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J. Mecinovic et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6192–6195
6194
troscopy, thermal analysis and Raman spectroscopy.25 The com-
bined use of these techniques revealed that oxamides preferentally
possess a trans geometry because of intramolecular hydrogen bond-
ing. Crystallographic analyses also revealed that when N-cyclopen-
tyl-N-(thiazol-2-yl)oxalamide forms a complex with methionine
aminopeptidase and a Co(II) ion, its oxamide moiety possesses a
trans geometry when chelates the Co(II) ion.26 Thus, the lack of
observed binding to PHD2 of 4 and 5 may, at least in part, be due
to the prefered trans geometry of the diamide moiety in these two
compounds. This issue does not arise with N-oxalylacids or 2-oxoac-
ids. A cyclic diamide 7 also did not form a complex with PHD2 (Sup-
plementary Fig. 2); the steric demand of 7 may play a role in its lack
of observed binding.
Overall, several new types of 2OG analogues have been evalu-
ated as inhibitors of PHD2. Among the newly identified inhibitors,
two new scaffolds may be promising for further development into
more potent inhibitors. 4,6-Dioxoheptanoic acid 10 and hydroxa-
mic acid 8 were shown to efficiently inhibit PHD2, whilst the other
tested 2OG analogues displayed weaker or no inhibition activity
against PHD2. In particular 10 could be modified by C-5 derivatisa-
tion. Binding studies employing non-denaturing ESI-MS analysis
provided further evidence that the more efficient inhibitors (1, 2,
3, 8, 10) formed strong complexes with PHD2, whereas weak inhib-
itors showed a decreased binding affinity. Thus, the results also
further validate the use of non-denaturing ESI-MS for assuming
binding affinities of small molecules to enzymes.23,28
Hydroxamic acids 8 and 9 displayed different affinities for bind-
ing to PHD2 by ESI-MS. Whilst 8 displayed a significant ability to
form a complex PHD2.Fe.8 at 28243 Da, the latter did not bind
(Supplementary Fig. 2). These observations are in agreement with
the turnover inhibition data showing that hydroxamic acid 8, un-
like 9, inhibited PHD2.
Acknowledgements
Financial support from the Newton-Abraham Fund, the Rhodes
Trust, the Commonwealth Scholarship Scheme, the Biotechnology
and Biological Sciences Research Council, and the Wellcome Trust
is gratefully acknowledged.
The ESI-MS results also revealed that 4,6-dioxoheptanoic acid
10 formed
a strong complex at 28268 Da corresponding to
PHD2.Fe.10, while its analogue 11, containing an amide instead
of a 4-carbonyl group, displayed a much reduced ability to form
a PHD2.Fe.11 complex (Fig. 2). The ability of 10 to form a strong
complex (by ESI-MS) and inhibit PHD2 can in part be explained
by its acidic C-5 methylene, that facilitates bidentate iron chelation
in an analogous manner to 2OG (Fig. 1), but via a 6-, rather than a
5-, membered chelate ring. Malonic acid derivatives 11–13 also
formed moderately stable complexes with PHD2. However, the
ethyl ester 14 formed only a very weak complex (Fig. 2). Notably,
the ESI-MS studies revealed that the tricarbonyl compound 15
underwent ferric iron-mediated cleavage to produce NOG in the
absence of PHD2 (see Ref. 27 for detailed studies on this reaction),
exemplifying the use of this technique for monitoring the
reaction.27
Supplementary data
Supplementary data (mass spectra and synthetic procedures)
associated with this article can be found, in the online version, at
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Figure 2. Non-denaturing ESI-MS spectra for PHD2 in the presence of equimolar
amounts of FeSO4 and inhibitors (a) 10; (b) 11; (c) 12; (d) 13; (e) 14; (f) 15. Peaks
present: (A) PHD2.Fe; (B) PHD2.Fe.10; (C) PHD2.Fe.11; (D) PHD2.Fe.12; (E)
PHD2.Fe.13; (F) PHD2.Fe.14; (G) PHD2.Fe.1.