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C. Bardelle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5717–5721
the appropriate benzoic acid derivatives (see anilines 32, 43 and
52). For aniline 48 (Scheme 2, used in inhibitor 11) the aniline
moiety was introduced via Curtius rearrangement of acid 46. Ani-
line 52 (present in inhibitor 8) was introduced via reduction of a
nitro group installed via nitration of commercially available
sulfone acid 49.
Introduction of a second sulfone group to 2 to give bis-sulfone 6
led to comparable enzyme activity15 but was clearly not detrimen-
tal. When the sulfone was combined with a second aminosulfona-
1.5
1
0.5
0
-0.5
-1
mide group as in 7, an increase in enzyme activity to 0.012 lM was
observed, a figure significantly more active than the contribution
from each substituent on its own (in 2 and 3), and appears to lend
support to the strategy for combining substituents with comple-
mentary binding modes. Similarly compound 8, comprising a
sulfone favouring binding towards Glu697, and a primary carbox-
amide favouring binding to Ile621 resulted in an inhibitor signifi-
cantly more potent than either substituent alone (compare to 2
and 5). Other examples of the synergistic nature of specific combi-
nations can be found in compounds 11 (combining a morpholinyl
-1.5
-2
1
2
3
4
5
6
7
Number H Donors
1.5
1
and aminosulfonamide to give a 0.002
bining morpholinyl with carboxamide to give a 0.013
l
M inhibitor) and 15 (com-
M inhibi-
l
0.5
0
tor) both of whose C-2 anilines might be expected to be potent
in combination, from the structural studies outlined above. Simi-
larly the one group for which there was no overall binding prefer-
ence, the sulfonamide (as in 1) also shows potent activity in the
single combination examined (here with aminosulfonamide as in
-0.5
-1
10) at 0.001 lM.
Surprisingly, it was observed that many other combinations
also led to potent inhibition, including combinations which singly
at least, were shown to prefer the same binding orientation. The
most striking example of this is bis-morpholine 14 that in 4 favours
a single conformation, but in combination leads to a very potent
-1.5
-2
3
3.5
4
4.5
5
inhibitor, at 0.002 lM nearly 300-fold more active than with a sin-
CLogP
gle substituent. It is noteworthy that this substantial potency in-
crease occurs in tandem with a 0.5 log unit decrease in ClogP
relative to 4, following introduction of a second morpholine. Mor-
pholine in combination with a sulfone (as in 9) was highly active
despite both substituents independently showing a favoured bind-
ing towards Glu697 and similarly bis-aminosulfonamide 12 and
bis-carboxamide 16 were also very potent. The inhibitor with car-
boxamide and aminosulfonamide groups, 13, showed enzyme
activity intermediate between the contributions from each substi-
tuent in isolation (3 and 5). The majority of 3,5-disubstituted ani-
lines examined are not only tolerated, but positively enhance
enzyme inhibition potency. Despite our initial assumption that
only substituents with complementary binding modes might be
expected to enhance potency, it is clear from the data that two
meta-substituents are favoured over one, and the observation that
the global protein structure does not change to accommodate the
different aniline orientations may in part explain this.
In comparison with the enzyme data, the activity in a cellular
assay of EphB4 inhibition16 for these compounds highlights a much
broader spread of data, ranging from potent inhibition for 9 and 14
to activity higher than the top concentration tested for 13 and 16.
Analysis of the correlation of the difference in enzyme to cell activ-
ity and parameters such as number of hydrogen-bond donors and
lipophilicity (ClogP) is informative (Fig. 2). The cell activity is seen
to drop off rapidly as a function of increasing number of hydrogen-
bond donors or decreasing ClogP.17 These data may be indicative of
a problem of permeability (since adequate lipophilicity is required
for permeation and higher numbers of donors may impair perme-
ability across the cell membrane) or efflux (since increased hydro-
gen bonding capability may increase recognition by transporters)18
or both. Compounds 7 (large cell drop-off) and 14 (small cell drop-
off) were examined in the cell assay in the presence of an efflux
inhibitor.19 Consistent with active efflux, the cell activity of 7
Figure 2. Plots of Log (enzyme to cell) difference versus number of hydrogen-bond
donors and ClogP for compounds 1–16. Closed circles represent 3,5-disubstituted C-
2 anilines, stars represent 3-substituted anilines. Points on top line are out of range
values.
was increased to 0.175
of the already potent 14 remained unchanged at
0.010 0.001
M.20
lM in this system, whereas the cell activity
l
In summary, crystallographic studies of a range of 3-substituted
anilinopyrimidine inhibitors of EphB4 have highlighted two alter-
native aniline conformations are available in the active site of the
kinase. In an attempt to exploit these two interactions simulta-
neously, a set of 3,5-disubstituted anilinopyrimidines has been
prepared and these show potent enzyme inhibition. The observed
range of cellular activities has been rationalised on the basis of
physicochemical and structural characteristics, and has been
linked, for some inhibitors, to a potential for efflux. Further studies
on optimisation of both the C-2 and C-4 anilines are underway and
will be reported in due course.
Acknowledgments
We thank Jonathan Wingfield for technical assistance, Eileen
McCall, Isabelle Green and Anna Valentine for construct design, in-
sect cell expression and protein preparation, and Claire Brassington
for crystallisation.
Supplementary data
Supplementary data associated with this article can be found, in