Synthesis and anticancer activity of 4-amino-5-oxo-8-(β-D-xylofuranosyl) pyrido[2,3-d]pyrimidine

Article abstract of DOI:10.1081/NCN-100002303

4-Amino-5-oxo-8-(β-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (4) was recently synthesized and evaluated in our laboratories for anticancer activities. This compound showed potent in vitro inhibitory effects on the growth of HTB-81 prostate cancer cells and

Full text of DOI:10.1081/NCN-100002303

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SYNTHESIS AND ANTICANCER ACTIVITY OF 4-
AMINO-5-OXO-8-(β-D-XYLOFURANOSYL) PYRIDO[2,3-
d]PYRIMIDINE
Zbigniew Pietrzkowski a , Jean-Luc Girardet b , Cathey Esler a & Guangyi Wang c
a Cancer Biology Laboratories, ICN Pharmaceuticals, Inc. , 3300 Hyland Avenue, Costa
Mesa, California, 92626, U.S.A.
b Chemistry, ICN Pharmaceuticals, Inc. , 3300 Hyland Avenue, Costa Mesa, California,
92626, U.S.A.
c Chemistry, ICN Pharmaceuticals, Inc. , 3300 Hyland Avenue, Costa Mesa, California,
92626, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Zbigniew Pietrzkowski , Jean-Luc Girardet , Cathey Esler & Guangyi Wang (2001) SYNTHESIS AND
ANTICANCER ACTIVITY OF 4-AMINO-5-OXO-8-(β-D-XYLOFURANOSYL) PYRIDO[2,3-d]PYRIMIDINE, Nucleosides, Nucleotides
and Nucleic Acids, 20:4-7, 323-328, DOI: 10.1081/NCN-100002303
To link to this article: http://dx.doi.org/10.1081/NCN-100002303
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 323–328 (2001)
SYNTHESIS AND ANTICANCER ACTIVITY
OF 4-AMINO-5-OXO-8-(β-D-XYLOFURANOSYL)
PYRIDO[2,3-d]PYRIMIDINE
Zbigniew Pietrzkowski,2 Jean-Luc Girardet,1 Cathey Esler,2
and Guangyi Wang1,∗
1Chemistry and 2Cancer Biology Laboratories,
ICN Pharmaceuticals, Inc. 3300 Hyland Avenue,
Costa Mesa, California 92626
ABSTRACT
4-Amino-5-oxo-8-(β-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (4) was recen-
tly synthesized and evaluated in our laboratories for anticancer activities. This
compound showed potent in vitro inhibitory effects on the growth of HTB-81
prostate cancer cells and Daudi-lymphoma. In vivo studies showed that the
compound could inhibit HTB-81 tumor growth in syngeneic mice by 93% at a
daily dose of 8.5 mg/kg for 10 days.
Certain nucleosides as anticancer drugs have been used clinically for many
years and new nucleoside analogues continue to show promise (1,2). However,
current nucleoside anticancer drugs are commonly associated with various ad-
verse effects (1). Apparently, there is a need to search for nucleoside analogues
that can selectively inhibit cancer cell proliferation. In the past decades, a large
number of sangivamycin (1) and toyocamycin (2) analogues have been synthe-
sized (3–8) as potential anticancer and antiviral agents. Unfortunately, no com-
pound in this group could be used clinically, primarily owing to their toxicity
to normal cells. Considered as sangivamycin analogues, 4-amino-5-oxo-8-(β-D-
ribofuranosyl)pyrido[2,3-d]pyrimidine-6-carboxamide (9) and 4-amino-7-oxo-8-
(β-D-ribofuranosyl)pyrido[2, 3-d]pyrimidine-5-carboxamide(10)weresynthesized,
∗Corresponding author.
323
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C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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PIETRZKOWSKI ET AL.
but their biological activity was not reported. To explore the possibility of pyrido[2,
3-d]pyrimidine nucleosides as anticancer and antiviral drugs, 4-amino-5-oxo-8-
(β-D-ribofuranosyl)-pyrido[2,3-d]pyrimidine (3) was synthesized at ICN Pharma-
ceuticals and identified as a very potent inhibitor of cancer cell proliferation (11).
However, 3 also showed a similar in vitro cytotoxicity profile to normal human cells.
The cytotoxicity of ribonucleoside analogues likely results from non-selective in-
hibition of RNA polymerases by their triphosphates, or from their non-selective
incorporation into RNAs (12,13). We anticipated that a selective inhibition of can-
cer cell proliferation might be achieved if a ribonucleoside could selectively enter
or be selectively metabolized in certain cancer cells. Recently, we synthesized
and evaluated a series of pyrido[2,3-d]-pyrimidine nucleosides having modified
sugars in search of selective inhibitors of cancer cells (14). 4-Amino-5-oxo-8-(β-
D-xylofuranosyl)pyrido[2,3-d]pyrimidine (4) was identified as a potent inhibitor of
some cancer cells with certain selectivity. In this communication, we report the
synthesis and anticancer activities of 4.
CHEMISTRY
Compound 5 (14) was treated with N,O-bis(trimethylsily)acetamide (BSA)
in 1,2-dichloroethane at 60◦C for 2 h and then the resulting silylated base was
condensed with 1-O-acetylated xylofuranose derivative 6 (14). Compound 7 was
formed readily and then slowly converted to 8 until an equilibrium was reached after
2-3 days. Compounds 7 and 8 were separated by flash chromatography and then
subjected to a treatment with methanolic ammonia to give 9 and 4, respectively.
By this approach, the by-product 9 was formed in a significant amount. In order
to avoid the formation of 7, compound 5 was acetylated to give 11, which was
silylated and then condensed with 6 to give 12 directly after several hours. By the
second approach, the N-1 coupled product was not formed and the purification of
the final product was simplified. It can be noted that the yield of the final product 4
by the second approach was not greatly improved. It was observed that the acetyl
on the N4 of the base was not stable enough under the condensation conditions.

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325
It is anticipated that the substitution of a benzoyl group for the acetyl would further
increase the yield of 4.
ANTICANCER ACTIVITIES
Our in vitro screening approach for anticancer activity was based on two
assays: MTS (colorimetric) and DNA-Hoechst33342 binding assay (fluorescent
method). Both assays were performed in parallel and under the same culture con-
ditions (treated for 72 h). These methods were very reliable for following the
growth of cells by mitochondrial activity (MTS Assay) or total amount of DNA
in the cell culture. For the cancer cells tested, these two methods produced very
similar results; however, in the case of normal cells, some discrepancies were
noticed. The results shown in Figure 1 indicate that compound 4 has potent in-
hibitory effects on the growth of Daudi-lymphoma (EC50 = 0.10 µM) and prostate

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PIETRZKOWSKI ET AL.
Figure 1. In vitro inhibitory effects of compound 4 on the growth of various cancer cells and normal
human fibroblasts. EC50-DNA is a concentration [µM] of the compound that caused a 50% reduction
of total DNA amount after 72 h treatment relative to untreated cells using fluorescent method (Hoechst
Dye). EC50-MTS is a concentration [µM] of the compound that caused a 50% reduction in absorbance
at 490 nm after 72 h treatment relative to untreated cells using MTS assay.
cancer HTB-81 cells (EC50 = 0.73 µM). The other types of cells tested were less
sensitive to the compound, with normal human fibroblasts being least sensitive
(EC50 = 20.7 µM).
Table 1 gives another set of data which shows the in vitro inhibitory effects
of compound 4 on the growth of various cancer cells based on an assay using
alamarBlue reaction. The EC50 values vary between 3.6-32 µM, indicating that
this compound is not equally potent towards all types of cancer cells. Moreover,
even among cell lines derived from the same types of tumors, compound 4 showed
different potency. Thus, human prostate cancer cells HTB-81 and PC-3 have differ-
ent sensitivities to the compound, with EC50 = 0.78 µM for HTB-81 and 16 µM
for PC-3. Another type of prostate cancer cell, LyLu, is even less sensitive to the
compound, with EC50 = 55 µM (not shown in Fig. 1 and Table 1). Similarly, two
Table 1. Inhibitory Effects of Compound 4 on
the Growth of Various Cancer Cells
Cancer Cell Line
EC50 [µM]
MCF7/Breast
T47D/Breast
HT29/Colon
ACHN/Kidney
K562/Leu
3.6
10.0
32.0
7.9
10.0
9.3
HC4/Liver
HepG2/Liver
PC6/Lung
SKMEL5/Melanoma
PC3/Prostate
9.5
12.0
16.0
12.0
EC50 is the concentration of the compound that
causes a 50% reduction in cell proliferation rel-
ative to untreated cells (alamarBlue reaction).

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327
Figure 2. In vivo inhibition of tumor growth by compound 4. % inhibition over untreated control:
a percentage by which tumor growth is inhibited by compound 4 compared to the untreated. % =
(U-T)/U, U is the average tumor weight of the untreated animals and T is the average tumor weight of
the treated animals. Animals were treated I.p. daily for 10 days with a dose of 8.5 mg/kg of compound
4. Human prostate cancer HTB-81 grew in nude mice; mouse melanoma B16 grew in syngeneic mice;
human prostate cancer LyLu grew in syngeneic rats.
breast cancer cell lines (CF-7 and T47D) also showed different sensitivities to the
compound. These results suggest that compound 4 may not be equally transported
to all types of cancer cells or may be metabolized at different rates in different
cancer cells.
Based on the in vitro inhibitory data, an in vivo study was initiated to evaluate
the inhibitory effects of compound 4 on tumor growths in animals. As can be seen
from Figure 2, compound 4 showed a strong inhibition on the growth of HTB-81
tumor in nude mice while less inhibition was observed for B16 tumor in syngeneic
mice and Lylu tumor in syngeneic rats. These results are proportional to the in vitro
inhibitory data (Fig. 1).
Figure 3 shows a dose-responsive inhibition of B16 tumor in syngeneic mice
by compound 4. Although the compound has an EC50 value of around 5 µM in
Figure 3. Compound 4 inhibits the growth of melanoma B16 in syngeneic mice in a dose-dependent
manner. A = 1.7 mg/kg and B = 8.7 mg/kg.

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PIETRZKOWSKI ET AL.
both total DNA and MTS assays, the in vivo study demonstrated that the compound
could effectively inhibit the tumor growth (also see Fig. 2). The tumor inhibition
by compound 4 at a dose of 8.7 mg/kg is significantly more effective than that at a
dose of 1.7 mg/kg.
In summary, synthesis of 4-amino-5-oxo-8-(β-D-xylofuranosyl)pyrido[2,3-
d]-pyrimidine (4) was explored. The compound showed potent in vitro inhibitory
effects on the growth of human prostate cancer HTB-81 and Daudi-lymphoma while
a moderate, broad-spectrum inhibition on various cancer cells was observed. An
effective inhibition (by 93%) of HTB-81 tumor growth in nude mice was achieved
at a dose of 8.5 mg/kg of compound 4. Further studies are underway to evaluate the
in vivo efficacy and toxicity of the compound.
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