A new synthetic analogue of thymidine, 7-(3-bromo-phenoxy)-thymidine, inhibits the proliferation of tumor cells

Article abstract of DOI:10.1016/j.bmcl.2004.10.039

The synthesis of new thymidine analogues and their biological activities are described. Modified thymidine analogues have been highlighted as useful agents for the treatment of cancer and viral diseases due to their potent biological activities. In the present study, we synthesized a new thymidine analogue, 7-(3-bromo-phenoxy)-thymidine (4a), as a potential lead for anti-tumor agent. Compound 4a potently inhibits HeLa cell proliferation with an IC ₅₀ of 15 μM.

Full text of DOI:10.1016/j.bmcl.2004.10.039

Bioorganic & Medicinal Chemistry Letters 15 (2005) 77–79
A new synthetic analogue of thymidine,
-(3-bromo-phenoxy)-thymidine, inhibits the
proliferation of tumor cells
7
a
Kyung Noo Kim, Jiyong Lee, Dong Hoon Kim, Jong-Shin Yoo and Ho Jeong Kwon
a
a
b
a,
*
a
Chemical Genomics National Research Laboratory, Department of Bioscience and Biotechnology,
Sejong University, Seoul 143-747, Korea
b
Korea Basic Science Institute, PO Box 41, Yusong, Daejon 305-333, Korea
Received 30 July 2004; revised 11 October 2004; accepted 12 October 2004
Available online 2 November 2004
Abstract—Modified thymidine analogues have been highlighted as useful agents for the treatment of cancer and viral diseases due to
their potent biological activities. In the present study, we synthesized a new thymidine analogue, 7-(3-bromo-phenoxy)-thymidine
(
ꢀ
4a), as a potential lead for anti-tumor agent. Compound 4a potently inhibits HeLa cell proliferation with an IC50 of 15lM.
2004 Elsevier Ltd. All rights reserved.
1. Introduction
2. Chemistry
1
–4
Thymidine is a component of thymonucleic acid and
modified thymidine nucleosides have been of great inter-
est as potential drugs for the treatment of viral diseases
Several hydrophobic substituents were introduced into
5-methyl group of thymidine and new thymidine ana-
logues were obtained by the synthetic sequence de-
scribed in Scheme 1.
5
,6
0
and cancer. As such, 5-R-2 -deoxyuracils (R = Et, Pr,
CHMe , CH OH, CH SMe) are known as antiviral
2
2
2
0
0
agents and 3 -azido-3 -deoxythymidine (AZT) is the first
7
The condensation of thymidine 1 with acetic anhydride
0
0
drug against human immunodeficiency virus (HIV). In
addition, several pyrimidine modified thymidine deriva-
tives have shown anticancer activities. These include 5-
provided the 3 ,5 -di-acetyl-thymidine 2 in 91% yield.
Compound 2 was isolated by flash column chromato-
graphy (hexane–ethyl acetate = 2:1) followed by recrys-
tallization (Fig. 1).
0
8–10
0
iodo-2 -deoxy-uridine,
1
5-trifluoromethyl-2 -deoxyl-
uridine, and its precursor compound, 5-fluoroura-
1
1
2,13
cil.
In particular, attention has been focused on
At the next reaction (b), bromination was achieved using
N-bromosuccinimide and benzoyl peroxide under reflux
(80–100ꢁC) condition in chloroform. Residual N-bromo-
succinimide and succinimide in chloroform were
the pyrimidine moiety of the compound for structure
activity related synthesis. Based on this idea, we have
synthesized several analogues of thymidine and investi-
gated the structure activity relationship of 5-methyl
group modified thymidine analogues for their antiprolif-
erative activity to cancer cells. In this paper, the syn-
thetic strategy and biological activity of a new
thymidine analogue, 7-(3-bromo-phenoxy)-thymidine
1
4
removed by flash column chromatography. Reaction
(c), in the presence of 1Mequiv of TEA, smoothly
proceeded to afford the corresponding compounds Ac-
4-(c–i) in 15–70% yield.
(
4a), are described.
Different reaction condition (d) was used for the synthe-
sis of Ac-4a, Ac-4b, and Ac-4j. The reaction was per-
formed in the presence of 1.05–1.2Mequiv of K CO
2
3
to afford the corresponding compounds Ac-4a, Ac-4b,
and Ac-4j in 35–90% yield. Finally, acetyl protecting
group of Ac-4-(a–j) was removed by NaOMe, resulting
Keywords: Thymidine; Antitumor activity.
*
Corresponding author. Tel.: +82 2 3408 3640; fax: +82 2 3408
334; e-mail: kwonhj@sejong.ac.kr
1
5,16
3
in deprotected thymidine analogues 4-(a–j).
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.039

78
K. N. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 77–79
O
N
O
N
O
HN
HN
HN
Br
O
O
O
O
N
O
a
O
b
AcO
HO
AcO
HO
AcO
AcO
1
2
3
c or d
O
N
O
HN
R
HN
R
O
O
N
O
O
e
HO
AcO
HO
AcO
4
Ac-4
reaction c
reaction d
R= Ac-4c, Ac-4d,
Ac-4e, Ac-4f,
Ac-4g, Ac-4h,
Ac-4i
R=Ac-4a, Ac-4b,
Ac-4j
Scheme 1. Reagent and conditions: (a) acetic anhydride, Py, DMAP, rt, 3h; (b) NBS, (BzO)
2
3 2
, CHCl , reflux, 4h; (c) R–OH, TEA, CHCl , rt, 7h;
(d) R–OH or R–SH, K
2
CO
3
, DMF, reflux, 5h; (e) NaOMe, MeOH, rt, 24h.
3. Biological activity
R
O
N
O
We investigated the effect of new thymidine analogues 4-
0
(
a–j), thymidine, and 2 -deoxy-uridine on the growth of
HeLa cells using MTT (1-(4, 5-dimethylthiazol-2-yl)-3,5-
HN
R
S
O
17,18
O
diphenylformazan) assay.
thymidine analogues 4-(a–j) exhibited higher inhibitory
As shown in Table 1,
HO
AcO
protecting
group
HO
activity toward HeLa cell proliferation than those of
0
2 -deoxy-uridine and thymidine. Among these thymi-
dine analogues, 4a potently inhibited the proliferation
Figure 1. Synthesis strategy of several analogues of thymidine.
Table 1. Cell growth inhibitory activity of 5-methyl group modified thymidine analogues
O
O
O
N
HN
HN
HN
R
O
N
O
N
O
O
O
O
HO
HO
HO
HO
-Deoxy-uridine
HO
Thymidine
Compd
HO
2
4-(a-j)
R
Compd
IC50 (lM)
R
IC50 (lM)
R
Compd
IC50 (lM)
Br
0
2
-Deoxy-uridine
NA
Thymidine
1
4000
4a
15
O
O
O
Br
OCH
3
OCH CH
2 3
4
4
b
e
25
4c
4f
200
250
4d
4g
250
200
O
O
O
O
NO2
250
4
h
50
4i
50
4j
500
O
O
S
NA=not active.

K. N. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 77–79
79
Barry, D. W.; Broder, S. Proc. Natl. Acad. Sci. U.S.A.
985, 82, 7096.
. Kern, E. R.; Overall, J. C.; Glasgow, L. A. J. Infect. Dis.
973, 128, 290.
. DeClercq, E.; Descamps, J.; Desomer, P.; Barr, P. J.;
Jones, A. S.; Walker, R. T. Proc. Natl. Acad. Sci. U.S.A.
1
1
20
00
1
8
9
1
8
6
4
2
0
0
0
0
0
1
979, 76, 2947.
10. DeClercq, E.; DeGreef, H.; Wildiers, J.; De Jonge, G.;
Drochmans, A.; Descamps, J.; De somer, P. Br. Med. J.
1980, 281, 1178.
1. Kauhman, H. E.; Heidelberger, C. Science 1964, 145,
585.
1
0
5
10
15
20
Concentration (µM)
12. Heidelberger, C.; Griesbach, L.; Cruz, O.; Schnitzer, R. J.;
Grunberg, E. Proc. Soc. Exp. Biol. Med. 1958, 97, 470.
13. Mukherjee, K. L.; Boohar, J.; Wentand, D.; Ans-fied, F.
J.; Heidelberger, C. Cancer Res. 1963, 23, 49.
14. Anderson, A. S.; Hwang, J. T.; Greenberg, M. M. J. Org.
Chem. 2000, 65, 4648.
Figure 2. Cell viability assay. 4a (5–20lM) was applied to the cell and
incubated for up to 72h. The cells were then stained with trypan blue
and counted by hemocytometer.
1
5. Synthetic procedure for 4a; (compound Ac-4a); Com-
pound 3 (370mg, 0.959mmol) was dissolved in DMF
of HeLa cell with IC50 value of 15lM, suggesting that
the increased hydrophobic property at 5-methyl group
of pyrimidine may contribute to enhance biological
activity of the compound. From this result, we speculate
that the position of bromide in phenoxy ring and hydro-
phobic property of the substitution can affect the biolog-
ical activity of analogues. In addition, it would be
interesting to isolate the specific binding protein for
(
5mL, dry) containing K CO (132.5mg, 0.959mmol), 3-
2 3
bromo-phenol (112.8lL, 0.913mmol) at 0ꢁC. The reac-
tion mixture was preceded at room temperature for 5h.
The reaction mixture was diluted with ethyl acetate
(20mL), washed with brine, dried (MgSO ), and concen-
4
trated in vacuo. The crude product was purified by flash
column chromatography (1.2:1 = n-hexane–EtOAc) to
give Ac-4a as a solid in 21% yield. (Compound 4a);
Compound Ac-4a was dissolved in CHCl2 (3mL) and
MeOH (2mL) containing NaOMe (12mg, 0.153mmol) at
4
the effect on the viability of the tumor cells using the try-
a. The selected candidate, 4a, was further studied for
1
9,20
pan blue exclusion method.
0
ꢁC. After 24h at room temperature, the reaction mixture
was neutralized at 0ꢁC using 10% HOAc in MeOH and
concentrated in vacuo. The crude product was directly
At the end of the assay the relative cell viability was cal-
culated by comparison with total cells counted. Com-
pound 4a showed no cytotoxicity against HeLa cells
even at the concentration ranges in which the prolifera-
tion of the cells was inhibited by the compound (Fig. 2).
purified
(20:1 = CH Cl –MeOH) to give 4a as a solid in 97% yield.
by
flash
column
chromatography
2
2
1
16. Analytical data for Ac-4a; H NMR (200MHz, CDCl ) d
3
7.69 (s, 1H), 7.11 (m, 3H), 6.80 (m, 1H), 6.24 (dd,
J = 5.8Hz), 4.79 (q, 2H, J = 1.0Hz), 4.34–4.26 (m, 4H),
2
HRMS (FAB, M+Na) Calcd C H O N Br: 519.0379,
.61–1.99 (m, 2H), 1.22 (dd, 6H, J = 3.6Hz, J = 14Hz).
In conclusion, we synthesized a new thymidine analogue
with increased hydrophobic property at 5-methyl group
of pyrimidine and this compound will be a promising
candidate for development of thymidine based anti-
tumor agent. Target identification using biotinylated
2
0
21
8
1
2
found = 519.0381: 4a; mp 74–76ꢁC. H NMR (200
MHz, DMSO-d ) d 8.54 (s, 1H), 7.64–7.60 (m, 2H), 7.52
d, 1H, J = 6.0Hz), 7.38 (dd, 1H, J = 1.0Hz, J = 4.0Hz),
6
(
4
.17 (q, 1H, J = 2.0Hz), 3.95 (s, 1H), 2.9–2.7 (m, 2H).
4a will be our next challenge for deciphering its mode
of action in anti-proliferative activity of tumor cells.
HRMS (FAB, M+Na) Calcd C16
found = 435.167.
17 6 2
H O N Br: 435.017,
1
1
7. Shim, J. S.; Kim, D. H.; Jung, H. J.; Kim, J. H.; Ahn, J.
W.; Yoo, J. S.; Rho, J. R.; Shin, J. H.; Kwon, H. J. Bioorg.
Med. Chem. 2002, 10, 2987.
Acknowledgements
8. HeLa (cervical carcinoma) cells were maintained in
DMEM and were grown at 37ꢁC in a humidified
This study was supported by the National Research Lab-
oratory Grant from the Ministry of Science and Techno-
logy, Republic of Korea and the Brain Korea 21 Project.
2
atmosphere of 5% CO . Cell proliferation was measured
by using MTT assay. Briefly, HeLa cells were inoculated
3
at a density of 4 · 10 cells/well in 96-well plates. Various
doses of thymidine analogues were added to each well and
inoculated for 72h. MTT (50lM, 2mg/mL) was added
and the plate was incubated for 4h. The absorbance of
MTT-formazan was measured using a 540nm filter-
equipped mircroplate reader (Bio-Tek instruments, Inc.,
Winooski, VT).
References and notes
1
2
3
4
. London, L. J. Biol. Chem. 1929, 83, 793.
. Tipson, L. J. Biol. Chem. 1935, 109, 623.
. Lemieux, R. U. Can. J. Chem. 1961, 39, 116.
. Tollin, P.; Wilson, H. R.; Young, D. W. Nature 1968, 217,
19. Jung, H. J.; Lee, H. B.; Kim, C. J.; Rho, J. R.; Shin, J. H.;
Kwon, H. J. J. Antibiot. 2003, 56, 492.
1
148.
20. The effect of thymidine analogues on the viability of
cancer cells was investigated. HeLa cells were inoculated
5
6
7
. Bhimani, R.; Troll, W.; Gtunberger, D.; Frenkel, K.
Cancer Res. 1993, 53, 4528.
. Boorstein, R. J.; Teebor, G. W. Cancer Res. 1989, 49,
4
at a density of 5 · 10 cells/well in a 24-well plate. The cells
were cultivated and exchanged with fresh media. Thymi-
dine analogues (5–20lM) was applied to each well and
incubated for up to 72h. The cells were then stained with
trypan blue and counted by hemocytometer.
1
509.
. Mitsuya, H.; Wrinhold, K. J.; Furman, P. A.; St. Clair,
M. H.; Lehrman, S. N.; Gallo, R. C.; Bolognesi, D.;