Synthesis and Anti-cancer Activity of Some Novel 5-Azacytosine Nucleosides

Article abstract of DOI:10.1081/NCN-120026872

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-L-threo-pentofuranose were coupled with 5-azacytosine to obtain α and β anomers of nucleosides. All four nucleosides were reduced to the corresponding

Full text of DOI:10.1081/NCN-120026872

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Synthesis and Anti-cancer Activity of Some Novel 5-
Azacytosine Nucleosides
Kamal N. Tiwari a , Loredana Cappellacci a , John A. Montgomery a & John A. Secrist III a b
a Southern Research Institute , Birmingham, Alabama, USA
b Southern Research Institute , P.O. Box 55305, Birmingham, AL, 35255-5305, USA
Published online: 19 Dec 2011.
To cite this article: Kamal N. Tiwari , Loredana Cappellacci , John A. Montgomery & John A. Secrist III (2003) Synthesis and
Anti-cancer Activity of Some Novel 5-Azacytosine Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 22:12, 2161-2170,
DOI: 10.1081/NCN-120026872
To link to this article: http://dx.doi.org/10.1081/NCN-120026872
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 12, pp. 2161–2170, 2003
Synthesis and Anti-cancer Activity of Some Novel
5-Azacytosine Nucleosides#
Kamal N. Tiwari, Loredana Cappellacci, John A. Montgomery,
*
and John A. Secrist III
Southern Research Institute, Birmingham, Alabama, USA
ABSTRACT
1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-d-erythro-pentofuranose and 2-deoxy-
1,3,5-tri-O-acetyl-4-thio-l-threo-pentofuranose were coupled with 5-azacytosine
to obtain a and b anomers of nucleosides. All four nucleosides were reduced
to the corresponding dihydro derivatives and deblocked to give target com-
pounds. All eight target compounds were evaluated in a series of human cancer
cell lines in culture. Only 20-deoxy-40-thio-5-azacytidine (3b) was found to be
cytotoxic in all the cell lines and was further evaluated in vivo. Details of the
synthesis and biological activity are reported.
Key Words: 5-Azacytosine; 40-Thionucleoside; Anti-cancer activity.
#In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*Correspondence: John A. Secrist III, Southern Research Institute, P.O. Box 55305,
Birmingham, AL 35255-5305, USA; Fax: (205) 581-2093; E-mail: secrist@sri.org.
2161
DOI: 10.1081/NCN-120026872
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Tiwari et al.
INTRODUCTION
A number of 20-deoxyribo and arabino nucleoside analogs have shown promise
as anticancer agents. For some years we have been pursuing the synthesis of a variety
of 40-thionucleosides as anticancer agents. In that regard, we have prepared 40-thio-
nucleosides with a variety of different carbohydrates attached to both the normal
purine and pyrimidine bases as well as analogs of those bases.[1–7,14] We have
reported the synthesis and antitumor activity of 1-(4-thio-b-d-arabinofuranosyl)
cytosine (40-thio-ara-C), which is currently in clinical trials. Preliminary results on
the mechanism of action of 40-thio-ara-C suggest that it is metabolized to its tripho-
sphate using deoxycytidine kinase as the first step, and that the triphosphate inhibits
DNA polymerase and is also incorporated into DNA.[8] The activity of 40-thio-ara-C
has encouraged us to continue research on related compounds.
One of the altered bases that has drawn attention for many years is 5-azacyosine.
This base, when present in 5-azacytidine,[11] 20-deoxy-5-azacytidine,[10] and ara-5-
azacytidine,[13] imparts anticancer activity, and these compounds have been exam-
ined clinically.[9] 5-Azacytidine, for example, after conversion to the corresponding
triphosphate by cellular kinases, is incorporated into RNA. It is also incorporated
into DNA after reduction at the diphosphate level to the 20-deoxynucleoside. In cells
in culture, 5-azacytidine has little effect on resting cells, being more toxic to cells in
the S phase of the cell cycle, indicating that its primary effect relates to DNA synth-
esis.[10] Incorporation of 20-deoxy-5-azacytidine into DNA results in hypomethyla-
tion of the DNA, as well as other methylation-related effects. Though insufficient
anticancer selectivity was found with these compounds, it is clear that some of the
biological effects caused by the 5-azacytosine moiety are quite desirable, if they
can be incorporated into a molecule with other favorable properties.
A problem associated with certain 5-azacytidine analogs, including those men-
tioned above, is the ready hydrolysis by ring opening of the base moiety in aqueous
solution. In order to combat that problem, Beisler et al. prepared the 5,6-dihydro
analog of 5-azacytidine,[11] which is much more stable hydrolytically, and examined
it for anticancer activity. It was found to have activity, though only at about one-
tenth of 5-azacytidine. Mechanistically, its activation and mode of action appeared
to be similar to those of 5-azacytidine.[12]
On the basis of the above information, we have prepared a series of 40-thionucleo-
sides incorporating both 5-azacytosine and 5,6-dihydro-5-azacytosine, in order to
assess their anticancer activity, as well as to compare the saturated and unsaturated
compounds in the same system. Specifically, we have prepared both the b and a anom-
ers of the nucleosides incorporating 5-azacytosine and 5,6-dihydro-5-azacytosine into
4-thio-2-deoxy-l-threo-pentofuranose and 4-thio-2-deoxy-d-erythro-pentofuranose.
Chemical synthesis and biological data on these compounds are presented herein.
CHEMISTRY
The synthesis of 1-O-acetyl-2-deoxy-4-thio-3,5-di-O-p-toluoyl-d-erythro-pento-
furanose 1 and 2-deoxy-1,3,5-O-acetyl-4-thio-l-threo-pentofuranose 6, as 1:1 mix-
ture of anomers have been performed as previously reported.[3,14]

5-Azacytosine Nucleosides
2163
Scheme 1.
Trimethylsilyl triflate catalyzed coupling[3,14] of thiosugar 1 and 6 with silylated
5-azacytosine afforded the corresponding nucleosides (2 and 7) as anomeric mixtures
(b:a ratio ꢀ1:2 and 2:1 respectively). Silica gel chromatography and fractional crys-
tallization of 2 and 7 afforded pure anomers. Reduction of the 5,6-double bond with
sodium borohydride of compounds 2a, 2b, 7a, 7b, afforded 4a, 4b, 9a, 9b, respec-
tively. Deprotection of compounds 2a, 2b, 7a, 7b, 4a, 4b, 9a, 9b, with sodium meth-
oxide afforded 3a, 3b, 8a, 8b, 5a, 5b, 10a, 10b, respectively (Sch. 1). We found, as
previously seen with other 5-azacytosine-containing nucleosides,[11] that 3a, 3b, 8a,
and 8b all were somewhat unstable in solution. After reduction of the 5,6 double
bond, however, stability was restored.
The anomeric configuration and point of attachment of sugar and pyrimi-
1
dine in all the nucleosides were confirmed by H NMR spectra using decoupling
experiments.[3,14]

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Tiwari et al.
Table 1. Cytotoxicity data: IC50 (mM).
CCRF-CEM
(leukemia)
CAKI-1
(renal)
DLD-1
(colon)
NCI-H23
(lung)
LOXIMVI
(melanoma)
SNB-7
(CNS)
Compound
3b
0.01
13.0
7.0
3.1
5.3
1.7
BIOLOGICAL DATA
The cytotoxicity of all eight analogs was determined against a panel of human
tumor cell lines.[15] Only 3b was cytotoxic to these cell lines (Table 1). The other
agents were not cytotoxic at the highest level tested (60 mM). On the basis of these
results, larger quantities of 3b were prepared for evaluation in an animal model.
The animal model chosen was the NCI-H23 non-small cell lung tumor implanted
subcutaneously in female nude mice. H23 was chosen because it is a solid tumor
model, rather than a leukemia, and because it is sensitive to a variety of agents.[16,17]
This model also has previously been shown in our laboratories to be sensitive to 40-
thio-ara-C.[18] In this study treatment began on day 15 when the median tumor size
ranged from 162 to 188 mg. Compound 3b was prepared in saline=tween 80 and was
administered intraperitoneally as a daily single injection on days 15–23. Modest
activity was observed at a tolerated dosage of 4.5 mg=kg=dose. Treated animals
had an increase in lifespan of 8 days beyond the control animals (T-C value of 8
days). Because of the inherent instability of this compound in solution, the same
study was performed in which compound 3b was prepared fresh daily. In this study,
similar modest activity was observed at a tolerated dosage of 6.7 mg=kg=dose (T-C
value of 10.9 days).
EXPERIMENTAL SECTION
Melting points were determined on a Mel-Temp apparatus and are uncorrected.
1H NMR spectra were recorded on a Nicolet NT-300 NB spectrometer operating at
300.635 MHz (1H). Chemical shifts are expressed in parts per million downfield from
tetramethylsilane. The NOE experiments were conducted on a degassed solution of
DMSO-d6. To minimize the effects 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 with a
Perkin-Elmer l 9 spectrometer by dissolving each compound in methanol or water
and diluting 10-fold with 0.1 N HCl, pH 7 buffer, or 0.1 N NaOH. Numbers in
parentheses are extinction coefficients (ꢁ10ꢂ3), sh ¼ shoulder. Microanalyses were
performed by Atlantic Microlab, Inc. (Atlanta, GA) or the Spectroscopic and
Analytical Laboratory of Southern Research Institute. Mass spectra were recorded
on a Varian=MAT 311A double-focusing mass spectrometer in the fast atom bom-
bardment (FAB) mode. HPLC analyses were carried out on a Hewlett-Packard
1100 series liquid chromatograph with a Phenomenex Sphereclone C18 column

5-Azacytosine Nucleosides
2165
(4.6 mm ꢁ 25 cm) and UV monitoring (254 nm). Flash chromatographic separations
were carried out by using 230–400 mesh silica gel from E. Merck. TLC was carried
out on Analtech precoated (250 mm) silica gel (GF) plates. Ion exchange chromato-
graphy was done on Dowex 50W-X8, 100–200 mesh ion exchange resin.
Cell Culture Cytotoxicity Data
All cell lines were grown in RPMI 1640 medium containing 10% fetal bovine
serum, sodium bicarbonate, and 2 mM L-glutamine. For in vitro evaluation cells
were plated in 96-well microtiter plates and then were exposed continuously to var-
ious concentrations of the compounds for 72 h at 37ꢃC. Cell viability was measured
using either the neutral red assay (absorbance read at 550 nm) or the sulforhodamine
B assay (absorbance read at 570 nm). The background absorbance mean was sub-
tracted from the data followed by conversion to percent of control. The drug concen-
trations producing survival just above and below the 50% level were used in a linear
regression analysis to calculate the IC50.
1-(2-Deoxy-3,5-di-O-toluoyl-4-thio-b-d-erythro-pentofuranosyl)-5-azacytosine (2b)
and Its a-Anomer (2a). To a suspension of 1-O-acetyl-3,5-di-O-toluoyl-4-thio-d-
erythropentofuranose (428 mg, 1 mmol) and 5-azaytosine (140.0 mg, 1.25 mmol) in
anhydrous acetonitrile (20 mL) were added consecutively hexamethyldisilazane
(HMDS, 162 mg, 1.0 mmol) and chlorotrimethylsilane (TMSCl, 434 mg, 4.0 mmol).
The mixture was stirred at room temperature for 0.5 h then cooled to –78ꢃC. Tri-
methylsilyltrifluoromethane sulfonate (267 mg, 1.2 mmol) was added, and the result-
ing solution was stirred at –78ꢃC for another 2.5 h, after which time the reaction was
essentially complete. The mixture was warmed to room temperature, concentrated
to a small volume (5 mL), diluted with methylene chloride (50 mL) and then washed
with water (20 mL) followed by saturated sodium bicarbonate and water. The
organic layer was dried over MgSO4 and evaporated to dryness. The residue was
purified by chromatography over silica gel (50 g. elution with CHCl3=MeOH 98:2)
to afford 2 (336 mg, 70%) as a colorless syrup TLC (95:5 CHCl3=MeOH Rf 0.45;
as 2:1 a,b mixture (HPLC); MS m=z 481 (M þ H)þ. Both the anomers were sepa-
rated by silica gel column using 200:1 ratio of silica gel and compound to obtain
1
2b (100 mg) and 2a (200 mg) as white solids. 2b MS m=z 481 (M þ H)þ. H NMR
(CDCl3) 8.60 (s, 1H, H-6), 7.96 (d, 2H, ortho H of toluoyl, J ¼ 8 Hz), 7.94 (d, 2H,
ortho H of toluoyl, J ¼ 8.0 Hz), 7.62 (s, 2H, NH2), 7.34 (t, 4H, meta H of toluoyl,
J ¼ 4 Hz), 6.20 (dd, 1H, H-10, J ¼ 8 and 1 Hz), 5.78–5.80 (m, 1H, H-30), 4.60–4.64
(m, 1H, H-50), 4.46–4.50 (m, 1H, H-50), 3.90–3.96 (m, 1H, H-40), 2.92–3.0 (m, 1H,
H-20), 2.60–2.68 (m, 1H, H-20), 2.40 (s, 3H, CH3), 2.38 (s, 3H, CH3).
1
2a MS m=z 481 (M þ H)þ. H NMR (CDCl3) 8.80 (s, 1H, H-6), 7.92 (d, 2H,
ortho H of toluoyl, J ¼ 8 Hz), 7.76 (d, 2H, ortho H of toluoyl, J ¼ 8.0 Hz), 7.54 (d,
2H, NH2, J ¼ 4 Hz), 7.36 (d, 2H, meta H of toluoyl, J ¼ 8 Hz), 7.30 (d, 2H, meta
H of toluoyl, J ¼ 8 Hz), 6.10 (dd, 1H, H-10, J ¼ 4 and 2 Hz), 5.66–5.70 (m, 1H,
H-30), 4.26–4.44 (m, 3H, H-40, H-50), 2.82–2.86 (m, 1H, H-20), 2.64–2.68 (m, 1H,
H-20), 2.20 (s, 3H, CH3), 2.18 (s, 3H, CH3).

2166
Tiwari et al.
1-(2-Deoxy-3,5-di-O-acetyl-4-thio-b-l-threo-pentofuranosyl)-5-azacytosine (7b)
and Its a-Anomer (7a). To a suspension of 1-O-acetyl-3,5-di-O-acetyl-4-thio-l-
thropentofuranose (276 mg, 1 mmol) and 5-azaytosine (140.0 mg, 1.25 mmol) in
anhydrous acetonitrile (20 mL) were added consecutively hexamethyldisilazane
(HMDS, 162 mg, 1.0 mmol) and chlorotrimethylsilane (TMSCl, 434 mg, 4.0 mmol).
The mixture was stirred at room temperature for 0.5 h then cooled to ꢂ78ꢃC.
Trimethylsilyltrifluoromethane sulfonate (267 mg, 1.2 mmol) was added, and the
resulting solution was stirred at ꢂ78ꢃC for another 2.0 h, after which time the reac-
tion was essentially complete. The mixture was warmed to room temperature, con-
centrated to a small volume (5 mL), diluted with methylene chloride (50 mL) and
then washed with water (20 mL) followed by saturated sodium bicarbonate and
water. The organic layer was dried over MgSO4 and evaporated to dryness. The resi-
due was purified by chromatography over silica gel (50 g. elution with
CHCl3=MeOH 98:2) to afford 2 (262 mg, 80%) as a colorless syrup TLC (96:4
CHCl3=MeOH Rf 0.50; as 2:1 a:b mixture (HPLC); MS m=z 329 (M þ H)þ. Both
the anomers were separated by silica gel column using using 200:1 ratio of silica
gel and compound to obtain 7b (160 mg) and 7a (75 mg) as white solids. 7b MS
1
m=z 329 (M þ H)þ. H NMR (CDCl3) 8.90 (s, 1H, H-6), 6.21 (t, 1H, H-10,
J ¼ 3.0 Hz), 5.64–5.70 (overlapping multiplets, 2H, H-30, H of NH2), 5.42 (broad
hump, 1H, H of NH2), 4.44–4.50 (m, 1H, H-50), 4.26–4.32 (m, 1H, H-50), 4.0–4.06
(m, 1H, H-40), 2.54–2.56 (m, 2H, H-20), 2.08 (s, 3H, CH3), 1.98 (s, 3H, CH3).
1
7a MS m=z 329 (M þ H) þ. H NMR (CDCl3) 8.52 (s, 1H, H-6), 6.70 (bm, 1H,
H-10), 6.32 (t, 1H, H of NH2, J ¼ 6 Hz), 5.70 (bs, 1H, H-6), 5.54–5.60 (m, 1H, H of
NH2), 4.31–4.36 (m, 1H, H-50), 4.14–4.26 (m, 2H, H-40, H-50), 2.76–2.84 (m, 1H, H-
20), 2.24–2.34 (m, 1H, H-20), 2.12 (s, 3H, CH3), 2.08 (s, 2H, CH3).
1-(2-Deoxy-4-thio-b-d-erythro-pentofuranosyl)-5-azacytosine (3b) and Its a-
Anomer (3a). To a solution of 2b (60 mg, 0.125 mmol) in anhydrous MeOH
(30 mL) was stirred at room temperature with a freshly prepared solution of sodium
methoxide (20 mg, 0.37 mmol) in MeOH (20 mL). A TLC aliquot (3 h, CHCl3:
MeOH, 85:15) showed complete consumption of starting material. The solution
was rendered neutral with Dowex 50Wꢄ8 (Hþ) ion exchange resin, and the resin
was filtered off with MeOH washing. The filtrate were combined and evaporated
to dryness. Preparative TLC (CHCl3:MeOH, 9:1) of the crude mixture gave white
solid, which was crystallized with ethanol to obtain 3b (15 mg, 50%), m.p. 211ꢃC;
1
MS m=z 245 (M þ H), H NMR (DMSO-d6) 8.70 (s, 1H, H-6); 7.54 (s, 2H, NH2),
6.08 (t, 1H, H-10, J ¼ 6.0), 5.31 (d, 1H, H-30, J ¼ 4 Hz), 5.16 (t, 1H, 50-OH, J ¼ 4 Hz),
4.30–4.34 (m, 1H, H-30), 3.50–3.66 (m, 2H, 50-H), 3.22–3.34 (m, 1H, H-40), 2.16–
2.32 (m, 2H, H-20). Anal. calcd for C8H12N4O3S: C, 39.33; H, 4.95; N, 22.95.
Found: C, 39.26; H, 4.84; N, 22.68.
Compound 3a was also made using the same procedure in 80% yield starting
from 150 mg of 2a m.p. 223ꢃC; MS m=z 245 (M þ H)þ, 1H NMR (DMSO-d6)
8.80 (s, 1H, H-6), 7.42 (d, 2H, NH2, J ¼ 1.5 Hz), 6.0 (dd, 1H, H-10, J ¼ 4.0 and
2.0 Hz), 5.40 (d, 1H, 30-OH, J ¼ 2 Hz), 5.05 (t, 1H, 50-OH, J ¼ 2 Hz), 4.40–4.44 (m,
1H, H-30), 3.54–3.60 (m, 1H, H-40), 3.30–3.42 (m, 2H, 50-H), 2.34–2.44 (m, 1H, H-20),
2.14–2.20 (m, 1H, H-20). Anal. calcd for C8H12N4O3S: 0.1 H2O C, 39.04; H, 5.0;
N, 22.78. Found: C, 38.99; H, 4.84; N, 22.62.

5-Azacytosine Nucleosides
2167
1-(2-Deoxy-4-thio-b-l-threo-pentofuranosyl)-5-azacytosine (8b) and Its a-Anomer
(8a). To a solution of 7b (60 mg, 0.183 mmol) in anhydrous MeOH (30 mL) was
stirred at room temperature with a freshly prepared solution of sodium methoxide
(20 mg, 0.37 mmol) in MeOH (20 mL). A TLC aliquot (3 h, CHCl3:MeOH, 85:15)
showed complete consumption of starting material. The solution was rendered neu-
tral with Dowex 50Wꢄ8 (Hþ) ion exchange resin, and the resin was filtered off with
MeOH washington. The filtrate were combined and evaporated to dryness. Prepara-
tive TLC (CHCl3:MeOH, 9:1) of the crude mixture gave white solid, which was crys-
tallized with ethanol to obtain 8b (33 mg, 74%), m.p. 211ꢃC; MS m=z 245(M þ H)þ.
1H NMR (DMSO-d6) 8.84 (s, 1H, H-6), 7.44 (d, 2H, NH2, J ¼ 6 Hz), 6.60 (dd, 1H,
H-10, J ¼ 8.0 and 2.0 Hz), 5.31 (d, 1H, H-30, J ¼ 4 Hz), 4.90–4.94 (m, 1H, 50-OH),
4.34–4.42 (m, 1H, H-30), 3.84–3.90 (m, 1H, 50-H), 3.60–3.68 (m, 2H, H-40, H-50),
2.20 2.34 (m, 2H, H-20). Anal. calcd for C8H12N4O3S: 0.1 H2O, 0.1 C2H5OH C,
39.28; H, 5.15; N, 22.36. Found: C, 39.32; H, 4.82; N, 22.35.
Compound 8a was also made using the same procedure in 82% yield starting
from 150 mg of 7a m.p. 223ꢃC; MS m=z 245 (M þ H)þ, 1H NMR (DMSO-d6)
8.64 (s, 1H, H-6), 7.44 (s, 2H, NH2), 6.20 (dd, 1H, H-10, J ¼ 12.0 and 8.0 Hz), 5.10
(d, 1H, H-30, J ¼ 4 Hz), 4.78–4.84 (m, 1H, 50-OH), 4.40–4.44 (m, 1H, H-30), 4.90–
4.98 (m, 1H, H-40), 3.70–3.80 (m, 1H, 50-H), 3.40–3.48 (m, 1H, H-50), 2.22–2.34
(m, 2H, H-20). Anal. calcd for C8H12N4O3S: 0.1 H2O C, 39.04; H, 5.0; N, 22.78.
Found C, 38.27; H, 4.84; N, 22.41.
1-(2-Deoxy-4-thio-b-d-erythro-pentofuranosyl)-5,6-dihydro-5-azacytosine (5b) and
Its a-Anomer (5a). A solution of 2b (96 mg, 0.20 mmol) in dry THF (20 mL) was
added solid sodium borohydride (64 mg, 1.68 mmol), and the mixture was stirred
at r.t. One hour aliquot showed complete reaction (CH3CN:NH4OH, 9:1). The sol-
vent was evaporated and the residue was diluted with cold MeOH (5 mL). The solu-
tion was quickly treated was Dowex 50Wꢄ8 strong cation exchange resin until
neutrilization. The resin was filtered, washed with MeOH and the filtrate was evapo-
rated to dryness to give crude which was purified by a small silica gel column to
afford pure 4b (80 mg, 80%) as white powder MS m=z 483 (M þ H)þ. This compound
was deblocked in a similar manner as described for compound 3 to afford compound
5b (white solid, 78%) and 5a (white solid, 70%) was made in a similar manner from
3a. Compound 5b MS m=z 247 (M þ H)þ. 1H NMR (DMSO-d6) 6.24 (dd, 1H, H-10,
J ¼ 8 and 1 Hz), 5.10 (bs, 1H, 30-OH), 5.12 (bs, 1H, 50-OH), 4.60 (d, 1H, H-6,
J ¼ 6 Hz), 4.42 (d, 1H, H-6, J ¼ 6 Hz), 4.28 (s, 1H, H-30), 3.40–3.42 (m, 1H, H-50),
3.10–3.14 (m, 1H, H-40), 2.0–1.98 (m, 1H, H-20), 1.60–1.64 (m, 1H, H-20). Anal. calcd
for C8H14N4O3S: 0.1 H2O, 0.1C2H5OH C, 38.97; H, 5.90; N, 22.18. Found: C, 38.96;
H, 5.81; N, 22.29.
1
5a MS m=z 247 (M þ H)þ. H NMR (DMSO-d6) 6.08 (t, 1H, H-10, J ¼ 4 Hz),
5.24 (d, 1H, 30-OH, J ¼ 4 Hz), 4.82 (t, 1H, 50-OH, J ¼ 4 Hz), 4.62 (d, 1H, H-6,
J ¼ 6 Hz), 4.54 (d, 1H, H-6, J ¼ 6 Hz), 3.90–3.96 (m, 1H, H-30), 3.62–3.64 (m, 1H,
H-40), 3.24–3.44 (m, 1H, H-50), 2.20–2.28 (m, 1H, H-20), 1.80–1.90 (m, 1H, H-20).
Anal. calcd for C8H14N4O3S: 0.1 H2O, 0.1C2H5OH C, 38.97; H, 5.90; N, 22.18.
Found: C, 38.90; H, 5.60; N, 22.25.
1-(2-Deoxy-4-thio-b-l-threo-pentofuranosyl)-5,6-dihydro-5-azacytosine (10b) and
Its a-Anomer (10a). Compound 10b (white solid, 78%) and 10a (white solid,

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Tiwari et al.
80%) were made in a similar manner fþrom 7b and 7a respectively as reported for
1
compound 5. 10b MS m=z 247 (M þ H) . H NMR (DMSO-d6) 6.10 (dd, 1H, H-10,
J ¼ 12.0 and 8.0 Hz), 4.70–4.90 (m, 2H, H-6), 4.44–4.50 (m, 1H, H-30), 3.98–4.02
(m, 1H, 50-H), 3.70–3.78 (m, 1H, H-50), 3.54–3.58 (m, 1H, H-40), 2.30–2.42
(m, 1H, H-20), 2.04–2.14 (m, 1H, H-20). Anal. calcd for C8H14N4O3S: 0.1 H2O,
0.1C2H5OH C, 38.97; H, 5.90; N, 22.18. Found: C, 38.90; H, 5.98; N, 22.15.
1
10a MS m=z 247 (M þ H)þ. H NMR (DMSO-d6) 6.26 (dd, 1H, H-10, J ¼ 12.0
and 6.0 Hz), 4.90–5.00 (m, 1H, H-6), 4.70–4.85 (m, 1H, H-6), 4.55–4.65 (m, 1H, H-
30), 3.90–3.98 (m, 2H, H-40, 50-H), 3.60–3.70 (m, 1H, H-50), 2.32–2.44 (m, 1H, H-20),
2.06–2.16 (m, 1H, H-20). Anal. calcd for C8H14N4O3S: 0.1 H2O, 0.1C2H5OH C,
38.97; H, 5.90; N, 22.18. Found: C, 39.0; H, 5.75; N, 22.30.
ACKNOWLEDGMENTS
This investigation was supported by the National Cancer Institute, National
Institutes of Health (PO1-CA34200). The authors are indebted to Dr. William R.
Waud for cytotoxicity and animal data, Dr. James M. Riordan, Mr. Mark Richard-
son and Mrs. Joan Bearden for spectral data, Mrs. Sheila Campbell for HPLC
analysis, Ms. Stephanie Brannon for help in preparation of this manuscript and
Mrs. Anita Shortnacy-Fowler for helpful suggestions.
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Received August 11, 2003
Accepted September 4, 2003