1336
L.-X. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1333–1337
and B10 versus B . On the other hand, B maintained
12
5. (a) Cozzarelli, N. R. Cell 1980, 22, 327. (b) Liu, L. F. Crit.
Rev. Biochem. 1983, 15, 1. (c) Kafiani, C. A.; Bronstein,
I. B.; Timofeen, A. V.; Gromova, I. I.; Terskikh, V. V.
Adv. Enzyme Regul 1986, 25, 439. (d) Zijlstra, J. G.; de
Jong, S.; de Vries, E. G. E.; Mulder, N. H. Med. Oncol.
Tumor Pharmacother. 1990, 7, 11.
6
moderate topoisomerase I inhibitory activity on both
wheat germ and calf thymus topoisomerase I. In addition,
the 6-substituent effect on topoisomerase I inhibition
was not evident.
6
¨
. (a) Zecher, W.; Krohnke, F. Chem. Ber. 1961, 94, 690. (b)
9
For the evaluation of antitumor cytotoxicity, four dif-
ferent human tumor cell lines were utilized: A-549
(
Krohnke, F. Angew. Chem., Int. Ed. 1963, 2, 380. (c)
hnke, F. Synthesis 1976, 1.
¨
Kro
¨
human lung carcinoma), HCT-15 (human colon adeno-
7
. Fukuda, M.; Nishio, K.; Kanzawa, F.; Ogasawara, H.;
Ishida, T.; Arioka, H.; Bojanowski, K.; Oka, M.; Saijo,
N. Cancer Res. 1996, 56, 789. The topoisomerase I inhib-
itory activity was carried out as follows: The activity of
DNA topoisomerase was determined by measuring the
relaxation of supercoiled DNA pBR322. For measure-
ment of topoisomerase activity, the reaction mixture was
comprised of 35 mM Tris–HCl (pH 8.0), 72 mM KCl, 5
carcinoma), SK-OV-3 (human ovary adenocarcinoma),
SK-MEL-2 (human malignant melanoma). Cytotoxicity
of prepared compounds against human cancer cell
lines indicated that moderate cytotoxicities (EC50 value
of 10.4–118.1 mM) were observed for all prepared
compounds except B (EC value of 0.01–0.03 mM).
4
50
2
mM MgCl , 5 mM DTT, 5 mM spermidine, 0.01%
In conclusion, we have designed an efficient synthetic
route to prepare thirteen 2,4,6-trisubstituted pyridine
derivatives and evaluated them for their inhibitory
activity of topoisomerase I. This was the first report of
the topoisomerase I inhibitory activity of 2,4,6-trisub-
stituted pyridine compounds. The structure–activity
relationship analysis revealed that the 2-thienyl-4-furyl-
pyridine skeleton exhibited strong topoisomerase I inhi-
bitory activity. Further structure–activity relationship
studies are on progress. This study may provide valuable
information to the researchers who are working on the
development of antitumor agents, especially that of
topoisomerase I inhibitors.
bovine serum albumin (BSA), 1 mg pBR322, and 2 U
DNA topoisomerase. Topoisomerase I inhibitors (pre-
pared compounds) were added to the above component
for measuring the inhibition of DNA relaxation.
ꢁ
The reaction mixture was incubated at 37 C for 30
min. The reactions were terminated by adding dye solution
comprising 1% SDS, 0.02% bromophenol blue and 50%
glycerol. The mixture was applied to 1% agarose gel and
electrophoresed for 1 h with a running buffer of Tris-ace-
tate EDTA. The gel was stained with ethidium bromide.
8
2 6
. The spectral data of B , B , B10 and B13:
B
2
: TLC (EtOAc:n-hexane=1:5, v:v), R =0.2, mp 83.1–
f
ꢁ
8
1
4.6 C.
H NMR (250 MHz, CDCl ) d 8.53 (dd, J=5.3, 0.8 Hz,
3
1
H, pyridine H-6), 7.87 (dd, J=1.5, 0.8 Hz, 1H, pyridine
H-3), 7.65 (dd, J=3.7, 1.1 Hz, 1H, thiophene H-5), 7.55
(dd, J=1.8, 0.6 Hz, 1H, furan H-5), 7.40 (dd, J=5.1, 1.1
Hz, thiophene H-3), 7.34 (dd, J=5.3, 1.5 Hz, pyridine H-
Acknowledgements
5
J=3.4, 0.6 Hz, 1H, furan H-3), 6.53 (dd, J=3.4, 1.8 Hz,
), 7.12 (dd, J=5.1, 3.7 Hz, 2H, thiophene H-4), 6.90 (dd,
This work was supported by a grant (KRF-2002-005-
E00019) from the Korea Research Foundation, R.O.K.
1
H, furan H-4).
1
3
3
C NMR (62.5 MHz, CDCl ) d 153.03, 151.21, 149.87,
1
1
44.72, 143.76, 138.07, 127.97, 127.60, 124.63, 116.06,
12.68, 112.08, 108.79.
References and notes
+
ESI LC/MS [MH] 228.
1
. (a) Mukkala, V. M.; Helenius, M.; Hemmila, I.; Kankare, J.;
Takalo, H. Helv. Chim. Acta 1993, 76, 1361. (b) Mukkala,
V. M.; Kwiatkowski, M.; Kankare, J.; Takalo, H. Helv.
Chim. Acta 1993, 76, 893. (c) Lowe, G.; Droz, A. S.; Park,
J. J.; Weaver, G. W. Bioorg. Chemistry 1999, 27, 477.
. (a) Jennette, K.; Lippard, S. J.; Vassiliades, G.; Bauer, W.
Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 3839. (b) Liu, H.
Q.; Cheung, T. C.; Peng, S. M.; Che, C. M. J. Chem. Soc.,
Chem. Commun. 1995, 1787. (c) McCoubrey, A.; Latham,
H. C.; Cook, P. R.; Rodger, A.; Lower, G. FEBS Letters
HPLC condition: column: C18 reverse phased, 1.5ꢂ150
mm, 5 mm, GL science, flow rate: 180 mL/min, injection
volume: 5 mL of 100 mM solution, mobile phase: 0.1%
formic acid in water (A), 0.1% formic acid in acetonitrile
(B), 10% B in A to 90% B in A for 10 min and retaining
for 10 min at 90% B in A, retention time: 15 min.
MS ionization condition: Sheath gas flow rate: 70 arb,
Aux gas flow rate: 20 arb, I spray voltage: 4.5 KV, Capil-
2
ꢁ
lary Temp.: 215 C, Capillary voltage: 21 V, Tube lens
offset: 10 V.
1996, 380, 73. (d) Vliet, P. M. V.; Toekimin, M. S.;
Haasnoot, J. G.; Reedijk, J.; Novakova, O.; Vrana, O.;
Brabec, V. Inorg. Chim. Acta 1995, 231, 57. (e) Carter,
P. J.; Cheng, C. C.; Thorp, H. H. J. Am. Chem. Soc. 1998,
The other compounds were followed the same above
conditions for ESI LC/MS, and showed almost the same
retention time for each compound.
´
´
´
B : TLC (EtOAc:n-hexane=1:5, v:v), R =0.3, mp 129.8–
6
f
ꢁ
120, 632.
130.8 C.
1
3
. Lowe, G.; Droz, A. S.; Vilaivan, T.; Weaver, G. W.;
Tweedale, L.; Pratt, J. M.; Rock, P.; Yardley, V.; Croft,
S. L. J. Med. Chem. 1999, 42, 999.
. (a) Kim, D. S. H. L.; Ashendel, C. L.; Zhou, Q.; Chang,
C. t.; Lee, E. S.; Chang, C.-j. Bioorg. Med. Chem. Lett.
H NMR (250 MHz, CDCl
3
) d 7.80 (s, 2H, pyridine H-3,
H-5), 7.57–7.55 (m, 3H, 2-furan H-5, 4-furan H-5, 6-furan
H-5), 7.17 (d, J=3.4 Hz, 2H, 2-furan H-3, 6-furan H-3),
6.96 (d, J=3.3 Hz, 1H, 4-furan H-3), 6.56–6.53 (m, 3H, 2-
furan H-4, 4-furan H-4, 6-furan H-4).
4
1
3
1
998, 8, 2695. (b) Xu, W. C.; Zhou, Q.; Ashendel, C. L.;
Chang, C. t.; Chang, C.-j. Bioorg. Med. Chem. Lett. 1999,
, 2279. (c) Zhao, L. X.; Kim, T. S.; Ahn, S. H.; Kim,
3
C NMR (62.5 MHz, CDCl ) d 153.63, 151.51, 149.66,
143.72, 143.29, 138.73, 112.10, 112.02, 110.85, 109.10,
108.87.
9
+
T. H.; Kim, E. k.; Cho, W. J.; Choi, H.; Lee, C. S.; Kim,
J. A.; Jeong, T. C.; Chang, C.-j.; Lee, E. S. Bioorg. Med.
Chem. Lett. 2001, 11, 2659.
ESI LC/MS [MH] 278.
B10: TLC (EtOAc:n-hexane=1:5, v:v), R
104.3 C.
=0.2, mp 102.5–
f
ꢁ