512
A. A. Thomas et al. / Bioorg. Med. Chem. Lett. 18 (2008) 509–512
Table 3. SAR for pyrazolothiamine analogs 14–16
References and notes
Compound Structure
HCT-116
CellTK
TPPK kcat
/
Km (nMÀ1 sÀ1
)
b
1. (a) Cascante, M.; Centelles, J. J.; Veech, R. L.; Lee, W.-N.
P.; Boros, L. G. Nutr. Cancer 2000, 36, 150; (b) Boros, L.
G.; Torday, J. S.; Lim, S.; Bassilian, S.; Cascante, M.; Lee,
W.-N. P. Cancer Res. 2000, 60, 1183.
a
EC50 (lM)
2. (a) Du, M. X.; Sim, J.; Fang, L.; Zheng, Y.; Koh, S.;
Stratton, J.; Pons, J.; Wang, J. J.-X.; Carte, B. J. Biomol.
Screen 2004, 9, 427; (b) Solovjeva, O. N.; Kochetov, G. A.
FEBS Lett. 1999, 462, 246; (c) Boros, L. G. Cancer Res.
1998, 58, 3188; (d) Boros, L. G.; Puigjaner, J.; Cascante,
M.; Lee, W.-N. P.; Brandes, J. L.; Bassilian, S.; Yusuf, F.
I.; Williams, R. D.; Muscarella, P.; Melvin, W. S.;
Schirmer, W. J. Cancer Res. 1997, 57, 4242; (e) Kochetov,
G. A.; Izotova, A. E.; Meshalkina, L. E. Biochem.
Biophys. Res. Commun. 1971, 43, 1198.
14
15
17
—
1.1
1.9 · 10À5
16
4.7
—
3. Voskoboyev, A. I.; Ostrovsky, Y. M. Ann. N.Y. Acad. Sci.
1982, 378, 161.
4. Kandiko, C. T.; Smith, D.; Yamamoto, B. K. Biochem.
Pharmacol. 1988, 37, 4375.
5. Timm, D. E.; Liu, J.; Baker, L.-J.; Harris, R. A. J. Mol.
Biol. 2001, 310, 195.
6. Gutowski, J. A.; Lienhard, G. E. J. Biol. Chem. 1976, 251,
2863.
a Average EC50, lM, for cellular assay performed in triplicate. Data
were within 4-fold of the mean.
s
b Average kcat/Km, nMÀ1 À1, for enzymatic assay performed in trip-
licate. Data were within 2-fold of the mean.
7. Kluger, R.; Gish, G.; Kauffman, G. J. Biol. Chem. 1984,
259, 8960.
thiamine transporters leading to improved cell potency.
Also, pyrazole analogs would be amenable to further
substitutions on the B-ring including extension of func-
tionalities into the substrate-binding region of TK,
which might allow for improved potency. As with the
thiazolones and deazathiamines, the pyrazoles possessed
TPPK activity (Table 3). Unfortunately, they demon-
strated only modest activity in the tumor cell assay
(HCT-116).
8. Though it is possible that the cellular activity of non-
pyrophosphates such as compound 3, N3P-TT, is a result
of their direct interaction with TK, we believe it is much
more likely that they are first converted by TPPK inside
the cell into their pyrophosphate forms. The pyrophos-
phate binding to TK is greater by several orders of
magnitude than the corresponding non-pyrophosphate
analogs (Table 1).
9. Synthesis of starting material N3PT is presented in the
preceding publication, Thomas, et al.
Taken together, these results support our hypothesis
that non-permanently charged thiamine mimetics are
competent substrates for TPPK, and that the resulting
pyrophosphates antagonize the activity of TK in vitro.
For the first time, low micromolar non-pyrophosphate
TK inhibitors were identified in a deazathiamine series.
Despite remarkable potencies in enzymatic assays, cellu-
lar potencies were modest to poor. An explanation for
the discrepancy between the two assays is not obvious,
although one might surmise that these analogs do not
achieve sufficient intracellular concentrations to com-
pete with endogenous thiamine pyrophosphate for TK.
Future work in this area will attempt to improve cell po-
tency by modifying their physiochemical properties and
incorporating functionalities which might enable recog-
nition by thiamine transporters.
10. Hawksley, D.; Griffin, D. A.; Leeper, F. J. J. Chem. Soc.,
Perkin Trans. 1 2001, 144.
11. Fevig, T. L.; Phillips, W. G.; Lau, P. H. J. Org. Chem.
2001, 66, 2493.
12. We would expect entropy differences related to structured
water around the more polar thiazolone ring versus the
thiophene ring. These entropy differences often lead to
increased protein–ligand interactions. Andrews, P. R. In
The Practice of Medicinal Chemistry; Wermuth, C. G.,
Ed.; Academic Press: London, 1996; p 349.
13. Prior to performing the TPPK/Apo-TK assay, each
compound was evaluated for its ability to be pyrophosph-
orylated by TPPK using thiamine as a control substrate.
The extent of substrate pyrophosphorylation relative to
thiamine pyrophosphorylation was determined prior to
competition with TPP in inhibiting TK activity according
to methods described in WO2005/095391.
14. Starting material (Z)-ethyl 5-(benzyloxy)-2-[(dimethyl-
amino)methylene]-3-oxopentanoate was prepared in two
steps from ethyl acetoacetate (50% yield). J. Org. Chem.
1986, 51, 106.
Supplementary data
15. Either N-methylated pyrazole regioisomer can be prepared
in a 3:1 regioselectivity by choice of methanol or diethyl
ether as solvent Menozi, G. et al. J. Heterocycl. Chem.
1987, 24, 1669.
Procedures and analytical data are available for com-
pounds 2, 3, 5, and 6–16. Supplementary data associated
with this article can be found, in the online version, at