4206
V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
Scheme 3. Reagents and conditions: (a) 3-(bromomethyl)benzaldehyde, CH3CN, Cs2CO3, 80 °C, 2 h; (b) THF/H2O, Pd(dppf)Cl2, Na2CO3, 80 °C, 2 h.
MLM stabilities despite the lower fraction unbound for 7d. In HeLa
cells, 7d inhibited both Aurora-A (p-T288 IC50 = 0.040 M) and
Aurora-B (p-HH3 IC50 = 0.330 M); potencies similar to those
l
l
observed with compound 7a. The direct attachment of a p-fluo-
rophenyl group to the N1-pyrazole was detrimental to Aurora inhi-
bition, with compound 7e inhibiting Aurora-A and Aurora-B with
IC50 values greater than 10 lM (Table 1). Cell growth inhibition
studies revealed that compounds 7b–d maintained the inhibitory
potencies seen with 7a; for example, 7d inhibited the growth of
SW620 and HCT116 cells (GI50 = 0.26 and 0.24
Likewise, 7b and 7c showed potent HCT116 cell growth inhibition
(GI50 = 0.20 and 0.13 M, respectively). We postulated that potent
lM, respectively).
l
cell growth inhibitory activity relative to biochemical Aurora-A
and Aurora-B modulation may be attributable to gain of off-target
kinase inhibition in this sub-series, although we acknowledge that
translation to more potent cellular inhibition of Aurora-A versus
Aurora-A biochemical potency may also be a contributing factor.
The potent cell-based activity prompted us to investigate kinome
selectivity profiles for this class of compound by screening 7a
Figure 2. Imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases co-crystallised
with Aurora-A: compound 15: PDB ID 2X6D;10 compound 16: PDB ID 4B0G.11
low oral bioavailability. In an attempt to improve the aqueous sol-
ubility, the Aurora inhibitory potency and kinase selectivity of this
sub-series, we explored the introduction of basic substituents such
as 1-methylpiperazine and pyrrolidine as well as replacement of
the phenyl ring with a more polar heterocycle. This approach
was guided by the ligand/protein interactions observed in the pro-
tein crystal structure of 7a, which showed that the N-benzyl sub-
stituent on the C7-pyrazole is oriented towards the P-loop
(Fig. 4). It was anticipated that the introduction of small sub-
stituents on the phenyl ring in 7a would be well tolerated without
altering its orientation in the kinase active site. Alternatively, we
envisaged that Thr217 in Aurora-A may be accessed via an appro-
priate pyrazole N1-benzyl derivatisation such as a bulky amido
substituent, which would form a favourable hydrogen bond inter-
action with the side chain hydroxyl of Thr217. Similar approaches
were previously applied by us in the design of selective inhibitors
of Aurora-A.12,16
and 7d in a 102-kinase panel at a concentration of 1 l
M.20
Indeed, both compounds inhibited a range of kinases including
ERK8, GSK3b, MLK1, JAK2, TrkA and VEGFR greater than 80%
(Table S1, Supplementary data) with Gini coefficients21 of 0.273
for 7a and 0.364 for 7d. These Gini coefficient values are signifi-
cantly lower to those of previously reported 7-(piperazin-1-yl)-
3H-imidazo[4,5-b]pyridine- and 7-phenoxy-3H-imidazo[4,5-b]
pyridine-based inhibitors of Aurora kinases.10,12 The Gini coeffi-
cient for compound 3 was reported as 0.719,12 and the correspond-
ing value for compound 1 is 0.560. To understand the potential for
further improvement, we obtained the crystal structure of 7a
bound to Aurora-A22 (Fig. 3, Table S3, Supplementary data). This
structure shows the ligand in the ATP binding site with the pyri-
dine nitrogen atom hydrogen bonded to the backbone NH of
Ala213 and the imidazole NH interacting with the carbonyl of
Ala213, consistent with previous reports of the hinge binding
mode for the imidazo[4,5-b]pyridine scaffold.10–12 Similar to crys-
tal structures of previous compounds 1, 15 and 16 (Figs. 1 and 2;
compounds 51 and 40c in Ref. 10, and compound 21a in Ref. 11,
respectively) the N-benzyl substituent on the C7-pyrazole is ori-
ented towards the P-loop, making Van der Waals contacts with
Val147 and Gly142 (Fig. 3).
In vivo mouse pharmacokinetic profiling of 7d revealed low oral
bioavailability (16%) with moderate clearance (0.016 L/h, 13.3 mL/
min/kg) and volume of distribution (0.02 L, 1.0 L/kg). Similar phar-
macokinetic parameters were observed for 7a with low oral
bioavailability (13%), moderate clearance (0.012 L/h, 10.0 mL/
min/kg) and volume of distribution (0.01 L, 0.5 L/kg). The mouse
plasma protein binding for 7a and 7d was determined as 99.48%
and >99.9%, respectively. We determined low kinetic solubility
for both 7a and 7d (<0.0001 mg/mL in phosphate buffer,
pH = 6.8)23 which may be a contributing factor to the observed
Replacement of the phenyl ring in 7a with 5-methylisoxazole
(compound 14d) led to a significant improvement in Aurora inhibi-
tion in our biochemical assays (Aurora-A IC50 = 0.035
IC50 = 0.075 M; Table 2) but promiscuous kinase inhibition
remained. In a panel of 105 kinases at a compound concentration
of 1 M, 22 proteins including ERK8, MLK1, JAK2, TrkA and
lM, Aurora-B
l
l
VEGFR were inhibited by P80% (Gini coefficient = 0.320,
Table S2, Supplementary data). Compound 14d inhibited the
growth of SW620 and HCT116 cells with GI50 values of 0.35 and
0.34 lM respectively, similar to the cell growth inhibition
observed with 7d. The crystal structure of 14d bound to Aurora-
A22,24,25 (Fig. 5, Table S3, Supplementary data) shows a similar
binding mode to that adopted by 7a; however, the P-loop of
Aurora-A adopts a different conformation, stabilised by interac-
tions with the 5-methylisoxazole; this P-loop conformation is very
similar to that previously observed for compound 1 (compound 51
in Ref. 10).
Bavetsias, Vassilios
