Month 2013
Convenient Synthesis of Toxoflavin that Targets b-Catenin/Tcf4 Inhibit Activities
(
DMSO-d , 300 MHz): d 3.00 (s, 3H), 3.02 (s, 3H), 4.64 (s, 1H);
6
1
3
C NMR (DMSO-d , 75MHz): d 26.428, 40.716, 72.695,
6
150.816, 154.266, 163.336; ms: m/z 171.09 (M + 1).
1,6-Dimethyl-pyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-dione
(
1). The compound 10 (34 g, 0.2 mol) was suspended in 300 mL
ꢀ
acetic acid and 30 mL water. The mixture was cooled to 0–5 C,
and 37% aqueous formaldehyde (18 mL, 0.22 mol) was added
slowly to the mixture, and the solution was stirred at this
temperature for 30min to give the intermediate 11 monitored by
TLC. Saturate sodium nitrite (15.2g, 0.22mol) aqueous solution
was added slowly to the mixture, keeping the temperature
ꢀ
below 5 C. The mixture was slowly warmed to room
temperature and stirred overnight. Diethyl ether (800 mL)
was added to the mixture, and the resulting precipitate was
filtered and dried to afford the crude product 1, which was
recrystallized twice from 1-propanol (300 mL) to afford the
Figure 2. Cell (HCT116) viability assay of toxoflavin.
bright yellow solid toxoflavin (1) (13 g, 33%), with 98.6%
1
purity (HPLC). H NMR (DMSO-d
6
, 600 MHz): d 3.23 (s,
indicate this to be a new, convenient, and practical method
for preparation of compound 1.
1
3
3
1
1
6
H), 3.95 (s, 3H), 8.97 (s, 1H); C NMR (DMSO-d ,
25 MHz): d 28.144, 42.328, 144.971, 146.375, 150.821,
54.059, 158.924; ms: m/z 194.09 (M + 1). HPLC conditions:
Waters XBridge BEH130 C18 4.6 Â 250 Â 5 mm; Detection:
EXPERIMENTAL
ꢀ
2
54 nm; Flow rate: 1.0 mL/min; Temperature: 25 C; Injection
All commercially available chemicals and solvents were
load: 5 mL; Concentration: 0.5 mg/mL; Run time: 25 min; Mobile
phase A: water (0.01% TFA); Mobile phase B: 90% acetonitrile/
water (0.01% TFA); Gradient program: time (min): 0 18 20 25; %
of mobile phase A: 100, 0, 100, 100; % of mobile phase B: 0,
1
purchased and used as received without further purification. H
13
NMR and C NMR spectra were recorded with a Bruker-BioSpin
00/600MHz spectrometer (Bruker BioSpin Corporation, Billerica,
3
MA) using TMS as an internal standard. The mass spectra were
obtained from a Thermo Q-Tof micro spectrometer (Thermo Fisher
Scientific, Waltham, MA). The HPLC results were generated using
a Waters 2489 UV/Visible Detector and Waters 1525 Binary HPLC
Pump (Waters Corporation, Milford, MA). The colon cancer cell
line HCT116 was obtained from American Type Culture Collection
and cultured in RPMI 1640 (Hyclone, Thermal Scientific) supple-
mented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL
100, 0, 0; Retention time of 1: 8.715min.
5
Luciferase reporter gene assay.
HCT116 cells (1 Â 10 )
were transfected with TOP-FLASH, containing Tcf4 binding
sites or FOP-FLASH harboring mutant Tcf4 binding sites,
respectively. Four hours post-transfection, compound was
added, and luciferase activities were determined after 24 h using
“Firefly Luciferase Assay Kit” (Biotium, Inc. Hayward, CA).
4
Cell viability assay. HCT116 cells were plated at 1 Â 10 /
ꢀ
penicillin, and 100 mg/mL streptomycin. Cells were maintained in a
well in 96-well plate and incubated overnight at 37 C. Cells
ꢀ
humidified 5% CO
2
atmosphere at 37 C.
were treated with different concentrations of taxoflavin and
incubated for 72 h. Cell viability was measured by a CellTiter-Blue
Cell Vability assay (Promega).
1-Methyl-2,4,6(1H,3H,5H)-pyrimidinetrione (8). Methylurea
(
148 g, 2 mol), malonic acid (208 g, 2 mol), and acetic anhydride
ꢀ
(380 mL, 4.1 mol) were mixed and stirred at 70 C for 2h. The
resulting acetic acid was removed under reduced pressure, and
4
00 mL ethanol was added to the residue. The mixture was then
Acknowledgments. This work was supported by grants from the
Connolly Endowment/Hendricks Fund and the LUNGevity
Foundation.
stirred and cooled in ice-water bath. The resulting precipitate was
filtered off and washed with 100 mL ethanol. Obtained pale solid
8
1
(233 g, 82%). H NMR (DMSO-d , 300 MHz): d 3.03 (s, 3H),
6
3.56 (s, 2H), 11.31 (s, 1H); ms: m/z 143.06 (M + 1).
6
-Chloro-3-methyl-2,4(1H,3H)-pyrimidinedione (9). To a
REFERENCES AND NOTES
stirred mixture of the pyrimidinetrione 8 (213 g, 1.5 mol) in
phosphorus oxychloride (600 mL, 6.4 mol) at 0 C, water
10 mL, 0.55 mol) was added slowly, and the mixture was then
stirred at 80 C for 4 h. The excess phosphorus oxychloride was
removed under reduced pressure, and the residue was poured
into ice water. The resulting precipitate was collected by suction
ꢀ
[
1] Machlowitz, R. R. A.; Fisher, W. P.; McKay, B. S.; Tytell, A.
A.; Charney, J. Antibiot Chemother 1954, 4, 259.
2] Levenberg, B.; Liton, S. N. J Biol Chem 1966, 241, 846.
3] Firsov, A. A.; Geodakyan, S. V.; Lichinitser, M. R.; Shutka, V.
Y. Antibiot Med Biotechhnol 1985, 30, 604.
(
ꢀ
[
[
filtration and washed with 100 mL water. Gave 9 (195 g, 81%)
[4] Nagamatsu, T.; Yamasaki, H.; Hirota, T.; Yamato, M.; Kido,
Y.; Shibata, M.; Yoneda, F. Chem Pharm Bull 1993, 41, 362.
1
as a white solid. H NMR (DMSO-d
6
, 300 MHz): d 3.07 (s,
[5] Nagamatsu, T.; Yamagishi, Y.; Yoneda, F. Japan Patent
3
H), 5.87 (s, 1H), 12.35 (s, 1H); ms: m/z 161.03 (M + 1).
-Methyl-6-(1-methylhydrazinyl)-2,4(1H,3H)pyrimidinedione
10). A mixture of the pyrimidinedione 9 (80 g, 0.5 mol) was
09255681, 1997.
3
[
[
6] Barker, N.; Clevers, H. Nat Rev Drug Discov 2006, 512, 997.
7] Lepourcelet, M.; Chen, Y.-N.; France, D. S.; Wang, H.; Crews,
(
suspended in ethanol (1 L) plus methylhydrazine (59 mL, 1.1 mol)
and heated at reflux for 2 h. After cooling the solution to room
temperature, the resulting precipitate was filtered and dried to
P.; Petersen, F.; Bruseo, C.; Wood, A. W.; Shivdasani, R. A. Cancer Cell
004, 5, 91.
[8] Wei, W.; Chua, M. S.; Grepper, S.; So, S. Int J Cancer 2010,
126, 2426.
2
1
give the faint yellow solid product 10 (56 g, 65%). H NMR
Journal of Heterocyclic Chemistry
DOI 10. 1002/jhet