Synthesis and biological activity of the new 5-fluorocytosine derivatives, 5′-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5′-carboxylic acid

Article abstract of DOI:10.1016/S0960-894X(01)00782-X

A series of 5-fluorocytosine derivatives, 5′-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5′-carboxylic acid, were synthesized and evaluated for their antitumor activity.

Full text of DOI:10.1016/S0960-894X(01)00782-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 483–486
Synthesis and Biological Activity of the New 5-Fluorocytosine
Derivatives, 50-Deoxy-N-alkyloxycarbonyl-5-fluorocytosine-50-
carboxylic Acid
Kwan-Hee Kim,* Ji-Young Kim, Keyong-Ho Lee, Moon-Jong Noh,
Youn-Chul Kim and Ho-Jin Park
Biomedical Research Institute, Kolon Central Research Park, 207-2 Mabuk-Ri, Guseong-Eup, Yongin-City,
Kyunggi-Do 449-797, Republic of Korea
Received 25September 2001; accepted 17 November 2001
Abstract—A series of 5-fluorocytosine derivatives, 50-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-50-carboxylic acid 6, were syn-
thesized and evaluated for their antitumor activity. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
5-fluorocytosine 1, derived from 5-fluorocytosine,
1,1,1,3,3,3,-hexamethyldisilazane, and catalytic amount
Since 5-fluorouracil (5-FU) was introduced as an anti-
tumor agent acting as thymidylate synthase inhibitor in
the 1960s, it is one of the most used and a standard
chemotherapeutic agent for some cancers. 5-FU, how-
ever, possesses toxicity and shows side effects. So, a
large number of its chemotherapy and derivatives with
promising antineoplastic activity and low toxicity, such
as 5-fluoro-1-(2-tetrahydrofuryl)-2,4 (1H,3H)-pyrimi-
dinedione (tegafur), 50-deoxy-5-fluoro-uridine (doxifluri-
dine), and N-pentyloxycarbonyl-50-deoxy-5-fluorocytidine
(capecitabine), have been developed in past decades
(Fig. 1).1
of ammonium sulfate in toluene,2 were coupled with b-d-
ribofuranose 1,2,3,5-tetraacetate 2 in acetonitrile in the
presence of tin(IV) chloride to afford 20,30,50-tri-O-
acetyl-5-fluorocytidine 3.3 This compound 3 was used
as a common intermediate herein. Then, alkyloxy-
carbonylation of 3 by means of N,N-diisopropylethyl-
amine and alkyl chloroformate in methylene chloride
gave 20,30,50-tri-O-acetyl-5-fluoro-N-(alkyloxycarbonyl)-
cytidine 4. The hydrolysis of triacetate 4 by sodium
methoxide in methanol gave triol, 5-fluoro-N-(alkyloxy-
carbonyl)cytidine 5. Finally, selective oxidation of
primary alcohol 5 with oxygen and platinum oxide gave
the carboxylic acid 6.4
In order to develop more active and less toxic analo-
gues, we also designed and synthesized new derivatives
of 5-fluorocytosine, 50-deoxy-N-alkyloxycarbonyl-5-
fluorocytosine-50-carboxylic acid 6, as new compounds
having potent antitumor activity and low toxicity.
Synthesis
A synthetic route for new 5-fluorocytosine derivatives,
50-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-50-carbox-
ylic acid 6 is outlined in Scheme 1. Trimethylsilylated
*Corresponding author. Tel.: +82-31-280-8670; fax: +82-31-280-
8999; e-mail: khkim@kolon.com
Figure 1. 5-FU and capecitabine.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00782-X

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K.-H. Kim et al. / Bioorg. Med. Chem. Lett. 12 (2002) 483–486
Scheme 1. Reagents and conditions: (a) SnCl4/CH3CN, 0 ꢁC to rt, 81%; (b) iPr2EtN/ROC(O)Cl/CH2Cl2, rt, 65–90%; (c) NaOMe/MeOH, rt, 80–
95%; (d) PtO2/O2/NaHCO3 buffer (pH 8–10), 56–77%.
Biological Activity
0. The test compound 6b and one reference compound,
capecitabine, were orally (po) administered daily for 2
weeks. Another reference compound, 5-FU, was intra-
venously (iv) administered daily also for 2 weeks.
Cytotoxicity of the 5-fluorocytosine derivatives 6a–f was
examined by using cells of human lung (A549), ovarian
(SK-OV-3), colon (HCT15), and melanoma (SK-MEL-
2) cancers and expressed in terms of inhibitory activity
(IC50, mg/mL) obtained by the MTT method.5 The
results are summarized in Table 1.
Table 1. Cytotoxicities of new 5-fluorocytosine derivatives against
tumor cells
Compd
R
IC50 (mg/mL)a
The in vivo antitumor activity of compounds 6 was
investigated with murine leukemia L1210 and the results
are listed in Tables 2 and 3.6 The activity was evaluated
in terms of increase of life span (ILS, %). The results in
Table 2 were carried out by the following protocol A.
L1210 cells were performed every 2 weeks into male
BDF1 mice, and then ascites cells were collected by
centrifugation. Collected cells were resuspended in
phosphate buffered saline (PBS) to a concentration of
1ꢀ106/mL. Then, L1210 cells (1ꢀ105/mL) were inocu-
lated intraperitoneally (ip) into male BDF1 mice on day
A549
SK-OV-3
HCT15
SK-MEL-2
5-FU
6a
6b
6c
6d
—
Ethyl
Pentyl
Allyl
Propargyl
Phenyl
Benzyl
0.26
0.49
0.17
0.040
0.0050.005 0.040
0.55
0.37
0.03
0.16
0.03
0.008
0.11
0.47
0.12
0.032
0.007
0.39
0.17
0.63
0.16
0.03
0.008
6e
6f
0.12
0.07
0.10
0.10
aActivities against tumor cells (A549, human lung; SK-OV-3, human
ovarian; HCT15, human colon; SK-MEL-2, human melanoma) were
measured by MTT assay after 3 days of incubation.

K.-H. Kim et al. / Bioorg. Med. Chem. Lett. 12 (2002) 483–486
485
Table 2. In vivo activities of new 5-fluorocytosine derivatives against
murine leukemia L1210 by protocol A
Table 3. In vivo activities of new 5-fluorocytosine derivatives against
murine leukemia L1210 by protocol B
Compd
(po)
Dosea
(mmol/kg/day)
MSTb
(day)
ILSb
(%)
Comp
(po)
Dosea
(mmol/kg/day)
MSTb
(day)
ILSb
(%)
Control
5-FU
8.8
Control
17.8
0.23
0.1514.3
16.5
62.5
87.5
Capecitabine
2.00
0.40
0.080
0.016
0.0032
22.8
20.1
20.2
19.2
18.9
28.1
12.9
13.5
7.9
Capecitabine
1.4521.20.5
0.67
0.13
9.6
9.4
9.1
6.8
6.2
6c
2.00
0.40
0.080
0.016
0.0032
5.1
16.2
25.7
24.8
22.1
ꢃ71.3
ꢃ9.0
44.4
39.3
24.2
6b
1.516.8
0.67
0.13
90.9
11.3
9.1
28.4
3.4
aL1210 cells (1ꢀ105) by protocol A were implanted intraperitoneally
(ip) into BDF1 male mice (6 weeks old) on day 0 and the mice were
divided into several groups (8 mice per group) on day 1. The 5-FU was
suspended in 0.5% CMC and intravenously (iv) administrated daily
for 2 weeks. The other test compounds were dissolved in saline and
administrated per os (po) daily for 2 weeks.
bSurvival number was monitored daily and the increase in life span
(ILS, %) was calculated from [(mean survival time of treated group)/
(mean survival time of control group)ꢃ1]ꢀ100.
aL1210 cells (1ꢀ106) by protocol B were implanted intraperitoneally
(ip) into BDF1 male mice (6 weeks old). The test compounds were
dissolved in saline and orally (po) administrated 15times for 3 weeks.
bSurvival number was monitored daily and the increase in life span
(ILS, %) was calculated from [(mean survival time of treated group)/
(mean survival time of control group)ꢃ1]ꢀ100.
8
compared to 115mg/kg of 5-FU. In case of 6d, the
LD50 value was 12 mg/kg for single dose and it is higher
than that of 6c.
The results in Table 3 were carried out by following
protocol B. L1210 cell line for implantation was main-
tained at 37 ꢁC under an atmosphere of 5% CO2 in a
2
75cm culture flask and subcultured once or twice per
As also shown in Table 3, the new 5-fluorocytosine
derivative 6c gave good antitumor activity over a broad
dose range compared to capecitabine.9 The compound
6c showed ILS ranging from 24.2% at 0.0032 mmol/kg/
day to 44.4% at 0.080 mmol/kg/day. ILS of capecita-
bine had a range of 6.2% at 0.0032 mmol/kg/day to
28.1% at 2.00 mmol/kg/day.
week in RPMI 1640 medium containing 10% fetal
bovine serum. The tumor cells were resuspended in PBS
to a concentration of 1ꢀ107/mL. Similarly, L1210 cells
(1ꢀ106/mL) were inoculated intraperitoneally (ip) into
male BDF1 mice on day 0. The test compound 6c and
the reference compound, capecitabine, were orally
administered (po) 15times for 3 weeks and the max-
imum doses were decided by considering acute toxicity.
Evidently, the in vivo antitumor activity of 6b and 6c is
better than that of 5-FU and capecitabine. Especially, in
case of 6c, it shows that the similar antitumor activity at
much lower dose range compared to that of capecitabine.
Results and Discussion
As shown in Table 1, all the new 5-fluorocytosine deri-
vatives 6 were potent to inhibit the proliferation of
A549, SK-OV-3, HCT15, and SK-MEL-2 cells. Among
the compounds, 6c and 6d showed excellent inhibitory
activity on the above cell lines, with IC50 values in the
range of 0.005ꢂ10.040 mg/mL. It is evident that the in
vitro potency of those bearing unsaturated alkyl groups
(6c, R=allyl; 6d, R=propargyl) is better than that of
those bearing saturated alkyl and aromatic groups (6a,
R=ethyl; 6b, R=pentyl, 6e, R=phenyl; 6f, R=benzyl).
We initially evaluated that the in vivo activity test of the
new 5-fluorocytosine derivative 6b and the reference
compounds, 5-FU and capecitabine. As shown in Table
2, the antitumor activity of 6b against L1210 leukemia
in mice showed the best ILS value at high dose range,
90.9% at 1.5mmol/kg/day, after po administration
compared to ILS of 5-FU and capecitabine, 87.5% at
0.23 mmol/kg/day (iv) and 42.0% at 1.5mmol/kg/day
(po), respectively.7
In conclusion, we have synthesized the new 5-fluoro-
cytosine derivatives, 50-deoxy-N-alkyloxy-carbonyl-5-
fluorocytosine-50-carboxylic acid 6, and they showed
better antitumor activity than that of 5-FU or capecita-
bine. In particular, 6c showed potent antitumor activ-
ities against L1210 leukemia and low toxicity. We
selected 6c for further pharmacological evaluation.
Acknowledgements
We would like to thank Mr. Young-Seok Park in Kolon
Central Research Park for the support of intermediate
synthesis.
References and Notes
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days in mice. This value for single dose is very low

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