C5-Modified nucleosides exhibiting anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2009.06.072

We describe (i) a simple method for the synthesis of C5-modified nucleosides from 5-iodo-2′-deoxyuridine and (ii) their activity against six types of human cancer cell lines (HCT15, MM231, NCI-H23, NUGC-3, PC-3, ACHN). We generated nitrile oxides in situ from oximes using a commercial bleaching agent; their cycloadditions with 5-ethynyl-2′-deoxyuridine yielded isoxazole derivatives possessing activity against the cancer cell lines. We synthesized several azides from benzylic bromides and their click reactions with 5-ethynyl-2′-deoxyuridine provided triazole derivatives.

Full text of DOI:10.1016/j.bmcl.2009.06.072

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4688–4691
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
C5-Modified nucleosides exhibiting anticancer activity
Yoon-Suk Lee a, Sun Min Park a, Hwan Mook Kim b, Song-Kyu Park b, Kiho Lee b, Chang Woo Lee b,
Byeang Hyean Kim a,
*
a Department of Chemistry, BK School of Molecular Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
b Bio-Evaluation Center, KRIBB, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
We describe (i) a simple method for the synthesis of C5-modified nucleosides from 5-iodo-20-deoxyuri-
dine and (ii) their activity against six types of human cancer cell lines (HCT15, MM231, NCI-H23, NUGC-3,
PC-3, ACHN). We generated nitrile oxides in situ from oximes using a commercial bleaching agent; their
cycloadditions with 5-ethynyl-20-deoxyuridine yielded isoxazole derivatives possessing activity against
the cancer cell lines. We synthesized several azides from benzylic bromides and their click reactions with
5-ethynyl-20-deoxyuridine provided triazole derivatives.
Article history:
Received 12 May 2009
Revised 16 June 2009
Accepted 18 June 2009
Available online 21 June 2009
Keywords:
Anticancer
Nucleoside
Triazole
Ó 2009 Elsevier Ltd. All rights reserved.
Isoxazole
Cycloaddition
About 13% of all human deaths throughout the world are caused
by cancers—diseases characterized by uncontrolled cell growth,
metastasis, and invasion.1 Although the risk of cancer increases
with age, people of all ages—even fetuses—can be affected by the
disease. The five most fatal cancers are lung, stomach, liver, colon,
and breast cancer. Several carcinogens, including tobacco smoke,
radiation, chemicals, and infectious agents, cause abnormalities
of the genetic material, which can induce the onset of cancer.1
For example, infections from hepatitis B or C are known to cause
liver cancer.2 Several nucleoside drugs have been developed as
cancer treatment agents: cladribine, clofarabine, capecitabine, cyt-
arabine, fludarabine, gemcitabine, decitabine (1a), and floxuridine
(1b).3 Indeed, nucleoside analogs were among the first oncology
drugs ever developed. Using Sonogashira coupling, Dembinski’s
group discovered that several C5-ethynyl-modified nucleosides
(1c) exhibit anticancer activity (Fig. 1).4 We became interested in
using such C5-ethynyl nucleosides as partners in [3+2] cycloaddi-
tions, which are powerful reactions for the modification of nucleo-
sides and nucleotides as well as for the synthesis of a diverse range
of heteroaromatic five-membered rings. Previously, we prepared a
small library of isoxazoles that we tested against 12 types of
viruses.5 In this present study we synthesized two scaffolds from
a series of C5-ethynyl-20-deoxyuridines—the isoxazoles 2a and tri-
azoles 2b—through reactions with oximes and azides, respectively
(Fig. 2). Click reactions have been used widely for the synthesis of
modified nucleosides and DNA strands ever since our group first
applied them to nucleoside chemistry in 2003.6 The Sharpless click
reaction is a very simple and useful transformation that does not
generate any sideproducts or byproducts, unlike other [3+2] cyc-
loadditions.7 The click reactions that have been applied to modify
the C-5 positions of pyrimidine nucleosides have also been used
to modify oligonucleotides.8 Several antiviral drugs, such as BVDU
((E)-5-(2-bromovinyl)-20-deoxyuridine) are C5-modified pyrimi-
dine nucleosides. Although the triazole nucleosides that we syn-
thesized through click chemistry appear to be inactive against
viruses, they exhibit no toxicity unlike their isoxazole congeners,
which displayed cytotoxicity against several human cell lines. Be-
cause the triazole nucleosides readily formed very good hydrogels,
we suspect that they might be useful as carrier in drug delivery
systems.
We have been exploring the behavior of our C5-modified
nucleosides in several biological studies; in this Letter, we describe
their anticancer activities. For the cell growth inhibition assays, we
tested all of our compounds against six human cancer cell lines of
R
O
N
O
N
NH2
N
F
NH
NH
N
HO
HO
HO
O
N
O
O
O
O
O
OH
OH
OH
1c
1a
1b
* Corresponding author. Tel.: +82 54 279 2115; fax: +82 54 279 3399.
E-mail addresses: bhkim@postech.ac.kr, bhkim@postech.edu (B.H. Kim).
Figure 1. Nucleoside anticancer drugs.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.072

Y.-S. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4688–4691
4689
R'
R'
displayed the greatest anticancer activity. The compounds substi-
tuted by smaller R groups—for example, a proton (8a) or an ethyl
group (8c)—displayed dramatically reduced anticancer activities.
Compounds 8f and 8g, which possess short alkyl group-substituted
isoxazole rings, exhibited no anticancer activity at all. In contrast,
the pentyl-substituted isoxazole 8h displayed minimal activity
against four of the cancer cell lines (MM231, NCI-H23, NUGC-3,
PC-3). The acetyl-free isoxazole nucleosides 9a and 9b provided
lower activities than any of the acetylated nucleosides 8. Indeed,
the hydrolyzed compound 9a had only about half the activity of
8e. Acetyl groups typically increase the lipophilicity of drugs,
thereby supporting their cell penetration; subsequent in-cell cleav-
age of the acetyl groups is mediated by carboxyesterase. Our assays
suggest that the anticancer activities of the pyrimidine isoxazole
nucleosides increased upon increasing the bulkiness of the nonpo-
lar substituent.
N
O
N
O
N
N
N
NH
NH
O
N
RO
RO
O
O
O
O
OR
OR
X
2a
2b
Figure 2. C5-ethynyl, -isoxazole, and -triazole nucleosides.
various origins—HCT15, MM231, NCI-H23, NUGC-3, PC-3, and
ACHN—and use the sulforhodamine B screening assay to determine
their degrees of cancer growth inhibition. The cells were cultured
in RPMI 1640 medium supplemented with 5% fetal bovine serum
at 37 °C in humidified air containing 5% CO2. The bioassay proto-
cols have been described previously9.
Among our triazole-appended nucleosides, compound 10c
exhibited the highest anticancer activity. Although we observed
no relationship between the activity and the size of the substitu-
ent on the triazole unit, we found that the acetyl-free triazole
nucleosides (e.g., 11) exhibited slight lower anticancer activities
than their acetylated congeners. In general, our triazole nucleo-
sides provided lower anticancer activities relative to those of
the isoxazole compounds. On the other hand, the triazole nucleo-
The experimental conditions that we used for the cycloaddi-
tions leading to the isoxazoles 8b, 8e–g, and 9b and the triazoles
have been reported previously.5,10 We prepared the requisite
dipolarophile, the acetyl-protected 5-ethynyl-20-deoxyuridine 5,
through Sonogashira coupling (87% yield) from C5-iodo-20-deoxy-
uridine and trimethylsilylacetylene.11,12 We obtained a series of
oximes 6 from the reactions of several aldehydes with hydroxyl-
amine; the azides 7 were acquired from the reactions of benzylic
bromides with sodium azide. We isolated the triazole nucleosides
after performing Sharpless click reactions of the azides with the
dipolarophile 5, using copper sulfate and sodiumascorbate as cata-
lysts in a mixed solvent of n-butanol and water (Scheme 1).7
Table 1 lists the anticancer activities of our 16 synthesized com-
pounds13 (see Fig. 3) against the six types of human cancer cell
lines. Three of the acetylated isoxazole nucleosides (8b, 8d, and
8e) inhibited the cancer growth rate by 50% or more at a concen-
sides exhibited no cytotoxicity at 300 lM concentration (CC50 for
three human cell lines). The cytotoxicity for three human cell
lines (Vero, MT-4, and HeLa) was determined during the antiviral
assay. The isoxazole compounds have cytotoxicity (CC50) in the
range of 3–300 lM. Therefore we believe that the triazole nucleo-
sides have a better chance as drug candidates. The triazole scaf-
fold contains an extra carbon atom between the triazole ring
and phenyl ring. In other words, this benzylic carbon atom
changes the relative positions of the five-membered heterocyclic
ring and the phenyl ring. Thus, to better understand the struc-
ture–activity relationships between the two scaffolds, it will be
necessary for us to prepare series of compounds with and without
the benzylic carbon atom.
tration of 10 lM. These three compounds, which feature isopro-
pyl-, n-butyl-, and tert-butyl-substituted phenyl-isoxazoles units,
exhibited their highest activities against the NUGC-3 (stomach)
and PC-3 (prostate) cancer cell lines. Among these three com-
pounds, 8e contains the most bulky (tert-butyl) group and
OH
O
R'
O
N
(e)
Br
N
O
N
(d)
N3
I
R''
H
NH
R'''
R''
H
NH
R'''
AcO
O
RO
7
(b)
O
6
O
O
R'' = pentyl
= octyl
R''' = H
OAc
OR
= ethyl
= isopropyl
4: R' = TMS
5: R' = H
R = H
= phenyl
= 4-ethylphenyl
= 4-n-butylphenyl
(a)
(c)
3: R = Ac
= 4-tert-butyl
R'''
R''
N
O
O
N
O
O
N
O
N
NH
NH
N
NH
NH
(f)
(g)
RO
N
RO
AcO
O
AcO
O
O
N
O
N
O
O
O
O
6
7
OAc
OAc
OR
OR
X
5
8: R = Ac
10: R = Ac
11: R = H
5
(h)
(h)
9: R = H
Scheme 1. Synthesis of compounds 3–11. Reagents and conditions: (a) Ac2O, pyridine, rt, 8 h (92% yield); (b) TMS–acetylene, Pd(PPh3)4, Et3N, DMF, 40 °C, 8 h (87% yield); (c)
KF, 10% MeOH in CH2Cl2 (80% yield); (d) HONH2ꢀHCl, 1 N NaOH, THF (1:1), rt, 6 h (90–99% yield); (e) NaN3, DMF, 40 °C, 6 h (86–97% yield); (f) 4% NaOCl, THF, slow dropwise
addition, rt, 10 h (59–80% yield); (g) CuSO4ꢀ5H2O, sodium ascorbate, n-BuOH, water, rt, 10 h (71–87% yield); (h) LiOH, MeOH/water (3:1), rt, 10 h (71–90% yield).

4690
Y.-S. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4688–4691
Table 1
Cancer growth rates of human tumor cell lines
HCT15
MM231
NCI-H23
NUGC-3
PC-3
ACHN
8a
8b
8c
8d
8e
8f
8g
8h
92.2
80.9
92.2
71.4
53.8
101.7
98.8
98.7
88.2
79.8
92.3
94.2
88.0
96.5
88.3
96.9
95.0
83.2
97.4
72.2
69.3
106.1
102.0
91.5
91.4
107.8
92.7
87.6
56.4
72.6
39.8
32.8
90.8
89.1
91.9
76.4
88.0
88.7
94.2
94.3
95.4
95.6
94.8
0.25
85.5
50.3
62.8
30.4
27.5
92.4
88.1
93.9
77.3
87.9
92.1
90.4
91.2
91.3
92.1
92.3
0.73
97.6
90.1
92.0
80.5
74.7
96.4
99.5
99.8
95.6
94.2
99.0
97.2
98.4
92.2
98.7
101.7
0.41
97.4
111.9
101.0
96.9
88.9
119.7
98.9
98.2
105.6
77.2
86.7
97.5
9a
9b
10a
10b
10c
10d
11a
11b
Doxorubicin
96.7
102.2
102.3
104.6
97.7
98.1
102.8
105.6
0.61
95.0
0.51
1.64
GI50 in (
lM)
SRB (sulforhodamine B) assay using adriamycin as a positive control. Human cancer cell lines: ACHN (renal), NCI-H23 (lung), MDA-MB-231 (breast), HCT-15 (colon), NUGC-3
(stomach), and PC-3 (prostate). The cell growth inhibition assay was determined at 10
l
g/mL. Doxorubicin was used as reference drug.
R
R
N
O
O
8a
8b
8c
8d
8e
R = H
N
N
O
NH
N
NH
= isopropyl
10a R = H
= ethyl
= n-butyl
= tert-butyl
AcO
AcO
10b
10c
10d
= tert-butyl
N
O
N
O
O
O
= ethyl
= isopropyl
OAc
OAc
R
N
R
O
N
O
NH
O
N
AcO
8f
8g
8h
R = methyl
= propyl
N
O
N
NH
O
HO
N
O
11a R = H
11b = tert-butyl
= pentyl
OAc
O
OH
R
N
O
N
O
NH
9a
9b
R = octyl
HO
O
O
= 4-tert-butyl phenyl
OH
Figure 3. Isoxazole (8 and 9) and triazole nucleosides (10 and 11) tested for their anticancer properties against six types of cancer cell lines.
4. Meneni, S.; Ott, I.; Sergeant, C. D.; Sniady, A.; Gust, R.; Dembinski, R. Bioorg.
Acknowledgment
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We thank KOSEF (Gene Therapy R&D and KNRRC programs) for
financial support.
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512.3, calcd 511.195. Compound 10a: 1H NMR (300 MHz, CDCl3) d 10.24 (br,
1H), 8.53 (s, 1H), 8.23 (s, 1H), 7.32–7.19 (m, 5H), 6.43 (t, J = 5.73 Hz, 1H), 5.47
(s, 2H), 5.25 (d, J = 6.00, 1H) 4.39–4.31 (m, 2H), 4.25 (br, 1H), 2.49–2.32 (m, 2H),
2.27 (s, 3H), 2.07 (s, 3H); 13C NMR (75 MHz, acetone-d6) d 170.5, 170.3, 161.8,
150.0, 145.0, 139.9, 135.2, 128.5, 128.3, 125.0, 123.1, 105.9, 87.9, 85.8, 71.1,
61.8, 53.9, 49.1, 21.1, 21.0; MS (EI+) m/z found 469.16, calcd 469.16. Compound
10c: 1H NMR (300 MHz, CDCl3) d 9.04 (br, 1H), 8.55 (s, 1H), 8.10 (s, 1H), 7.24–
7.17 (m, 4H), 6.45 (t, J = 5.69 Hz, 1H), 5.46 (s, 2H), 5.28 (d, J = 6.23, 1H) 4.42–
4.34 (m, 2H), 4.28 (br, 1H), 2.65–2.57 (m, 2H), 2.47–2.33 (m, 2H), 2.31 (s, 3H),
2.10 (s, 3H), 1.19 (t, J = 7.37, 3H); 13C NMR (75 MHz, acetone-d6) d 170.7, 170.4,
162.0, 150.1, 145.1, 139.9, 137.1, 133.2, 128.7, 128.6, 123.1, 105.9, 88.0, 86.0,
71.3, 61.8, 53.9, 49.1, 40.4, 28.5, 21.2, 21.0, 15.4. MS (EI+) found m/z 497.19,
calcd 497.191. Compound 11a: 1H NMR (300 MHz, CD3OD) d 8.59 (s, 1H), 8.28
(s, 1H), 7.33 (br, 5H), 6.31 (t, J = 6.61 Hz, 1H), 5.59 (s, 2H), 4.41 (br, 1H), 3.94 (br,
1H), 3.84–3.71 (m, 2H), 2.31–2.20 (m, 2H); MS (EI+) m/z found 385.138, calcd
385.139.
13. Selected spectroscopic data. 8c: 1H NMR (300 MHz, acetone-d6) d 10.46 (br, 1H),
8.84 (s, 1H), 7.76–7.67 (m, 2H), 7.28–7.25 (m, 2H), 6.97 (s, 1H), 6.29 (t,
J = 6.21 Hz, 1H), 4.50 (br, 2H), 3.97 (br, 1H), 3.83–3.79 (m, 2H), 2.64–2.57 (m,
2H), 2.33–2.30 (m, 2H), 2.25 (s, 3H), 2.12 (s, 3H), 1.15 (t, J = 9.12 Hz, 3H); 13C
NMR (75 MHz, CDCl3) d 170.8, 170.6, 163.6, 161.8, 159.4, 149.4, 147.0, 137.6,
128.8, 127.4 105.0, 101.6, 86.1, 83.4, 74.6, 64.1, 29.1, 21.1, 15.6; MS (EI+) m/z
found 483.16, calcd 483.164. Compound 8d: 1H NMR (300 MHz, CDCl3) d 9.74
(br, 1H), 8.69 (s, 1H), 7.73 (d, J = 7.97 Hz, 2H), 7.25–7.23 (m, 3H), 6.42 (t,
J = 6.01 Hz, 1H), 5.27 (d, J = 5.76 Hz, 1H), 4.48–4.43 (m, 1H), 4.35–4.32 (m, 2H),
2.64–2.54 (m, 3H), 2.30–2.28 (m, 1H), 2.26 (s, 3H), 2.11 (s, 3H), 1.61–1.54 (m,
2H), 1.37–1.30 (m, 2H), 0.97 (t, J = 7.22 Hz, 3H); 13C NMR (75 MHz, CDCl3) d
170.9, 170.6, 163.6, 161.8, 159.7, 149.6, 145.7, 137.6, 129.3, 127.2, 105.0, 101.6,
86.1, 83.4, 74.7, 64.2, 38.8, 35.9, 33.7, 22.7, 21.2, 14.2; MS (EI+) m/z found