to the hydrophobic pocket described earlier, as a consequence
possibly increasing its inhibitory activity.
In conclusion, the results indicate that 4a binds the active site
metal of the HIF hydroxylase (at least for FIH) via its oxo-
carboxylate functionality rather than its bipyridyl group. They also
indicate that the tricyclic ring system can be reduced in size to achieve
a quinoline structure, suitable for further modification with a view
to obtaining selective inhibitors of individual HIF hydroxylases.
We thank the European Commission (A.C-G. Marie Curie Pro-
gramme MEIF-CT-2003-500525), a Glasstone Fellowship (K.S.H),
BBSRC and the Wellcome Trust for financial support. We also
thank Michael Nagle for assistance with the quantum mechanical
calculations and Benoit M. Lienard for helpful discussions.
Scheme 2 Reagents and conditions: (i) heat, 1 h, 80–85%; (ii) Ph2O,
reflux, 1–1.5 h, 70–75%; (iii) 10% KOH, reflux, 0.5–1 h, 95%. R 5 H for
5a–8a; NHCOMe for 5b–7b; NH2 for 8b; NHCOPh for 5c–8c; CONHPh
for 5d–8d; CONHBn for 5e–8e and CN for 5f–8f.
Notes and references
1 R. K. Bruick, Genes Dev., 2003, 17, 2614; M. Safran and W. G. Kaelin,
J. Clin. Invest., 2003, 111, 779; G. L. Semenza, Physiology, 2004, 19, 176;
C. J. Schofield and P. J. Ratcliffe, Nat. Rev. Mol. Cell. Biol., 2004, 5, 343.
2 P. Jaakkola, D. R. Mole, Y. M. Tian, M. I. Wilson, J. Gielbert,
S. J. Gaskell, A. von Kriegsheim, H. F. Hebestreit, M. Mukherji,
C. J. Schofield, P. H. Maxwell, C. W. Pugh and P. J. Ratcliffe, Science,
2001, 292, 468; M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando,
M. Ohh, A. Salic, J. M. Asara, W. S. Lane and W. G. Kaelin, Science,
2001, 292, 464.
3 R. K. Bruick and S. L. McKnight, Science, 2001, 294, 1337;
A. C. R. Epstein, J. M. Gleadle, L. A. McNeill, K. S. Hewitson,
J. O’Rourke, D. R. Mole, M. Mukherji, E. Metzen, M. I. Wilson,
A. Dhanda, Y. M. Tian, N. Masson, D. L. Hamilton, P. Jaakkola,
R. Barstead, J. Hodgkin, P. H. Maxwell, C. W. Pugh, C. J. Schofield
and P. J. Ratcliffe, Cell, 2001, 107, 43; L. A. McNeill, K. S. Hewitson,
J. Gleadle, L. E. Horsfall, N. J. Oldham, P. Maxwell, C. W. Pugh,
P. J. Ratcliffe and C. J. Schofield, Bioorg. Med. Chem. Lett., 2002, 12,
1547.
4 D. Lando, D. J. Peet, D. A. Whelan, J. J. Gorman and M. L. Whitelaw,
Science, 2002, 295, 858; L. A. McNeill, K. S. Hewitson,
T. D. W. Claridge, J. F. Seibel, L. E. Horsfall and C. J. Schofield,
Biochem. J., 2002, 367, 571.
5 M. Ivan, T. Haberberger, D. C. Gervasi, K. S. Michelson, V. Gu¨nzler,
K. Kondo, H. Yang, I. Sorokina, R. C. Conaway, J. W. Conaway and
W. G. Kaelin, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 13459.
6 K. S. Hewitson, L. A. McNeill, M. V. Riordan, Y. M. Tian,
A. N. Bullock, R. W. D. Welford, J. M. Elkins, N. J. Oldham,
S. Battacharya, J. Gleadle, P. J. Ratcliffe, C. W. Pugh and C. J. Schofield,
J. Biol. Chem., 2002, 277, 26351; D. Lando, D. J. Peet, J. J. Gorman,
D. A. Whelan, M. L. Whitelaw and R. K. Bruick, Genes Dev., 2002, 16,
1466; P. C. Mahon, K. Hirota and G. L. Semenza, Genes Dev., 2001, 15,
2675.
Fig. 5 The effect of 1 mM test compounds 2–4 and 6–8 on the activity of
FIH.
The 4-oxo-7-substituted-quinoline-3-carboxylic acid derivatives
were synthesized following the same general route (Scheme 2)
employing an addition–elimination reaction of diethylethoxy-
methylene malonate with aniline derivatives (for their syntheses
see supplementary information){ to give enamines (6b to 6f)
followed by cyclisation to the corresponding quinolines (7b to 7f)
and saponification to give acids 8b to 8f. In the hydrolysis of 7f, a
trace amount of di-acid was also observed resulting from the
hydrolysis of the nitrile group. Compounds 6 to 8 were then
screened alongside 4 in assays against FIH (Fig. 5) using a
fluorescence based assay.13
7 J. M. Elkins, K. S. Hewitson, L. A. McNeill, J. F. Seibel,
I. Schlemminger, C. W. Pugh, P. J. Ratcliffe and C. J. Schofield,
J. Biol. Chem., 2003, 278, 1802.
8 D. R. Mole, I. Schlemminger, L. A. McNeill, K. S. Hewitson,
C. W. Pugh, P. J. Ratcliffe and C. J. Schofield, Bioorg. Med. Chem.
Lett., 2003, 13, 2677.
9 M. A. McDonough, L. A. McNeill, M. Tilliet, C. A. Papamica¨el,
Q. Y. Chen, B. Banerji, K. S. Hewitson and C. J. Schofield, J. Am.
Chem. Soc., 2005, 127, 7680.
10 T. J. Franklin, W. P. Morris, P. N. Edwards, M. S. Large and
R. Stephenson, Biochem. J., 2001, 353, 333.
11 M. Hirsila¨, P. Koivunen, V. Gu¨nzler, K. I. Kivirikko and J. Myllyharju,
J. Biol. Chem., 2003, 278, 30772.
12 H. R. Snyder and F. H. Freier, J. Am. Chem. Soc., 1946, 68, 1320;
J. K. Shah and A. E. Coats, J. Med. Chem., 1977, 20, 1001; F. C. K. Chiu,
R. T. C. Brownlee and D. R. Phillips, Tetrahedron, 1994, 50, 889.
13 L. A. McNeill, L. Bethge, K. S. Hewitson and C. J. Schofield, Anal.
Biochem., 2005, 336, 125.
14 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart,
R. K. Belew and A. J. Olson, J. Comput. Chem., 1998, 19, 1639.
15 H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat,
H. Weissig, I. N. Shindyalov and P. E. Bourne, Nucleic Acids Res., 2000,
28, 235.
As expected, the ester derivatives 3 and 7 displayed little activity
against FIH, as in general did the diesters (2 and 6); the low levels
of activity observed for some of these compounds (e.g. 6b and 6e)
may have resulted from hydrolysis under the incubation condi-
tions. For all the 7-substituted quinoline-3-carboxylic acids (8b to
8f), only the cyanide 8f showed a better activity than the
7-unsubstituted quinoline 8a, with an IC50 of 150 mM.
Docking studies revealed that, as for 4a, 4b and 8a, only one
binding mode was found for 8f (Fig. 4). In contrast, similar
analysis for compounds 8c, 8d and 8e implied more than one
binding mode; most of which predict binding overlapping with the
site occupied by Val-802, Asn-803 and Ala-804 of the HIF
substrate (see supplementary information).{ The reduced activity
for 8c and 8d may thus reflect competition with the HIF-a
substrate used in the assays (as well as with 2OG). The flexibility of
the N-benzylamide 8e may allow it to avoid HIF by conforming
5440 | Chem. Commun., 2005, 5438–5440
This journal is ß The Royal Society of Chemistry 2005