work
in other 4ꢁ-thionucleoside series, we have developed an efficient route for
the synthesis of the requisite sugar intermediate needed to make these kinds
of nucleosides. All of the new compounds have been evaluated for their cyto-
toxicity in a panel of human tumor cell lines, and also against certain viruses.
CHEMISTRY
Our goal in this L-xylofuranose series, as with other 4ꢁ-thionucleoside
analogs, was to prepare a series of compounds that included natural and
modified purines chosen on the basis of biological evaluations within
the series and biological information from our laboratories and others
suggesting compounds with desirable properties. Thus, we had need of
a versatile carbohydrate precursor for nucleoside synthesis in this series
that would be readily accessible and that would give acceptable yields of
both nucleoside anomers, because we often examine both the isomers if
they are readily available in sufficient quantity. In this particular series
examination of both anomers is of particular relevance if possible because
of the change of configuration of both C-2ꢁ and C-4ꢁ from the standard
nucleoside building blocks.
Employing a synthetic strategy similar to those we have previously used
for other 4-thiofuranose precursors,[25–27] we have developed a five step

Synthesis of Purine Nucleosides
1897
FIGURE 1
sequence to the key intermediate 6 (Figure 1). Conversion of D-arabinose
to methyl 2,3,5-tri-O-benzyl-D-arabinofuranoside (3) was accomplished in
two steps by the usual method. Conversion to dibenzyl dithioacetal 4
employing benzyl mercaptan and stannic chloride proceeded in 48% yield
after chromatographic purification. Cyclization at C-4 involving a single
inversion,[27] thus converting the D-arabino to the L-xylo configuration, was
accomplished employing triphenylphosphine, iodine, and imidazole in 60%
yield. The final step, replacement of the benzylthio group at C-1 by an
acetoxy group, involved treatment of 5 with mercuric acetate in acetic acid

1898
K. N. Tiwari et al.
at room temperature. The overall yield of 6 from 1, including four column
purifications, was 25%, and afforded a ca. 1:1 mixture of , anomers.
A series of purine nucleoside analogs were prepared through the cou-
pling of 6 and 2,6-dichloropurine. A Lewis acid catalyzed reaction utiliz-
ing stannic chloride in acetonitrile was found to be an efficient method to
achieve this coupling, and 30% and 25% yields of and anomers of 7 were
obtained after chromatographic purification/separation. After treatment
with ethanolic ammonia to produce the respective blocked 2-chloroadenine
nucleosides 8α and 8β, removal of the O-benzyl groups was accomplished
with boron trichloride in dichloromethane at −50◦C to yield the final nucle-
oside targets 9α (55%) and 9β (45%). Treatment of 7α and 7β with sodium
azide in 95% aqueous ethanol at reflux produced the corresponding 2,6-
diazido intermediates 10α and 10β, which were subjected to reduction with
stannous chloride in dichloromethane to afford the blocked diaminopurine
nucleosides 11α (80%) and 11β (82%) respectively. Deblocking of 11α and
11β with boron trichloride in dichloromethane produced the target di-
amino nucleosides 12α (71%) and 12β (75%), respectively. The conversion
of 12β to the corresponding guanine nucleoside 13 (45%) was accomplished
by treatment with adenosine deaminase under standard conditions. The
deamination was slow, requiring 72 h at room temperature for completion,
even with a high concentration of enzyme present. On the other hand, 9-(4-
thio- -L-xylofuranosyl)-9H-purine-2,6-diamine (12α) was resistant to deami-
nation when treated with adenosine deaminase for several days.
BIOLOGICAL DATA
The cell culture cytotoxicity of all five target compounds (9α, 9β, 12α,
12β, and 13) was determined against our standard panel of human cancer
cell lines, which includes CCRF-CEM (leukemia), CAKI-1 (renal), DLD-1
(colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS) cells.
No significant cytotoxicity was seen for any of the target compounds at
concentrations up to 100 M.
Compounds that do not show significant cytotoxicity in our panel of
human cancer cell lines are typically evaluated in a series of antiviral assays.
In this case, the same five compounds were examined in standard assays for
herpes simplex type 1 (HSV-1), human cytomegalovirus AD169 (HCMV),
respiratory syncyntial virus (RSV), influenza A/PR/8, and human herpes
virus type 8 (HHV8). Activity was seen in only two of these viruses, HCMV
and HHV8. Data on active compounds only are presented in Table 1. It
should be noted that 12α has no detectable amount of 12β in it, while 12β
has 1% of 12α in it. Given that 12α is only tenfold more potent than 12β,
the amount of 12α present as a minor impurity does not account for all of
the antiviral activity of 12β.

Synthesis of Purine Nucleosides
1899
TABLE 1 Antiviral Data
Compound
Virusa,b
IC50 (µM)
TC50 (µM)
Ganciclovir
9β
12α
12β
Cidofovir
13
HCMV
HCMV
HCMV
HCMV
HHV8
HHV8
8.0
>10
>10
>10
>10
>25
>10
0.17
0.051
0.49
1.8
2.5
aHCMV was grown in MRC-5 cells.
bHHV8 was grown in TPA-induced BCBL-1 cells.
EXPERIMENTAL SECTION
Melting points were determined on a Mel-Temp apparatus and are un-
1
corrected. H NMR spectra were recorded on a Nicolet NT-300 NB spec-
trometer operating at 300.635 MHz (1H). Chemical shifts are expressed in
parts per million downfield from tetramethylsilane. Decoupling methodol-
ogy and NOE experiments were utilized to establish the carbohydrate con-
figurations as well as the anomeric configuration. The NOE experiments
were conducted on a degassed solution of DMSO-d6. To minimize the ef-
fects of magnetic perturbations with the sample non-spinning, eight FIDs
were acquired with the decoupler set to a desired frequency and eight FIDs
were recorded with the decoupler off resonance. The process was repeated
until 800 FIDs had been acquired. UV absorption spectra were determined
on a Perkin-Elmer Lambda 9 spectrometer by dissolving each compound in
methanol or water and diluting 10-fold with 0.1 N HC1, pH 7 buffer, or 0.1 N
NaOH. Numbers in parentheses are extinction coefficients (×10−3), sh =
shoulder. Microanalyses were performed by Atlantic Microlab, Inc. (Atlanta,
Georgia) or the Molecular Spectroscopy Section of Southern Research In-
stitute. Analytical results indicated by elemental symbols were within 0.4%
of the theoretical values. Mass spectra were recorded on a Varian/MAT
311A double-focusing mass spectrometer in the fast atom bombardment
(FAB) mode. Routine HPLC analyses were carried out on a Hewlett-Packard
HP1084B liquid chromatograph with a Waters Associates Bondapak C18
column (3.9 mm × 30 cm) and UV monitoring (254 nm). HPLC purity de-
terminations were carried out on a Sphereclone C18 5 column using 0.01M
NH4H2PO4 (pH 5.1): CH3OH in a 20 min linear gradient with a flow rate
of 1.0 mL/min. All preparative chromatographic separations were carried
out by flash chromatography using 230–400 mesh silica gel from E. Merck.
TLC was carried out on Analtech precoated (250 m) silica gel (GF) plates.
2,3,5-Tri-O-benzyl-D-arabinose Dibenzyl Dithioacetal (4). D-Arabinose
(1, 25 g, 167 mmol) was stirred for 5 h in 0.5% hydrogen chloride
in methanol (675 mL) at room temperature and then neutralized with

1900
K. N. Tiwari et al.
Amberlite IRA-400 OH anion exchange resin. The filtrate and washings
were combined and evaporated to dryness and the crude product was
purified by silica gel chromatography (CHCl3/MeOH, 92:8) to afford 26.0 g
of methyl D-arabinofuranoside (2, 94% yield) as / (1:1) mixture. MS 164
(M)+.
To an ice-cold solution of 2 (20 g, 120.8 mmol) in dry tetrahydrofuran
(500 mL) was added sodium hydride (60% dispersion in mineral oil, 29.6 g,
720 mmol) and the reaction mixture was stirred for 15 min under N2. To
this reaction mixture was added solid tetrabutylammonium iodide (0.72 g,
1.92 mmol) followed by a drop wise addition of benzyl bromide (73.2 g, 418
mmol). The reaction mixture was stirred for 3 days at room temperature.
After the addition of methanol (50 mL) the solution was evaporated
under reduced pressure, and the crude product was purified by silica gel
chromatography (cyclohexane/EtOAc, 9:1) to afford pure methyl 2,3,5-tri-
1
O-benzyl-D-arabinofuranoside (3, 44 g, 83% yield). MS 435 (M+H)+. H
NMR (CDCl3) 7.26–7.18 (m, 30H, aromatic Hs), 4.92 (d, 1H, H-1, J1,2
=
4.3 Hz), 4.82 (d, 1H, H-1, J1,2 = 0.9 Hz), 4.60–4.14 (m, 10H, PhCH2s), 4.31
(m, 1H, H-4), 4.10 (dt, 1H, H-4, J4,5a = 3.7 Hz, J4,5b = 6.5 Hz, J3,4 = 6.2
Hz), 3.98 (t, 1H, H-3, J3,4 = 6.9 Hz, J2,3 = 5.6 Hz), 3.95 (dd, 1H, H-3, J3,4
=
6.2 Hz, J2,3 = 2.5 Hz), 3.90 (dd, 1H, H-2, J2,3 = 5.6 Hz), 3.86 (t, 1H, H-2,
J2,3 = 2.5 Hz), 3.63 (dd, 1H, H-5a, J4,5a = 4.5 Hz, J5a,5b = 10.4 Hz), 3.61
(dd, 1H, H-5a, J4,5a = 3.7 Hz, J5a,5b =10.7 Hz), 3.54 (dd, 1H, H-5b, J4,5b
7.5 Hz), 3.40 (dd, 1H, H-5b, J4,5b = 6.5 Hz).
=
To a solution of 3 (42 g, 97 mmol) in dichloromethane (1000 mL) were
added benzyl mercaptan (49.6 g, 400 mmol) and stannic chloride (4.93 g,
18.9 mmol), and the reaction mixture was stirred at room temperature
overnight. After neutralization with 5% aqueous NaHCO3 (750 mL), the
organic layer was separated and the aqueous layer was extracted with
dichloromethane (500 mL). The combined organic layers were evaporated,
and crude 4 was purified by silica gel chromatography (cyclohexane/EtOAc,
99:1) to afford 4 (7.0 g, 48%) of sufficient purity to carry forward. MS 657
(M+Li)+. 1H NMR (CDCl3) 7.28–7.25 (m, 19H, aromatic Hs), 7.09–7.02 (m,
4H, aromatic Hs), 6.99–6.96 (m, 2H aromatic Hs), 4.78 (d, 1H, PhCHH, J =
11.1 Hz), 4.68 (two overlapping ds, 2H, PhCHH, PhCHH, J = 11.1 Hz, J =
11.2 Hz), 4.36 (d, 1H, PhCHH, 11.2 Hz), 4.31 (d, 1H, PhCHH, J = 11.9
Hz), 4.28 (d, 1H, PhCHH, J = 11.9 Hz), 4.02 (dd, 1H, H-2, J1,2 = 3.0 Hz,
J2,3 = 7.5 Hz), 3.68–3.60 (m, 4H, two PhCH2s), 3.56 (d, 1H, H-1, J1,2 = 3.0
Hz), 3.30–3.22 (m, 2H, H-4, H-5a), 3.12–3.08 (m, 1H, H-5b), 2.08 (d, 1H,
4-OH, J = 6.2 Hz).
2,3,5-Tri-O-benzyl-1-O-acetyl-4-thio-L-xylofuranose (6). To a solution of
4 (6.5 g, 10 mmol) in dry 2:1 toluene/acetonitrile (150 mL) were added
triphenylphosphine (7.85 g, 30 mmol), iodine (6.35 g, 25 mmol) and

Synthesis of Purine Nucleosides
1901
imidazole (2.72 g, 40 mmol). The reaction mixture was stirred at 90◦C for
24 h, after which time the solution was evaporated to dryness. The crude
product was purified by silica gel chromatography (cyclohexane/EtOAc,
4:1) to afford benzyl 2,3,5-tri-O-benzyl-1,4-dithio-L-xylofuranoside as a syrup
1
(5, 3.75 g, 60%). MS 543 (M+H)+. H NMR (CDCl3) 7.32–7.14 (m, 20H,
aromatic Hs), 4.62–4.39 (m, 6H, three PhCH2Os), 4.32 (m, 1H, H-1), 4.14
(m, 2H, H-2, H-3), 3.81 (s, 2H, PhCH2S-), 3.73 (dd, 1H, H-5a, J4,5a = 7.4
Hz, J5a,5b = 9.3 Hz), 3.51 (dd, 1H, H-5b, J4,5b = 7.1 Hz), 3.40 (m, 1H, H-4).
To a suspension of mercuric acetate (4.64 g, 11.45 mmol) in acetic
acid (50 g) was added 5 (2.71 g, 5 mmol), and the resulting mixture was
stirred at room temperature for 2 h. The reaction mixture was diluted with
dichloromethane (150 mL) and washed successively with water, saturated
aqueous NaHCO3 and 5% aqueous KCN solution. The organic layer was
dried over Na2SO4 and concentrated. Chromatography of the crude prod-
uct using cyclohexane:ethyl acetate (98:2) as eluent gave a mixture of and
(1:1) anomers of 6 (1.85 g, 77%) as a colorless syrup. MS 479 (M+H)+. 1H
NMR (CDCl3) 7.31–7.20 (m, 15H, aromatic Hs), 6.03 (d, 0.25H, H-1, J1,2
=
4.0 Hz), 5.95 (d, 0.75H, H-1, J1,2 = 2.8 Hz), 4.78–4.40 (m, 6H, PhCH2s),
4.21 (dd, 0.75H, H-2, J2,3 = 5.4 Hz), 4.10–4.06 (m, 0.5H, H-2, H-3), 4.00 (t,
0.75H, H-3, J3,4 = 6 Hz), 3.72–3.65 (m, 1.25H, H-4, H-5a, H-5a), 3.49–3.36
(m, 1.75H, H-5b, H-4, H-5b), 2.05 (s, 3H, CH3- and CH3-).
9-(2,3,5-Tri-O-benzyl-4-thio-α and -β-L-Xylofuranosyl)-2,6-dichloropurine
(7α and 7β). To a stirred mixture of 6 (0.956 g, 2 mmol) and 2,6-dichloro-
purine (0.568 g, 3 mmol) in acetonitrile (50 mL) at room temperature
was added a solution of stannic chloride in dichloromethane (3 mL of
1.0 M) over 1 min and stirring was continued for 2 h. The reaction mixture
was quenched by pouring it into a mixture of 50 mL of dichloromethane
and 25 mL of saturated NaHCO3. The organic phase was dried (MgSO4)
and concentrated. The residue was purified by silica gel chromatography
(cyclohexane/EtOAc, 9:1) to afford 7α (364 mg, 30%) eluting first followed
by 7β (291 mg, 25%).
Compound 7α: MS 608 (M+H)+,1H NMR (CDCl3) 8.88 (s, 1H, H-8)
7.30–7.14 (m, 13H, aromatic Hs), 6.92 (m, 2H, aromatic Hs), 6.18 (d, 1H, H-
1ꢁ, J1,2 = 4.9 Hz), 4.65 (d, 1H, PhCHH, J = 11.6 Hz), 4.52 (s, 2H, PhCH ),
4.48 (s, 2H, PhCH2 ), 4.44 (d, 1H, PhCHH-, J = 11.6 Hz), 4.32–4.23 2(m,
2H, H-2ꢁ, H-3ꢁ), 3.65 (dd, 1H, H-5ꢁ a, J4,5a = 4.6 Hz, J5a,5b = 10.0 Hz), 3.61
(dd, 1H, H-5ꢁ b, J4,5b = 4.8 Hz), 3.49 (m, 1H, H-4ꢁ, J3,4 = 5.0 Hz).
Compound 7β: MS 608 (M+H)+1H NMR (CDCl3) 8.46 (s, 1H, H-8),
7.32–7.19 (m, 13H, aromatic Hs), 7.00 (m, 2H, aromatic Hs), 6.05 (d, 1H,
H-1ꢁ, J1,2 = 2.1 Hz), 4.67 (s, 2H, PhCH2 ), 4.56 (d, 1H, PhCHH-, J = 12.0
Hz), 4.48 (d, 1H, PhCHH-, J = 12.0 Hz), 4.46 (d, 1H, PhCHH-, J = 11.9
Hz), 4.34 (d, 1H, PhCHH-, J = 11.9 Hz), 4.24 (t, 1H, H-2ꢁ, J1,2 = 2.1 Hz,

1902
K. N. Tiwari et al.
J2,3 = 2.5 Hz), 4.20 (t, 1H, H-3ꢁ, J3,4 = 2.5 Hz), 4.05 (m, 1H, H-4ꢁ), 3.80
(dd, 1H, H-5ꢁ a, J3,4 = 8.2 Hz, J5a,5b = 9.5 Hz), 3.54 (dd, 1H, H-5ꢁ b, J4,5b
=
6.7 Hz.
2-Chloro-9-(4-thio-α-L-xylofuranosyl)adenine (9α). A mixture of com-
pound 7α (608 mg, 1 mmol) and saturated ethanolic ammonia (100 mL)
was heated at 50◦C in a glass-lined stainless steel pressure vessel for 48 h.
The reaction mixture was evaporated to dryness to afford a solid (8α) that
was dissolved in dichloromethane (50 mL) and was added dropwise to a so-
lution of 1 M BCl3 in CH2Cl2 (100 mL) at −50◦C. Solid precipitated from
the solution near the end of addition. The reaction in a tightly sealed flask
was stored at −20◦C for 16 h. The resulting clear solution was evaporated
to dryness at −20◦C to give a dark residue. A solution of this material in
ice-cold CH2Cl2 (25 mL) was evaporated to dryness four times to provide a
foam. Ice-cold saturated aqueous NaHCO3 (20 mL) was added to the foam,
and the mixture was stirred vigorously until the pH remained stable (pH 7–
8). Water (150 mL) was added to form a clear solution that was extracted
with two portions of CH2Cl2 (50 mL, 25 mL) to remove color and impuri-
ties. The colorless aqueous layer was held briefly under vacuum to remove
residual CH2Cl2 before being applied to a column (13 × 190 mm) of Bio
Beads SM-4 (100–200 mesh) equilibrated in water. Water elution with frac-
tions monitored at 254 nm provided pure 9α (175 mg, 55%);TLC, 3:1:0.1
1
CHCl3-MeOH-NH4OH, Rf 0.55; m.p. 140◦C; MS 318 (M+H)+, H NMR
(DMSO-d6) 8.24 (s, 1H, H-8), 7.76 (br s, 2H, NH2), 5.90 (d, 1H, H-1ꢁ, J1,2
=
5.2 Hz), 5.72 (d, 1H, 2ꢁ -OH, J = 4.3 Hz), 5.49 (d, 1H, 3ꢁ -OH, J = 4.6 Hz),
5.15 (t, 1H, 5ꢁ -OH, J = 5.1 Hz), 4.09-4.07 (m, 2H, H-3ꢁ, H-2ꢁ), 3.77 (dd, 1H,
H-5ꢁ a, J4,5a = 4.3 Hz, J5a,5b = 11.2 Hz), 3.73 (dd, 1H, H-5ꢁ b, J4,5b = 6.5
Hz), 3.19 (mm, 1H, H-4ꢁ, J3,4 = 6.4 Hz). HPLC purity 96.4%. HRMS calcd
for C10H12N5O3ClS (M+Na)+: 340.0242. Found: 340.0246.
2-Chloro-9-(4-thio-β-L-xylofuranosyl)adenine (9β). This compound was
prepared from 7 by the same procedure as reported for 9α in 45% yield;
TLC, 3:1:0.1 CHCl3 -MeOH-NH4OH, Rf 0.45; m.p. 235: MS 318 (M+H)+1
H
NMR (DMSO-d6) 8.46 (s, 1H, H-8), 7.80 (br s, 2H, NH2), 5.79 (d, 1H, 2ꢁ
-OH, J = 5.5 Hz), 5.65 (d, 1H, 3ꢁ -OH, J = 5.0 Hz), 5.62 (d, 1H, H-1ꢁ, J1,2
=
7.1 Hz), 4.95 (dd, 1H, 5ꢁ -OH, J5a,OH = 4.6 Hz, J5b,OH = 6.0 Hz), 4.43 (m,
1H, H-2ꢁ, J2,3 = 7.7 Hz), 3.87 (m, 1H, H-5ꢁ a, J4,5a = 3.8 Hz, J5a,5b = 10.9
Hz), 3.74 (m, 1H, H-3ꢁ, J3,4 = 8.2 Hz), 3.62 (m, 1H, H-4ꢁ), 3.42 (m, 1H, H-
5ꢁ b, J4,5b = 8.3 Hz). HPLC purity 97.8%. HRMS calcd for C10H12N5O3ClS
(M+Na)+: 340.0242. Found: 340.0239.
9-(2,3,5-Tri- O-benzyl-4-thio- α-L-xylofuranosyl)-9H-purine-2,6-diamine
(11α). A solution of 7α (303 mg, 0.5 mmol) and sodium azide (162.5 mg,

Synthesis of Purine Nucleosides
1903
2.5 mmol) in 20 mL of 95% ethanol was heated at reflux for 2 h. The
solvent was removed in vacuo, and the residue was partitioned between
dichloromethane and water. The organic phase was dried (MgSO4) and
concentrated in vacuo to yield 290 mg of a yellowish solid (10α) [TLC:
CHCl3/MeOH, 97:3; Rf 0.45; mass spectrum, m/z 621 (M+H)+], which was
redissolved in 20 mL of dichloromethane and 2 mL of methanol. This solu-
tion was treated with stannous chloride (190 mg, 1 mmol) and the resulting
suspension was stirred for 30 min. After evaporation of solvent, purifica-
tion was accomplished by silica gel chromatography (CHCl3/MeOH, 97:3)
to afford 11α (214 mg, 75%), which was suitable for deblocking. MS 569
(M+H)+. 1H NMR (CDCl3) 8.04 (s, 1H, H8), 7.30–7.50 (m, 13H, aromatic
Hs), 7.01 (m, 2H, aromatic Hs), 6.44 (d, 1H, H-1ꢁ, J1,2 = 5.3 Hz), 5.36 (bs,
2H, NH2), 4.68 (d, 1H, PhCHH, J = 11.7 Hz), 4.67 (bs, 2H, NH2), 4.58–
4.50 (m, 4H, two PhCH2s), 4.32(dd, 1H, H-3ꢁ, J3,4 = 6.3 Hz), 4.22 (dd, 1H,
H-2ꢁ, J2,3 = 7.0 Hz), 3.72–3.62 (m, 2H, H-5ꢁ a, H-5ꢁ b), 3.53 (m, 1H, H-4ꢁ).
9-(2,3,5-Tri- O-benzyl-4-thio- β-L-xylofuranosyl)-9H-purine-2,6-diamine
(11β). This compound was prepared in 78% yield by the same procedure as
reported above for 11α but starting from 7β, affording material suitable for
1
deblocking. MS 569 (M+H)+. H NMR (CDCl3) 7.89 (s, 1H, H-8), 7.35–
7.24 (m, 13H, aromatic CHs), 7.13–718 (m, 2H, aromatic CHs), 6.00 (d,
1H, H-1ꢁ, J = 4.0 Hz), 5.44 (bs, 2H, NH2), 4.74 (bs, 2H, NH2), 4.64 (d, 1H,
PhCHH-, J = 12.1 Hz), 4.52–4.49 (m, 4H, PhCH2s), 4.41 (t, 1H, H-2ꢁ, J =
3.2 Hz), 4.19 (t, 1H, H-3ꢁ, J3,4 = 4.0 Hz), 4.01 (ddd, 1H, H-4ꢁ), 3.76 (dd,
1H, H-5ꢁ b, J4,5b = 7.4 Hz, J5a,5b = 9.6 Hz), 3.54 (dd, 1H, H-5ꢁ a, J4,5a
=
6.7 Hz).
9-(4-Thio-α-L-xylofuranosyl)-9H-purine-2,6-diamine (12α). An ice-cold
solution of 11α (218 mg, 0.38 mmol) in CH2Cl2 (2.6 mL) was added
dropwise to a solution of 1 M BCl3 in CH2Cl2 (30 mL) at −50◦C. Solid
precipitated from the solution near the end of addition. The reaction in a
tightly sealed flask was stored at −20◦C for 16 h. The resulting clear solution
was evaporated to dryness at −20◦C to give a dark residue. A solution of this
material in ice-cold CH2Cl2 (25 mL) was evaporated to dryness four times
to provide a foam. Ice-cold saturated aqueous NaHCO3 (20 mL) was added
to the foam, and the mixture was stirred vigorously until the H remained
stable (pH 7–8). Water (150 mL) was added to form a clear solution that was
extracted with two portions of CH2Cl2 (50 mL, 25 mL) to remove color and
impurities. The colorless aqueous layer was held briefly under vacuum to
remove residual CH2Cl2 before being applied to a column (13 × 190 mm)
of Bio Beads SM-4 (100–200 mesh) equilibrated in water. Water elution with
fractions monitored at 254 nm provided pure 12α that was crystallized from
hot MeOH (81 mg, 71%). TLC, 3:1:0.1 CHCl3-MeOH-NH4OH, Rf 0.48; m.p.

1904
K. N. Tiwari et al.
140◦C; MS 299 (M+H)+) 1H NMR (DMSO-d6) 7.88 (s, 1H, H8), 6.66 (s, 2H,
NH2), 6.40 (d, 1H, H-1ꢁ, J1,2 = 4 Hz), 5.82 (bs, 2H, NH2), 4.20(dd, 1H, H-3ꢁ,
J3,4 = 6.3 Hz), 4.06 (bdd, 2H, H-2ꢁ H-4ꢁ), 3.72–3.82 (m, 1H, H-5ꢁ), 3.48–
3.54 (m, 1H, H-5ꢁ). HPLC purity, 99.3%. HRMS calcd for C10H14N6O3S
(M+H)+: 299.0921. Found: 299.0923.
9-(4-Thio-β-L-xylofuranosyl)-9H-purine-2,6-diamine (12β). This com-
pound was prepared in 75% yield (crystallized from water) by the same
procedure as reported above for 12α but starting from 11β. TLC, 3:1:0.1
1
CHCl3-MeOH-NH4OH, Rf 0.43 m.p. 130◦C; MS 299 (M+H)+, H NMR
(DMSO-d6) 8.08 (s, 1H, H8), 6.68 (s, 2H, NH2), 5.80 (bs, 2H, NH2), 5.60 (d,
1H, H-1ꢁ, J1,2 = 4 Hz), 4.41 (t, 1H, H-2ꢁ, J = 3.2 Hz), 4.16 (t, 1H, H-3ꢁ, J =
4.0 Hz), 3.60 9m. 1H. H-4ꢁ), 3.78 (d, 1H, H-5ꢁ), 3.70 (d, 1H, H-5ꢁ). HPLC
purity, 94.7%. HRMS calcd for C10H14N6O3S (M+H)+: 299.0921. Found:
299.0923. Anal. calcd. for C10H14N6O3S: H2O: C 37.96; H 5.10; N 26.58.
Found: C 38.14; H 4.94; N 26.56.
9-(4-Thio-β-L-xylofuranosyl)guanine (13). To a solution of 12β (50 mg,
0.17 mmol) in 20 mL of water was added 100 u of adenosine deaminase
type VIII (40 L). The reaction was stirred for 72 h, the solution was boiled
for 3 min to deactivate the enzyme, and the suspension was treated with
charcoal and filtered through Celite. The filtrate was concentrated to give
gelatinous 13 which was dissolved in hot water (4 mL) and filtered through
0.45- m filter (25 mm, Gelman Acrodisc GHP-GF). The clear filtrate was
lyophilized to provide 13 as a fluffy white solid which was crystallized from
water (20 mg, 40%): TLC, 3:1:0.1 CHCl3-MeOH-NH4OH, Rf 0.40; m.p.
1
260◦C; MS 300 (M+H)+, H NMR (DMSO-d6) 10.5 (bs, 1H, N-3H) 8.02
(s, 1H, H-8), 6.48 9s, 2H, NH2), 6.00 (d, 1H, 2ꢁ-OH, J = 4.0 Hz), 5.58 (d,
1H, 3ꢁ-OH), 4.90 (t, 1H, 5ꢁ-OH, J = 3.0 Hz), 4.35 (t, 1H, H-2ꢁ, J = 3.2
Hz), 4.18 (t, 1H, H-3ꢁ, J3,4 = 4.0 Hz), 3.80 (dd, 1H, H-5ꢁ b, J4,5b = 7.4 Hz,
J5a,5b = 9.6 Hz), 3.68 (dd, 1H, H-5ꢁ a, J4,5a = 6.7 Hz) 3.60 (ddd, 1H, H-
4ꢁ). HPLC purity, 95.6%. HRMS calcd for C10H13N5O4S: 322.0581. Found:
322.0581.
REFERENCES
1. Brockman, R.W.; Schabel, F.M., Jr.; Montgomery, J.A. Biologic activity of 9-β-D-arabinofuranosyl-
2-fluoroadenine, a metabolically stable analog of 9-β-D-arabinofuranosyladenine. Biochem. Phar-
macol. 1977, 26, 2193–2196.
2. Gandhi, V.; Plunkett, W. Cellular and clinical pharmcology of fludarabine. Clinical Pharmacoki-
netics 2002, 41, 93–103.
3. Montgomery, J.A.; Shortnacy-Fowler, A.T.; Clayton, S.D.; Riordan, J.M.; Secrist, J.A., III. Synthesis
and biologic activity of 2 -fluoro-2-halo derivatives of 9-β-D-arabinofuranosyladenine. J. Med.
Chem. 1992, 35, 397–401.

Synthesis of Purine Nucleosides
1905
4. Waud, W.R.; Schmid, S.M.; Montgomery, J.A.; Secrist, J.A., III. Preclinical antitumor activity of
2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (Cl-F-ara-A). Nucleosides, Nucleotides
& Nucleic Acids 2000, 19, 447–460.
5. Parker, W.B.; Shaddix, S.C.; Chang, C.H.; White, E.L.; Rose, L.M.; Brockman, R.W.; Shortnancy,
A.T.; Montgomery, J.A.; Secrist, J.A., III; Bennett, L.L., Jr. Effects of 2-chloro-9-(2-deoxy-2-
fluoro-β-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human
ribonucleotide reductase and DNA polymerases by its 5-triphosphate. Cancer Res. 1991, 51, 2386–
2394.
6. Parker, W.B.; Secrist, J.A., III; Waud, W.R. Purine nucleoside antimetabolites in development for
the treatment of cancer. Curr. Opin. Investig. Drugs 2004, 5, 592–596.
7. Sternberg, A. Clofarabine. Curr. Opin. Investig. Drugs 2003, 4, 1479–1487.
8. Cooper, T.; Kantarjian, H.; Plunkett, W.; Gandhi, V. Clofarabine in adult acute leukemias: Clinical
success and pharmacokinetics. Nucleosides, Nucleotides & Nucleic Acids 2004, 23, 1417–1423.
9. DeVita, V.T., Jr.; Hellman, S.; Rosenberg, S.A. Cancer Principles & Practice of Oncology, 7th Ed.,
vol. 1. Lippincott Williams & Wilkins: Philadelphia, PA, 2005; 358–374.
10. Faderl, S.; Gandhi, V.; Kantarjian, H.; Plunkett, W. New nucleoside analogues in clinical
development. Cancer Chemotherapy and Biological Response Modifiers 2002, 20, 37–58.
11. Gunaga, P.; Moon, H.R.; Choi, W.J.; Shin, D.H.; Park, J.G.; Jeong, L.S. Recent advances in 4 -
thionucleosides as potential antiviral and antitumor agents. Curr. Med. Chem. 2004, 11, 2585–
2637.
12. Tiwari, K.N.; Shortnacy-Fowler, A.T.; Cappellacci, L.; Parker, W.B.; Waud, W.R.; Montgomery, J.A.;
Secrist, J.A., III. Synthesis of 4 -thio-β-D-arabinofuranosylcytosine (4 -thio-ara-C) and comparison
of its anticancer activity with that of ara-C. Nucleosides, Nucleotides & Nucleic Acids, 2000, 19,
329–340.
13. Parker, W.B.; Shaddix, S.C.; Rose, L.M.; Waud, W.R.; Shewach, D.S.; Tiwari, K.N.; Secrist, J.A., III.
Metabolism of 4 -thio-β-D-arabinofuranosylcytosine in CEM cells. Biochem. Pharmacol. 2000, 60,
1925–1932.
14. Someya, H.; Shaddix, S.C.; Tiwari, K.N.; Secrist, J.A., III; Parker, W.B. Phosphorylation of 4 -
thio-arabinofuranosylcytosine and its analogs by human deoxycytidine kinase. J. Pharmacol. Exp.
Ther. 2003, 304, 1314–1322.
15. Richardson, K.A.; Vega, T.P.; Richardson, F.C.; Moore, C.L.; Rohloff, J.C.; Tomkinson, B.; Bendele,
R.A.; Kuchta, R.D. Polymerization of the triphosphates of araC, 2 ,2 -difluorodeoxycytidine and
OSI-7836 (T-araC) by human DNA polymerase alpha and DNA primase. Biochem. Pharmacol.
2004, 68, 2337–2346.
16. Richardson, F.; Black, C.; Richardson, K.; Franks, A.; Wells, E.; Karimi, S.; Sennello, G.; Hart,
K.; Meyer, D.; Emerson, D.; Brown, E.; LeRay, J.; Nilsson, C.; Tomkinson, B.; Bendele, R.
Incorporation of OSI-7836 into DNA of calu-6 and h460 xenograft tumors. Cancer Chemother.
Pharmacol. 2005, 55, 213–221.
17. Someya, H.; Waud, W.R.; Parker, W.B. Long intracellular retention of 4 -thio-arabinofuranosylcyto-
sine 5 -triphosphate as a critical factor for the anti-solid tumor activity of 4 -thio-arabinofuranosyl-
cytosine. Cancer Chemother. Pharmacol. 2005, 1–9.
18. Whistler, R.L.; Doner, L.W.; Nayak, U.G. 4-Thio-D-arabinofuranosylpyrimidine nucleosides. J. Org.
Chem. 1971, 36, 108–110.
19. Parker, W.B.; Shaddix, S.C.; Rose, L.M.; Waud, W.R.; Shewach, D.S.; Tiwari, K.N.; Secrist, J.A., III.
Metabolism of 4 -thio-β-D-arabinofuranosylcytosine in CEM cells. Biochem. Pharmacol. 2000, 60,
1925–1932.
20. Someya, H.; Shaddix, S.C.; Tiwari, K.N.; Secrist, J.A., III; Parker, W.B. Phosphorylation of 4 -
thio-arabinofuranosylcytosine and its analogs by human deoxycytidine kinase. J. Pharmacol. Exp.
Ther. 2003, 304, 1314–1322.
21. Someya, H.; Waud, W.R.; Parker, W.B. Correlation of in vitro cytotoxicity using high concen-
trations for short periods with in vivo antitumor activity of cytosine arabinoside (araC) and
4 -thio-arabinofuranosyl cytosine (T-araC). Proc. Am. Ass. Cancer Res. 2003, 44, 629.
22. Someya, H.; Parker, W.B. Comparison of intracellular elimination of 2 ,2 -difluoro-2 -deoxy-
cytidine (gemcitabine, dFdC) 5 -triphosphate (dFdCTP) with that of 4 -thio-arabinofuranosylcyto-
sine (T-araC, OSI-7836) 5 -triphosphate (T-araCTP). Proc. Am. Ass. Cancer Res. 2004, 45, 865.

1906
K. N. Tiwari et al.
23. Someya, H.; Parker, W.B. Greater substrate activity of 4 -thio-arabinofuranosylcytosine (T-araC) 5 -
monophosphate (T-araCMP) with UMP/CMP kinase as a likely factor for the longer intracellular
retention of its triphosphate. Proc. Am. Ass. Cancer Res. 2005, 46, 4104.
24. Secrist, J.A., III; Tiwari, K.N.; Riordan, J.M.; Montgomery, J.A. Synthesis and biological activity of
2 -deoxy-4 -thio pyrimidine nucleosides. J. Med. Chem. 1991, 34, 2361–2366.
25. Secrist, J.A., III; Tiwari, K.N.; Shortnacy-Fowler, A.T.; Messini, L.; Riordan, J.M.; Montgomery, J.A.
Synthesis and biological activity of certain 4 -thio-D-arabinofuranosylpurine nucleosides. J. Med.
Chem. 1998, 41, 3865–3871.
26. Tiwari, K.N.; Shortnacy-Fowler, A.T.; Cappellacci, L.; Parker, W.B.; Waud, W.R.; Montgomery,
J.A.; Secrist, J.A., III. Synthesis and structure activity relationship of 5-substituted 4 -thio-β-D-
arabinofuranosylcytosines. Nucleosides, Nucleotides & Nucleic Acids 2000, 19, 2005–2017.
27. Tiwari, K.N.; Montgomery, J.A.; Secrist, J.A., III. The synthesis and biological activity of 1-(2-deoxy-
4-thio-α-L-threo-pentofuranosyl)thymine. Nucleosides Nucleotides 1993, 12, 841–846.