over 
 the past decade there has been extensive interest in aurora 
 kinases as anticancer targets .
aurora proteins , a family of three 
 kinases designated as aurora - a , -b , and -c , play key and distinct 
 roles in different stages of mitosis and are overexpressed in a wide range of human malignancies .
research toward the discovery of aurora kinase modulators 
 as cancer 
 therapeutics led to the identification of a significant number of 
 small - molecule inhibitors of aurora kinases , the majority of which 
 inhibit all three isoforms a , b , and c. compounds displaying isoform selectivity have also been reported , 
 including azd1152 , a selective aurora - b 
 inhibitor , and 1,2,3,4 , and 5 , which selectively 
 inhibit aurora - a ( figure 1 ) .
to isoform selectivity , the ideal inhibitor profile ( i.e. , selective 
 isoform inhibition versus pan - aurora inhibition ) is still unclear , 
 but the ongoing clinical evaluation of aurora inhibitors may provide 
 new insights to better answer this question . however , it should be 
 noted that the aurora kinases have distinct cellular functions and 
 their inhibition has divergent effects on cells undergoing mitosis .
aurora - a is involved in the initiation of mitosis 
 by promoting centrosome maturation and bipolar mitotic spindle formation , 
 and its inhibition leads to increased g2/m accumulation and spindle 
 defects .
aurora - b is 
 part of the chromosomal passenger complex ( cpc ) , which also includes 
 borealin , survivin , and the inner centromere protein ( incenp ) , and 
 plays a critical role in the regulation of chromosome alignment and 
 also in cytokinesis .
inhibition of aurora - b 
 leads to abrogation of the spindle assembly checkpoint , so that cells 
 rapidly undergo chromosome segregation even in the presence of spindle 
 defects .
the effects of aurora - b inhibition 
 mask the effects of aurora - a inhibition , and therefore , selective 
 inhibitors of both aurora - a and aurora - b are needed to carry out detailed 
 cell - based and in vivo studies on this kinase family .
the role of aurora - c during mitosis is not well understood ; 
 however , it is established that aurora - c plays a specific role in 
 spermatogenesis .
aurora - a alone forms a complex with n - myc , an oncoprotein that induces 
 neuroblastoma , which protects n - myc from proteosomal degradation , and aurora - a - selective inhibitors mln8054 and mln8237 disrupt the 
 aurora - a / n - myc complex .
significantly , 
 the aurora - a / n - myc complex was not disrupted by the aurora - a - selective 
 inhibitor mk-5108 , which belongs to a different chemotype .
this demonstrates the importance of having access 
 to a range of structurally diverse selective aurora isoform inhibitors , 
 in addition to the existing pan - aurora modulators . they can serve 
 as useful small - molecule chemical tools to further explore the cellular 
 function of aurora proteins toward their eventual therapeutic application 
 in cancer treatment
we have previously reported the discovery 
 of novel imidazo[4,5-b]pyridine derivatives as potent , 
 orally bioavailable inhibitors 
 of aurora kinases ( figure 2 ) , with a compound in this series being identified as a dual aurora / flt3 
 preclinical development candidate . in 
 parallel to this work , we also investigated the design and synthesis 
 of aurora - a - selective inhibitors based on the imidazo[4,5-b]pyridine scaffold .
herein , we report our medicinal chemistry 
 program aimed at the identification of imidazo[4,5-b]pyridine- and 7-azaindole - based inhibitors displaying a high degree 
 of selectivity for aurora - a over aurora - b .
the synthesis of 7-azaindoles 16a  d is shown in schemes 2 and 3 , with the key step being formation 
 of the azaindole ring 
 via a pd - catalyzed heteroannulation reaction . starting 
 from 8 , access was readily gained to the 
 alkyne 10 using standard boc protection followed by a 
 sonogashira cross - coupling reaction ( scheme 1 ) .
5-chloro-3-iodo-2-aminopyridine ( 11 ; scheme 2 ) was obtained from 2-amino-5-chloropyridine 
 upon treatment with n - iodosuccinimide and p - toluenesulfonic acid .
azaindole ring formation ( compound 12 ) was effected by reacting 11 with alkyne 10 in the presence of dabco and pd(pph3)2cl2 ( scheme 2 ) .
the silyl / iodo 
 exchange proceeded in good yield using n - iodosuccinimide 
 as previously described .
the final products 16a , b were then obtained 
 via a suzuki cross - coupling reaction ( scheme 2 ) .
starting 
 from key intermediate 14 , access to the acetamide 
 derivatives 16c , d ( scheme 3 , table 1 ) was accomplished 
 by acetylation of the amino functionality in 14 followed 
 by the introduction of 1-methylpyrazol-4-yl and 4-methoxylphenyl via 
 a suzuki cross - coupling reaction ( scheme 3 ) .
the imidazo[4,5-b]pyridine derivatives 21a  f , 28a  c , and 40a  f ( tables 2 , 3 , and 5 ) 
 and 34 ( table 4 ) were prepared 
 by reacting 
 1,3-dimethyl-1h - pyrazole-4-carbaldehyde with the 
 requisite 2-amino-3-nitropyridine precursor in the presence of na2s2o4 as reported previously ( schemes 49 ) .
the key 2-amino-3-nitropyridine intermediates 20a , c , d , f were obtained by nucleophilic displacement of the c4-chloride of 
 4,5-dichloro-3-nitropyridin-2-amine ( 18 ) with the appropriately 
 substituted pyrrolidine ( schemes 4 and 5 ) or piperazine ( scheme 6 ) in the presence of diisopropylethylamine in 2-propanol . 

intermediates 20b and 20e were obtained 
 by procedures analogous to those described for their enantiomeric 
 forms 20a and 20d , respectively .
starting 
 from 4,5-dichloro-3-nitropyridin-2-amine ( 18 ) , the benzylamino 
 derivative 27 and n - phenylbenzamide 
 derivative 29 were readily prepared by a nucleophilic 
 substitution reaction , though for the synthesis of 29 the temperature had to be raised to 90 c to ensure efficient 
 displacement by the poorly nucleophilic aniline ( scheme 7 ) .
access to 
 the 2-amino-3-nitropyridine derivative 31 was achieved 
 upon treatment of 4-hydroxy - n - phenylbenzamide 
 ( 30 ) , prepared as previously described , with kobu in dmf followed 
 by the addition of 4,5-dichloro-3-nitropyridin-2-amine ( scheme 8) .
the c2-unsubstituted analogue of 28c , compound 35 , was prepared from 31 by 
 first reducing the nitro group with na2s2o4 and subsequently condensing the resulting diamine product 33 with trimethyl orthoformate under acidic conditions ( scheme 8) .
for the synthesis of the deschloro analogue of 28c ( compound 34 ) , the 2-amino-3-nitropyridine 
 derivative 32 , obtained from 30 and 4-chloro-3-nitropyridin-2-amine via an snar substitution reaction , 
 was condensed with 1,3-dimethyl-1h - pyrazole-4-carbaldehyde 
 in a manner similar to that described for 28c ( scheme 8) .
the 2-amino-3-nitro-4-phenoxypyridine 
 derivatives 39a  f , precursors to 40a  f ( scheme 9 , table 5 ) , were accessed by reacting 4,5-dichloro-3-nitropyridin-2-amine 
 ( 18 ) with the requisite 4-hydroxybenzamide using potassium tert - butoxide as the base in dmf ( scheme 9 ) .
4-hydroxybenzamide derivatives 38a , b and 38d  f were all prepared from 4-acetoxybenzoic 
 acid ( 36 ) in a two - step 
 sequence ( scheme 9 ) ; 4-hydroxy - n-(pyridin-3-yl)benzamide ( 38c ) was commercially available . 

amide bond formation was effected via acid chloride carboxyl activation , 
 and ester hydrolysis was accomplished under alkaline conditions ( scheme 9 ) .
aurora - a , 
 -b , and -c kinases share a high degree of primary sequence 
 homology ; there are only three sequence differences in the atp - binding 
 site . in aurora - a , these residues are 
 leu215 ( arg in aurora - b / c ) and thr217 ( glu in aurora - b / c ) on the c - lobe 
 extension of the hinge region and arg220 ( lys in aurora - b / c ) on the 
 d helix .
the majority of reported aurora - a - selective inhibitors 
 exploit the thr217 atp - binding site sequence difference to drive selectivity 
 for aurora - a inhibition over aurora - b inhibition .
crystallographic analysis of aurora - a in complex 
 with small - molecule ligands provides insight into the attractiveness 
 of targeting the thr217 residue to elicit aurora isoform selectivity . 

for example , in the case of the imidazo[1,2-a]pyrazine 
 derivative 3 ( figure 1 ) , it was 
 proposed that the observed selectivity for aurora - a inhibition is 
 driven by the interaction of a sulfonamide with the side chain of 
 thr217 in aurora - a and a steric clash with the equivalent glu residue 
 in aurora - b / c . in the crystal structure 
 of 3
bound to aurora - a , the leu215 side chain points 
 away from the active site , whereas the arg220 is highly mobile and 
 disordered .
exploitation of these two 
 residues ( i.e. , leu215 and arg220 ) to drive selectivity for aurora - a 
 inhibition over aurora - b / c inhibition is therefore considered to be 
 challenging . we have previously reported the discovery of imidazo[4,5-b]pyridine derivatives as inhibitors of aurora kinases , 
 and compounds in this series were cocrystallized with aurora - a , providing 
 us with a clear insight into the interactions of this class of compounds 
 with aurora kinases . the structural knowledge 
 on the ligand 
protein interactions 
 across the imidazo[1,2-a]pyrazine and imidazo[4,5-b]pyridine series formed the basis of a design principle for imidazo[4,5-b]pyridine - derived aurora - a - selective inhibition .
overlay 
 of the imidazo[4,5-b]pyridine - based inhibitor 6(27 ) with the selective inhibitor 3(19 ) bound to aurora - a ( figure 3 ) indicated that thr217 could be accessed via an 
 appropriate c7-imidazo[4,5-b]pyridine derivatization 
 ( e.g. , compounds 21c in scheme 5 and 21d , f in scheme 6 ) or elaboration 
 through the n1-position of the imidazopyridine ring ( e.g. , 7-azaindole 
 structures 16a  d in schemes 2 and 3 ) . with regard to the 
 former approach
, we recognized that thr217 targeting for aurora - a - selective 
 inhibition via c7-imidazo[4,5-b]pyridine derivatization 
 could be a challenging task due to the conformational flexibility 
 of the proposed c7-pyrrolidine- and -piperidine - based substituents . 

for the latter approach , docking of c3-n - benzylmethanesulfonamide - derivatized 
 7-azaindoles into the active site of aurora - a indicated a mode of 
 binding to the hinge region similar to that observed for the imidazo[4,5-b]pyridine - based inhibitors of aurora and an interaction of the sulfonamide with thr217 .
overlay of 6 ( pdb code 2x6d ) with 3 ( pdb code 2xng ) bound to the atp - binding site of aurora - a . 
 the hinge - binding residue ala213 and the thr217 residue are highlighted .
it was also envisaged that the 
 introduction of a 4-amino - n - phenylbenzamide or 4-hydroxy - n - phenylbenzamide 
 substituent at the 7-position of the imidazo[4,5-b]pyridine ring could be beneficial for aurora - a isoform selectivity . 

a 4-amino - n - phenylbenzamide fragment is present in 
 2-anilinopyrimidine - based aurora - a - selective inhibitors reported by 
 aliagas - martin et al .
docking of structure 28c ( scheme 8) into the active site 
 of aurora - a ( pdb code 3h0z ) and a comparison with 2 bound to aurora - a indicated a good overlay of the n - phenylbenzamide fragments ( figure 4 ) .
( a ) docking of 28c into the active site of aurora - a 
 ( pdb code 3h0z ) and ( b ) overlay of 28c with the aurora - a - selective inhibitor 2 .
our initial efforts focused on the evaluation of the 7-azaindole 
 scaffold ( table 1 ) in relation to aurora inhibitory potency and aurora - a isoform selectivity . 
 assuming a mode of binding to the hinge region similar to that observed 
 for the imidazo[4,5-b]pyridine - based inhibitors , p - methoxyphenyl and 1-methylpyrazol-4-yl were chosen 
 as optimal representative r substituents for the 7-azaindole 
 scaffold .
1-methylpyrazol-4-yl derivatives 16a and 16c displayed higher aurora - a and -b inhibitory 
 potencies compared with their p - methoxyphenyl counterparts 16b and 16d ( table 1 ) . 

on the other hand , both the sulfonamide and the acetamide groups appear 
 to have similar aurora - a inhibitory effects ( 16a vs 16c and 16b vs 16d , table 1 ) . regarding selectivity for aurora - a inhibition , 
 although a trend to more potent aurora - a inhibition was seen with 
 all compounds listed in table 1 , the level 
 of the observed isoform selectivity was not considered significant .
attempts to cocrystallize compounds of this 7-azaindole 
 class with 
 aurora - a failed to provide quality crystals ; as a result we were unable 
 to rationalize these results on a structural basis . at this point
, 
 we revisited the docking of 16a , b into the active site 
 of aurora - a , which indicated an additional binding mode to that previously 
 observed .
the preferred binding mode for 16a was similar 
 to that observed with the imidazo[4,5-b]pyridine - based 
 inhibitors of aurora with the 2-aryl / heteroaryl substituent , in both 
 imidazo[4,5-b]pyridine and 7-azaindole scaffolds , 
 pointing to the solvent - accessible area ( see figure 5a and compound 6 in figure 3 ) .
however , an additional flipped binding mode was 
 observed in docking experiments with compound 16b compared 
 to 16a ( compare figure 5b with 
 figure 5a ) .
it should also be noted that the p - methoxyphenyl derivatives 16b and 16d exhibited low aurora - a inhibitory potencies .
taking all these findings 
 into account , we decided to terminate our interest in the 7-azaindole 
 scaffold and focus our efforts on evaluating the imidazo[4,5-b]pyridine - based approaches for aurora - a - selective inhibition .
( a ) docking 
 of 16a into the active site of aurora - a 
 ( pdb code 2x6d)and ( b ) docking of 16b into the active site of aurora - a ( pdb code 2x6d ) . targeting aurora - a thr217 
 for selective aurora - a isoform inhibition 
 was continued by investigation of c7-imidazo[4,5-b]pyridine derivatization , i.e. , introduction of pyrrolidine- and 
 piperidine - based substituents ( table 2 ) .
this approach afforded highly potent inhibitors 
 of both aurora - a and -b ( 21a  f , table 2 ) , with all compounds displaying similar inhibitory 
 effects against these two aurora isoforms . on this
protein interactions was gained by cocrystallization 
 of 21c and 21a with aurora - a .
the complexes 
 formed crystals in space group p6122 , 
 with one aurora / compound complex per asymmetric unit .
results 
 are mean values of two independent 
 determinations ( sd ) unless specified otherwise .
a superscript 
 roman  a  indicates results from a single experiment 
 ( each concentration run in duplicate ) .
compound 21c occupies the atp - binding 
 site , with the 
 pyridine n and imidazole nh forming hydrogen bonds to ala213 in the 
 hinge region of aurora - a consistent with previous reports ( figure 6a ) .
the amide substituent of the 
 pyrrolidine in 21c points to thr217 but is neither in 
 the right orientation nor close enough ( 5.9  distance between 
 the oh of thr217 and the amide carbonyl of compound 21c ) to form a strong hydrogen bond with the side chain of 
 thr217 .
likewise , compound 21a binds to the hinge region 
 as previously observed with closely related derivatives in this series . 

however , the substituted pyrrolidine ring adopts an unfavorable conformation 
 for interaction with thr217 , with the acetamido group pointing away 
 from this residue ( figure 6b ) .
these crystal 
 structures were consistent with the lack of selectivity for compounds 21a and 21c , and we therefore , investigated alternative 
 substitutions at c7 .
crystal structures of c7-imidazo[4,5-b]pyridine 
 compounds bound to aurora - a : ( a ) compound 21c , ( b ) compound 21a .
we continued our attempts 
 to target thr217 for aurora - a - selective 
 inhibition via c7-imidazo[4,5-b]pyridine derivatization 
 by the introduction of a benzylamino substituent ( compound 28a , table 3 ) and 
 4-amino - n - phenylbenzamide substituent ( compound 28b , table 3 ) .
the benzylamino derivative 28a inhibited aurora - a and -b with similar ic50 values ( table 3 ) .
however , the n - phenylbenzamide derivative 28b was a significantly 
 more potent inhibitor of aurora - a compared to aurora - b , the ic50 values were determined as 0.075 and 4.12 m , respectively 
 ( table 3 ) .
a similar trend was observed in 
 hela cervical cancer cells ; 28b inhibited the autophosphorylation 
 of aurora - a at t288 ( a cell - based biomarker for aurora - a inhibition ) with an ic50 value of 0.32 m and histone h3 phosphorylation 
 at s10 ( a cell - based biomarker for aurora - b inhibition ) with a significantly higher ic50 value of 18.6 m 
 ( 58-fold difference ) .
subsequent replacement of the c7-nh linker in 28b with an oxygen gave compound 28c , which displayed 
 a higher degree of selectivity in inhibiting aurora - a over aurora - b 
 in biochemical assays ( table 3 ) and in cells . 

in the biochemical assays , the ic50 values for inhibition 
 of aurora - a and -b were determined as 0.067 and 12.71 m , respectively 
 ( table 3 ) . in hela cervical cancer cells
, 28c inhibited the autophosphorylation of aurora - a at t288 
 ( p - t288 ic50 = 0.16 m ) 480-fold more potently compared 
 with aurora - b inhibition ( p - hh3 ic50 = 76.84 m ) . 

a similar trend was also observed in hct116 human colon carcinoma 
 cells ; the ic50 values for the inhibition of autophosphorylation 
 of aurora - a at t288 and histone h3 phosphorylation at s10 were determined 
 as 0.065 and 24.65 m , respectively .
the selectivity of 28c against the kinome was also determined by profiling in 
 a 110-kinase panel at a concentration of 1 m . in this panel ,
28c inhibited only three kinases at a level higher than 80% , 
 namely , aurora - a , vegfr ( vegfr1 ) , and gsk3b ( table s3 , supporting information ) . on this basis ,
compound 28c is a highly selective kinase inhibitor with a gini coefficient calculated as 0.719 . in an attempt to understand 
 possible reasons for the observed inhibition of vegfr1 and gsk3b by 28c ,
we examined conserved residues in the binding site between 
 aurora - a and vegfr1 and between aurora - a and gsk3b .
we identified 
 e211 , y212 , g216 , and l263 ( residue numbers for aurora - a ) as conserved 
 binding motifs between aurora - a and vegfr1 and residues l210 , y212 , 
 p214 , and t217 ( residue numbers for aurora - a ) as conserved binding 
 motifs between aurora - a and gsk3b . however , determination of whether 
 these conserved regions influence the inhibition of vegfr1 and gsk3b 
 would require additional crystallography beyond the scope of this 
 study .
the antiproliferative activity in human tumor cell lines was 
 also studied , and 28c inhibited the growth of hct116 
 human colon carcinoma cells with a gi50 value of 2.30 m . 

we have previously shown that compounds of the imidazo[4,5-b]pyridine class exhibit potent dual aurora - a / flt3 inhibition , and consistent with this finding , we observed 
 flt3 inhibition in vitro ( ic50 = 0.162 m ) and potent antiproliferative activity in mv4 - 11 
 human aml cells ( gi50 = 0.299 m ) .
the results 
 are mean values of at 
 least two independent determinations ( sd ) unless specified otherwise . 
 a superscript roman 
a  indicates results from a single 
 experiment ( each concentration run in duplicate ) .
compound 28c displayed 
 high mouse and liver microsomal 
 stability ( after a 30 min incubation with mouse and human liver microsomes , 
 only 22% and 9% of 28c was metabolized , respectively ) . 

this promising stability prompted in vivo mouse pharmacokinetic profiling 
 that revealed 28c as a highly orally bioavailable compound 
 ( f = 100% ) with moderate clearance ( 0.053 l / h , 44.16 
 ml / min / kg ) and volume of distribution ( 0.036 l , 1.8 l / kg ) .
the 
 effects of the 6-cl and c2-pyrazolyl substituents on aurora - a 
 inhibition were subsequently studied by preparing compounds 34 and 35 , respectively ( scheme 8 , table 4 ) .
both 34 and 35 were significantly less 
 potent inhibitors of aurora - a compared to 28c ( tables 3 and 4 ) , indicating the requirement 
 for both c6-cl and c2-aromatic or -heteroaromatic substituents and 
 consistent with previously reported sars .
having identified 28c as a highly selective 
 aurora - a 
 inhibitor , our efforts focused on replacing the aniline moiety in 28c , a potential toxicophore , with a range 
 of aliphatic and heteroaryl amines ( table 5 ) .
all replacements were 
 well tolerated in relation to aurora - a inhibitory potency , and the 
 selectivity for aurora - a over aurora - b inhibition was generally maintained 
 ( table 5 ) .
compounds were also tested for the 
 cellular inhibition of both aurora - a and -b , and 40a inhibited 
 aurora - a in hct116 cells significantly more potently compared to aurora - b 
 ( p - t288 ic50 = 0.095 m versus p - hh3 ic50 = 4.93 m , 52-fold difference ) .
likewise , 40c was a more potent inhibitor of aurora - a than aurora - b in hela cells 
 ( p - t288 ic50 = 0.28 m versus p - hh3 ic50 = 19.72 m , 70-fold difference ) .
a similar trend was seen 
 with 40b ; in hela cells it inhibited aurora - a more potently 
 compared to aurora - b ( p - t288 ic50 = 0.58 m versus 
 p - hh3 ic50 = 19.74 m , 34-fold difference ) .
compound 40f displayed the highest potency inhibiting aurora - a in the 
 biochemical assay ( ic50 = 0.015 m , table 5 ) , with aurora - b inhibition being determined as 
 3.05 m ( table 5 ) . in hela cells ,
40f inhibited aurora - a 346 times more potently compared to 
 aurora - b ( p - t288 ic50 = 0.070 m versus p - hh3 ic50 = 24.24 m ) .
profiling of 40f in a 50-kinase 
 panel at a concentration of 1 m revealed a highly selective 
 inhibitor ; only one kinase , namely , vegfr ( vegfr1 ) , was inhibited 
 higher than 80% ( table s4 , supporting information ) .
compound 40f exhibited high mouse and liver microsomal 
 stability ( after a 30 min incubation with mouse and human liver microsomes , 
 28% and 22% of 40f was metabolized , respectively ) .
however , 
 an in vivo pharmacokinetic profiling in mouse revealed a lower oral 
 bioavailability ( 14% ) compared to that for 28c ( 100% ) .
the results are 
 mean values of at 
 least two independent determinations ( sd ) .
many attempts to cocrystallize 28c and 40f with aurora - a were unsuccessful . 
 however , the docking of 28c into the active site of aurora - a 
 suggested that the aniline moiety 
 resides in close proximity to thr217 ( figure 4 ) . on this basis
, we probed whether thr217 ( glu in aurora - b ) is the 
 main residue governing the selectivity for aurora - a inhibition .
testing 
 of 28c against the aurora - a wild type and its t217e mutant 
 expressed in hela cells revealed that the aurora - a t217e mutant was 
 significantly less sensitive to inhibition ( 40-fold ) compared to the 
 aurora - a wild type ( p - t288 ic50 = 4.11 and 0.107 m , 
 respectively ) .
subsequently , both 28c and 40f were tested against the aurora - a wild type and its t217e , l215r , 
 and r220k mutants in hct116 cells ( table 6 , 
 figure 7 , and figure s1 in the supporting information ) .
both 28c and 40f inhibited the aurora - a l215r and r220k mutants 
 with ic50 values similar to those seen for the aurora - a 
 wild type ( table 6 , figure 7 , and figure s1 ) . on the other hand ,
the aurora - a t217e mutant 
 was significantly less sensitive to inhibition by 28c and 40f compared to the wild type ( 33-fold and 64-fold , 
 respectively ; table 6 , figure 7 , and figure s1 ) .
this body of evidence suggests that the 
 thr217 residue ( glu in aurora - b / c ) plays an important role in governing 
 the observed selectivity for aurora - a inhibition . in the above experiment
, 
 the inhibition of aurora - b by 40f was also investigated 
 by measuring the reduction in the phosphorylation of histone h3 at 
 s10 . as shown in figure s2 ( supporting information ) , inhibition of histone h3 phosphorylation at s10
was only achieved 
 at high concentrations of 40f ( partial inhibition at 
 25 m and complete inhibition at 50 m ) .
interestingly , 
 at concentrations where phosphorylation of aurora - a was completely 
 inhibited ( for example , at 1.5 m ) , there was an increase in 
 histone h3 phosphorylation ( figure s2 ) , most likely due to an increase 
 in the percentage of mitotic cells as previously reported for other 
 aurora - a - selective inhibitors .
however , at higher 
 concentrations , histone h3 phosphorylation was inhibited , indicating 
 onset of aurora b inhibition ( figure s2 ) .
inhibition of aurora - a wild - type and mutant proteins expressed 
 in hct116 cells by compound 40f . in conclusion ,
structural knowledge of imidazo[4,5-b]pyridine and imidazo[1,2-a]pyrazine - based 
 ligand  aurora - a 
 protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine - derived aurora - a - selective inhibition .
this led 
 to the discovery of imidazo[4,5-b]pyridine derivatives 
 displaying a high degree of selectivity for inhibition of aurora - a 
 over aurora - b in both biochemical and cellular assays .
in particular , 28c and 40f were highly selective in inhibiting 
 aurora - a in both hela cervical cancer and hct116 human colon carcinoma 
 cells .
testing of 28c and 40f against the 
 aurora - a wild type and its t217e , l215r , and r220k mutants suggested 
 that the aurora - a thr217 residue ( glu in aurora - b / c ) plays an important 
 role in governing the observed selectivity for aurora - a inhibition . 

in addition , 28c and 40f are a different 
 chemotype compared to other known aurora - a - selective inhibitors , and 28c is highly selective within the tested kinome , with a kinase 
 profiling differing from that of mk-5108 and mln8237 . these compounds could 
 therefore serve as useful small - molecule tools to further explore the function of aurora - a in cells ; indeed , the 
 need for structurally diverse inhibitors displaying aurora isoform 
 selectivity has recently been highlighted by brockmann et al . in their study , the aurora - a - selective inhibitors 
 mln8054 and mln8237 disrupted the aurora - a / n - myc complex and induced 
 apoptosis in neuroblastoma cell lines and tumors , a phenotype different 
 from that seen with the structurally differentiated aurora - a - selective 
 inhibitor mk-5108 from a different chemotype .
these data suggest differences in the mechanism of action and also 
 indicate a specific target patient population for treatment with an 
 aurora - a - selective inhibitor .
aurora - a and aurora - b kinase ic50 values were determined as follows : kinase activity was measured 
 in a microfluidic assay which monitors the separation of a phosphorylated 
 product from its substrate .
the assay was performed on an ez reader 
 ii ( perkinelmer life sciences , waltham , ma ) using separation buffer 
 ( no . 760367 , perkinelmer life sciences ) containing cr-8 ( 500 nm , no . 
 760278 , perkinelmer life sciences ) .
for each compound , a 10 mm stock 
 concentration in 100% dmso was used .
sunnyvale , ca ) to generate 
 duplicate 8 pt dilution curves directly into 384 lv polystyrene plates 
 ( no .
corning , ny ) to give final assay concentrations 
 in the range of 0.25 pm to 200 m in either 1.0% or 2% ( v / v ) 
 dmso . for the aurora - a kinase
the enzyme reaction ( total volume 
 10 l ) was carried out with 5 nm n - terminal his - tagged aura , 
 which was expressed and purified as previously described , 1.5 m fluorescently labeled peptide ( no . 
 760365 , fl - peptide 21 , perkinelmer , sequence 5fam - lrraslg - conh2 ) , 20 m atp in assay buffer ( 50 mm tris ( ph 7.4 ) , 200 
 mm nacl , 5 mm mgcl2 , 2 mm dtt , 0.1% tween 20 ) , and the 
 test compound or dmso control .
for the aurora - b kinase the enzyme 
 reaction ( total volume 10 l ) 
 was carried out with 1 nm full - length aurb / incenp complex ( no . 05 - 102 , 
 carna biosciences , inc . , kobe , japan ) , 1.5 m fluorescently 
 labeled peptide ( no
. 760365 , fl - peptide 21 , perkinelmer , sequence 
 5fam - lrraslg - conh2 ) , 15 m atp in assay buffer ( 50 
 mm tris ( ph 7.4 ) , 200 mm nacl , 5 mm mgcl2 , 2 mm dtt , 0.1% 
 tween 20 ) , and either the test compound or a dmso control . for 
 both enzymes
the reaction was carried out for 60 min at room 
 temperature and stopped by the addition of 10 l of stop buffer 
 containing 100 mm hepes - buffered saline ( ph 7.3 ) , 20 mm edta , and 
 0.05% ( v / v ) brij-35 .
the reader provides a software package (  reviewer  ) 
 which converts the peak heights into percent conversion by measuring 
 both product and substrate peaks .
the percent inhibition was calculated 
 relative to the total wells ( containing enzyme ) and blank wells ( containing 
 no enzyme plus dmso ) .
ic50 values were calculated from 
 a four - parameter logistics fit of percentage inhibition versus log 
 concentration using graphpad prism 5 ( graphpad software , inc .
, la 
 jolla , ca ) or the studies package ( dotmatics , bishops stortford , u.k . ) .
compound 28c was 
 profiled against a panel of 110 kinases at the international 
 centre for protein kinase profiling , division of signal transduction 
 therapy , university of dundee .
compound 40f was profiled 
 against a panel of 50 kinases ( mrc - ppu express screen ) also at the 
 international centre for protein kinase profiling .
gi50 values ( 50% cell 
 growth inhibitory concentration ) were determined as previously described .
determination of cellular inhibition of aurora - a 
 ( at t288 ) and histone h3 ( at s10 ) has been described previously .
briefly , cells were transfected with the myc - tagged 
 aurora - a wild type or mutants and treated with different concentrations 
 of the inhibitors for 2 h followed by lysis in lds sample buffer . 

the samples were sonicated and boiled , and the proteins were separated 
 by lds  page and transferred to nitrocellulose membranes .
the 
 upper and lower parts of the membranes were probed with anti - p - t288 
 ( aurora - a autophosphorylation / activation ) and anti - p - histone h3 ( aurora - b 
 substrate phosphorylation ) antibodies , respectively . the blots were 
 quantified and ic50 values determined using graphpad prism . 

mammalian cell expression constructs encoding aurora - a t217e , l215r , 
 and r220k mutants with n - terminal myc tags were produced using quikchange 
 mutagenesis ( stratagene ) . the protein 
ligand 
 cocrystal 
 ( pdb 2x6d ) was 
 prepared in moe ( moleular operating environment ) using protonate3d , 
 and the waters were removed .
gold ( genetic 
 optimization for ligand docking ) was used for all docking experiments 
 using the chemscore ( chemscore_kinase ) scoring function .
the docked poses were analyzed on the basis 
 of their chemscore and also the consistency of the docked poses .
the 
 higher scored poses were selected as the preferred solutions if the 
 majority of these solutions were consistent in binding mode .
the binding 
 site was defined as those atoms within 6  of the cocrystallized 
 ligand in pdb 2x6d and the cocrystal ligand removed .
the gold algorithm was set to 
 explore an extensive binding mode space by setting the search 
 efficiency to 200% , or very flexible .
cocrystals 
 of aurora - a bound to compounds 21a and 21c were generated as previously described .
structures were solved by molecular replacement 
 using aurora - a ( pdb code 1mq4 ) as a model .
ligand fitting and model rebuilding were 
 carried out using coot , and refinement 
 was carried out using phenix .
the compounds ( 10 
 m ) were incubated with male cd1 mouse liver microsome ( 1 mg 
 ml ) protein in the presence of nadph ( 1 mm ) , udpga 
 ( 2.5 mm ) , and mgcl2 ( 3 mm ) in phosphate - buffered saline 
 ( 10 mm ) at 37 c .
control incubations were generated by the omission of nadph and udpga 
 from the incubation reaction .
the compounds ( 10 
 m ) were incubated with mixed - gender pooled human liver microsome 
 ( 1 mg ml ) protein in the presence of nadph ( 1 
 mm ) , udpga ( 2.5 mm ) , and mgcl2 ( 3 mm ) in phosphate - buffered 
 saline ( 10 mm ) at 37 c .
control incubations were generated by the omission of nadph 
 and udpga from the incubation reaction .
mice ( female 
 balb / c ) were dosed po or iv with 28c ( 5 mg kg ) in 10% dmso and 5% tween 20 in saline .
after administration , the 
 mice were sacrificed at 5 , 15 , and 30 min and 1 , 2 , 4 , 6 , and 24 h. 
 blood was removed by cardiac puncture and centrifuged to obtain plasma 
 samples .
the plasma samples ( 100 l ) were added to the analytical 
 internal standard ( is ; olomoucine ) , followed by protein precipitation 
 with 300 l of methanol .
following centrifugation ( 1200 g , 30 min , 4 c ) , the resulting supernatants were analyzed 
 for 28c levels by lc / ms using a reversed - phase x - bridge 
 c18 ( waters , 50  2.1 mm ) analytical column and positive ion 
 mode esi mrm on a waters 2795 liquid chromatography system coupled 
 to a quattro ultima triple - quadrupole mass spectrometer ( waters ) .
mice ( female 
 balb / c ) were dosed po or iv with 40f ( 5 mg kg ) in 10% dmso and 5% tween 20 in saline .
after administration , the 
 mice were sacrificed at 5 , 15 , and 30 min and 1 , 2 , 4 , 6 , and 24 h. 
 blood was removed by cardiac puncture and centrifuged to obtain plasma 
 samples .
the plasma samples ( 100 l ) were added to the analytical 
 is ( olomoucine ) , followed by protein precipitation with 300 l 
 of methanol .
following centrifugation ( 1200 g , 30 
 min , 4 c ) , the resulting supernatants were analyzed for 40f levels by lc / ms using a reversed - phase polar rp ( phenomenex , 
 50  2 mm ) analytical column and positive ion mode esi mrm on 
 a waters 2795 liquid chromatography system coupled to a quattro ultima 
 triple - quadrupole mass spectrometer ( waters ) . commercially available 
 starting 
 materials , reagents , and dry solvents were used as supplied .
flash column chromatography was performed using merck silica gel 
 60 ( 0.0250.04 mm ) .
column chromatography was also performed 
 on a flashmaster personal unit using isolute flash silica columns 
 or a biotage sp1 purification system using biotage flash silica cartridges . 

samples were prepared as solutions in a deuterated solvent and referenced 
 to the appropriate internal nondeuterated solvent peak or tetramethylsilane . 

chemical shifts were recorded in parts per million (  ) downfield 
 of tetramethylsilane .
lc / ms analysis , method a : analysis was 
 performed on a waters lct 
 with a waters alliance 2795 separation module and waters 2487 dual - wavelength 
 absorbance detector coupled to a waters / micromass lct time - of - flight 
 mass spectrometer with an esi source .
analytical separation was carried 
 out at 30 c on a merck chromolith speedrod column ( rp-18e , 50 
  4.6 mm ) using a flow rate of 2 ml / min in a 3.5 min gradient 
 elution with detection at 254 nm .
the mobile phase was a mixture of 
 methanol ( solvent a ) and water containing formic acid at 0.1% ( solvent 

gradient elution was 1:9 ( a / b ) to 9:1 ( a / b ) over 2.25 min , 9:1 
 ( a / b ) for 0.75 min , then reversion back to 1:9 ( a / b ) over 0.3 min , 
 and finally 1:9 ( a / b ) for 0.2 min .
lc / ms analysis , method b : analysis 
 was performed on an agilent 1200 series hplc instrument with a diode 
 array detector coupled to a 6210 time - of - flight mass spectrometer 
 with a dual multimode apci / esi source .
analytical separation was carried 
 out at 30 c on a merck chromolith speedrod column ( rp-18e , 50 
  4.6 mm ) using a flow rate of 2 ml / min in a 4 min gradient elution 
 with detection at 254 nm .
the mobile phase was a mixture of methanol 
 ( solvent a ) and water containing formic acid at 0.1% ( solvent b ) . 

gradient elution was 1:9 ( a / b ) to 9:1 ( a / b ) over 2.5 min , 9:1 ( a / b ) 
 for 1 min , then reversion back to 1:9 ( a / b ) over 0.3
min , and finally 
 1:9 ( a / b ) for 0.2 min . for lc / ms analyses , if the method is not stated , 
 then method a was followed .
lc / hrms analysis was performed on 
 an agilent 1200 series hplc instrument 
 with a diode array detector coupled to a 6210 time - of - flight mass 
 spectrometer with a dual multimode apci / esi source .
analytical separation 
 was carried out at 30 c on a merck chromolith speedrod column 
 ( rp-18e , 50  4.6 mm ) using a flow rate of 2 ml / min in a 4 min 
 gradient elution with detection at 254 nm .
the mobile phase was a 
 mixture of methanol ( solvent a ) and water containing formic acid at 
 0.1% ( solvent b ) .
gradient elution was 1:9 ( a / b ) to 9:1 ( a / b ) over 
 2.5 min , 9:1 ( a / b ) for 1 min , then reversion back to 1:9 ( a / b ) over 
 0.3 min , and finally 1:9 ( a / b ) for 0.2 min .
the following references 
 masses were used for hrms analysis : caffeine [ m + h ] 195.087652 , 
 ( hexakis(1h,1h,3h - tetrafluoropentoxy)phosphazene [ m + h ] 922.009798 ) , 
 and hexakis(2,2-difluoroethoxy)phosphazene [ m + h ] 622.02896 
 or reserpine [ m + h ] 609.280657 .
analytical hplc 
 analysis was performed on a thermo - finnigan surveyor 
 hplc system or an agilent technologies 1200 series hplc system at 
 30 c using a phenomenex gemini c18 column ( 5 m , 
 50  4.6 mm ) and 10 min gradient of 10%  90% meoh/0.1% 
 formic acid , visualizing at 254 , 309 , or 350 nm .
the purity of the 
 final compounds was determined by analytical hplc as described above 
 and is 95% unless specified otherwise . to a mixture of 4,5-dichloro-3-nitropyridin-2-amine 

( 0.152 g , 0.73 mmol ) and 2-propanol ( 13.0 ml ) was added ( s)-3-acetamidopyrrolidine ( 0.098 g , 0.77 mmol ) followed by n , n - diisopropylethylamine ( 0.17 ml , 0.97 
 mmol ) .
the reaction mixture was placed in an oil bath preheated at 
 45 c and stirred at this temperature for 18 h ; it was then allowed 
 to cool to room temperature and diluted with 2-propanol ( 8 ml ) .
the 
 precipitate was collected by filtration , washed with 2-propanol ( 2 
  5 ml ) and diethyl ether ( 5 ml ) , and dried to give the title 
 compound as a yellow solid ( 0.133 g , 61% ) : h nmr ( 500 
 mhz , dmso - d6 ) 1.80 ( s , 3h , coch3 ) , 1.83 ( m , 1h , pyrrolidine ch ) , 2.08 ( m , 1h , pyrrolidine ch ) , 3.18 
 ( dd , j = 5.1 , 10.5 hz , 1h , pyrrolidine ch ) , 3.56 
 ( m , 2h , pyrrolidine ch ) , 3.64 ( m , 1h , pyrrolidine ch ) , 4.20 ( m , 1h , 
 pyrrolidine ch ) , 6.85 ( s , 2h , nh2 ) , 7.88 ( s , 1h , pyridine 
 6-h ) , 8.09 ( d , j = 6.1 hz , 1h , conh ) ; lc / ms ( esi , m / z ) tr = 1.32 
 min , 300 , 302 [ ( m + h ) , cl isotopic pattern ] . to a mixture of ( s)-n-(1-(2-amino-5-chloro-3-nitropyridin-4-yl)pyrrolidin-3-yl)acetamide 
 ( 0.045 g , 0.15 mmol ) and ethanol ( 3.5 ml ) was added 1,3-dimethyl-1h - pyrazole-4-carbaldehyde ( 0.020 g , 0.16 mmol ) followed 
 by a freshly prepared aqueous solution of na2s2o4 ( 1 m , 0.65 ml , 0.65 mmol ) .
the reaction mixture was 
 stirred at 80 c for 20 h ; it was then concentrated in vacuo . 

the residue was absorbed on silica gel ( 1.7 g ) , and the free - running 
 powder was placed on a 10 g isolute silica column .
elution with dichloromethane / ethyl 
 acetate ( 1:1 , v / v ) , 4% methanol in dichloromethane / ethyl acetate ( 1:1 , 
 v / v ) , and 6% methanol in dichloromethane / ethyl acetate ( 1:1 , v / v ) 
 afforded a solid residue which was triturated with diethyl ether . 

the precipitate was collected by filtration and washed with diethyl 
 ether , water ( 4  2 ml ) , and diethyl ether ( 3  3 ml ) to 
 afford the title compound as a pale yellow solid ( 0.022 g , 39% ) : h nmr ( 500 mhz , dmso - d6 ) 1.81 
 ( s , 3h , coch3 ) , 1.85 ( m , 1h , pyrrolidine ch ) , 2.08 ( m , 
 1h , pyrrolidine ch ) , 3.83 ( s , 3h , pyrazole
n - ch3 ) , 3.92 ( m , 1h , pyrrolidine ch ) , 4.16 ( m , 1h , pyrrolidine 
 ch ) , 4.25 ( m , 3h , pyrrolidine ch ) , 7.86 ( s , 1h ) and 8.12 ( s , 2h ) ( pyrazole 
 5-h , imidazo[4,5-b]pyridine 5-h and conh ) , 12.78 
 ( s , 1h , imidazo[4,5-b]pyridine nh ) ; lc / ms ( esi , m / z ) tr = 1.90 
 min , 374 , 376 [ ( m + h ) , cl isotopic pattern ] ; hrms m / z found 374.1496 , calcd for c17h21cln7o ( m + h ) 374.1491 ; hplc tr = 6.18 min , 94% at  = 354 nm . to a solution of ( s)-tert - butyl 3-(aminomethyl)pyrrolidine-1-carboxylate ( 0.30 
 g , 1.50 mmol ) in anhydrous ch2cl2 ( 4 ml ) cooled 
 in an ice bath under argon was added dipea ( 0.213 g , 0.29 ml , 1.65 
 mmol ) followed by a dropwise addition of acetic anhydride ( 0.168 g , 
 1.65 mmol ) .
the reaction mixture was stirred for 4 h under argon while 
 being allowed to warm to room temperature .
the clear solution was 
 diluted with ethyl acetate ( 50 ml ) , washed with saturated nahco3 ( 20 ml ) , brine ( 20 ml ) , 1 m aq hcl ( 20 ml ) , and brine ( 20 
 ml ) , then dried ( na2so4 ) , and concentrated in 
 vacuo .
the product was obtained as a colorless oil ( 0.300 g , 83% ) : h nmr ( 500 mhz , dmso - d6 ) 1.39 
 ( s , 9h , c(ch3)3 ) , 1.53 ( m , 1h , pyrrolidine ch ) , 
 1.80 ( s , 3h , coch3 ) , 1.85 ( m , 1h ) , 2.23 ( m , 1h ) , 2.89 ( dd , j = 7.2 , 10.8 hz , 1h ) , 3.00 ( m , 1h ) , 3.07 ( m , 1h ) , 3.16 
 ( m , 1h ) , and 3.32 ( m , obscured by water peak ) ( pyrrolidine ch and 
 ch2nh ) , 7.89 ( s , 1h , 
 conh ) ; lc / ms ( esi , m / z ) compound 
 is non - uv - absorbent , 265 ( m + na ) .
a solution of ( s)-tert - butyl 3-(acetamidomethyl)pyrrolidine-1-carboxylate 
 ( 0.263 g , 1.09 mmol ) in dichloromethane ( 7 ml ) and trifluoroacetic 
 acid ( 3.0 ml ) was stirred at room temperature for 1.5 h. solvents 
 were removed in vacuo and azeotroped with toluene to leave a colorless 
 oily residue , ( r)-n-(pyrrolidin-3-ylmethyl)acetamide 
 as a tfa salt . this material ( supposedly 1.08 mmol )
was dissolved 
 in 2-propanol ( 14 ml ) , and to this solution was added 4,5-dichloro-3-nitropyridin-2-amine 
 ( 0.214 g , 1.03 mmol ) followed by n , n - diisopropylethylamine ( 0.503 g , 0.68 ml , 3.89 mmol ) .
the reaction 
 mixture was placed into an oil bath preheated to 45 c and stirred 
 at this temperature for 20 h ; a nearly clear solution was obtained . 

approximately half of the solvent volume was then removed in vacuo 
 to give a precipitate which was collected by filtration and washed 
 with 2-propanol and diethyl ether .
the product was obtained as a yellow 
 solid ( 0.205 g , 64% ) : h nmr ( 500 mhz , dmso - d6 ) 1.62 ( m , 1h , pyrrolidine ch ) , 1.79 ( s , 3h , coch3 ) , 1.95 ( m , 1h , pyrrolidine ch ) , 2.32 ( m , 1h , pyrrolidine 
 ch ) , 3.03 ( m , 1h ) , 3.12 ( m , 1h ) , 3.21 ( dd , j = 6.3 , 
 10.4 hz , 1h ) , and 3.383.52 ( m , 3h ) ( pyrrolidine ch and ch2nh ) , 6.84 ( s , 2h , nh2 ) , 7.87 ( s , 1h , pyridine 6-h ) , 7.93 ( br t , j = 5.4 
 hz , 1h , conh ) ; lc / ms ( esi , m / z ) tr = 1.26 min , 314 , 316 [ ( m + h ) , 
 cl isotopic pattern ] . to a mixture of ( s)-n-((1-(2-amino-5-chloro-3-nitropyridin-4-yl)pyrrolidin-3-yl)methyl)acetamide 
 ( 0.055 g , 0.18 mmol ) and ethanol ( 3.5 ml ) was added 1,3-dimethyl-1h - pyrazole-4-carbaldehyde ( 0.024 g , 0.19 mmol ) followed 
 by a freshly prepared aqueous solution of na2s2o4 ( 1 m , 0.75 ml , 0.75 mmol ) , and the reaction mixture 
 was stirred at 80 c for 20 h. the reaction mixture was then 
 concentrated in vacuo , the residue was absorbed on silica gel ( 1.7 
 g ) , and the free - running powder was placed on a 10 g isolute silica 
 column which was eluted with dichloromethane / ethyl acetate ( 1:1 , v / v ) , 
 5% methanol in dichloromethane / ethyl acetate ( 1:1 , v / v ) , and 9% methanol 
 in dichloromethane / ethyl acetate ( 1:1 , v / v ) .
trituration of the resultant 
 residue with diethyl ether gave a precipitate which was collected 
 by filtration , washed with diethyl ether , water , and diethyl ether , 
 and then dried .
the title compound was obtained as an off - white solid 
 ( 0.016 g , 23% ) : h nmr ( 500 mhz , dmso - d6 ) 1.64 ( m , 1h , pyrrolidine ch ) , 1.81 ( s , 3h , coch3 ) , 2.01 ( m , 1h , pyrrolidine ch ) , 2.35 ( m , 1h , pyrrolidine 
 ch ) , 3.83 ( s , 3h , pyrazole
n - ch3 ) , 3.13 
 ( t , j = 6.3 hz , 2h ) , 3.85 ( m , 1h ) , and 4.14 ( m , 3h ) 
 ( pyrrolidine ch and ch2nh ) , 7.86 ( s , 1h , pyrazole 5-h or imidazo[4,5-b]pyridine 
 5-h ) , 7.94 ( br t , j = 5.1 hz , 1h , conh ) , 8.12 ( s , 
 1h , imidazo[4,5-b]pyridine 5-h or pyrazole 5-h ) , 
 12.76 ( s , 1h , imidazo[4,5-b]pyridine nh ) ; lc / ms ( esi , m / z ) tr = 1.78 
 min , 388 , 390 [ ( m + h ) , cl isotopic pattern ] ; hrms m / z found 388.1649 , calcd for c18h23cln7o ( m + h ) 388.1647 . to a mixture 
 of 4,5-dichloro-3-nitropyridin-2-amine 
 ( 0.104 g , 0.50 mmol ) and 2-propanol ( 7.0 ml ) was added 4-aminobenzanilide 
 ( 0.114 g , 0.54 mmol ) followed by n , n - diisopropylethylamine ( 0.105 ml , 0.078 g , 0.60 mmol ) .
the reaction 
 mixture was heated at 45 c for 18 h , then the temperature was 
 raised to 90 c , and stirring was continued at this temperature 
 for 7 h. the reaction mixture was then allowed to cool to room temperature 
 and diluted with 2-propanol ( 8.0 ml ) ; a precipitate was obtained which 
 was collected by filtration , washed with 2-propanol ( 2  5 ml ) 
 and diethyl ether ( 2  5 ml ) , and dried to afford the title compound 
 as an orange solid ( 0.070 g , 37% ) : h nmr ( 500 mhz , dmso - d6 ) 7.02 ( d , j = 8.7 hz , 2h , 
 arh ) , 7.08 ( t , j = 7.2 hz , 1h , p - phh ) , 7.26 ( s , 2h , nh2 ) , 7.33 ( t , j = 
 8.3 hz , 2h , m - phh ) , 7.75 ( d , j = 
 7.8 hz , 2h , arh ) , 7.86 ( d , j = 8.7 hz , 2h , arh ) , 
 8.18 ( s , 1h , pyridine 6-h ) , 9.14 ( s , 1h , nhar ) , 10.07 ( s , 1h , nhar ) ; 
 lc / ms ( esi ,
m / z ) tr = 2.31 min , 384 , 386 [ ( m + h ) , cl isotopic 
 pattern ] . to a mixture 
 of 4-((2-amino-5-chloro-3-nitropyridin-4-yl)amino)-n - phenylbenzamide ( 0.065 g , 0.17 mmol ) and ethanol ( 5.5 ml ) was added 
 1,3-dimethyl-1h - pyrazole-4-carbaldehyde ( 0.024 g , 
 0.19 mmol ) followed by a freshly prepared aqueous solution of na2s2o4 ( 1 m ; 0.90 ml , 0.90 mmol ) .
the reaction 
 mixture was stirred at 80 c for 22 h , then allowed to cool to 
 room temperature , and concentrated in vacuo .
the residue was absorbed 
 on silica gel ( 1.7 g ) , and the free - running powder was placed on a 
 10 g isolute silica column which was eluted with ch2cl2 , dichloromethane / ethyl acetate ( 1:1 , v / v ) , 2% methanol in 
 dichloromethane / ethyl acetate ( 1:1 , v / v ) , and finally 3% methanol 
 in dichloromethane / ethyl acetate ( 1:1 , v / v ) .
the title compound was 
 obtained as an off - white solid after trituration with diethyl ether / ch2cl2 ( 1:1 , v / v ) ( 0.012 g , 15% ) : h nmr 
 ( 500 mhz , dmso - d6 ) 2.22 ( s , 3h , pyrazole 
 3-me ) , 3.82 ( s , 3h , pyrazole n - me ) , 7.08 ( t , j = 7.4 hz , 1h , p - phh ) , 7.19 ( d , j = 7.2 hz , 2h , arh ) , 7.33 ( t , j = 7.8 
 hz , 2h , m - phh ) , 7.78 ( d , j = 7.9 
 hz , 2h , arh ) , 7.89 ( d , j = 8.7 hz , 2h , arh ) , 8.16 
 ( s , 1h , pyrazole 5-h or imidazo[4,5-b]pyridine 5-h ) , 
 8.17 ( s , 1h , pyrazole 5-h or imidazo[4,5-b]pyridine 
 5-h ) , 8.74 ( s , 1h , nhar ) , 9.97 ( s , 1h , nhar ) , 13.03 ( s , 1h , imidazo[4,5-b]pyridine nh ) ; lc / ms ( esi , m / z ) tr = 2.36 min , 458 , 460 [ ( m + h ) , cl isotopic pattern ] ; hrms m / z found 458.1493 , calcd for c24h21cln7o ( m + h ) 458.1491 . to a solution 
 of 4-hydroxy - n - phenylbenzamide ( 0.068 g , 
 0.32 mmol ) in anhydrous dmf ( 2.3 ml ) was added potassium tert - butoxide ( 0.036 g , 0.32 mmol ) .
the solution was stirred at room 
 temperature for 20 min under argon , and then 4,5-dichloro-3-nitropyridin-2-amine 
 ( 0.062 g , 0.30 mmol ) was added portionwise over a 5 min period . stirring 

was continued at room temperature for 1 h and 10 min under argon ; 
 the reaction mixture was then partitioned between ethyl acetate ( 110 
 ml ) and h2o ( 20 ml ) .
the organic layer was washed with 
 h2o ( 15 ml ) and brine ( 20 ml ) , dried ( na2so4 ) , and concentrated in vacuo to give a yellow solid .
this 
 was triturated with dichloromethane , the precipitate was collected 
 by filtration and washed with dichloromethane to obtain the title 
 compound as a yellow solid ( 0.087 g , 76% ) : h nmr ( 500 
 mhz , dmso - d6 ) 7.09 ( t , j = 7.4 hz , 1h , p - phh ) , 7.13 ( d , j = 8.8 hz , 2h , arh ) , 7.34 ( t , j = 7.7 hz , 2h , m - phh ) , 7.62 ( br s , 2h , nh2 ) , 7.75 ( d , j = 7.7 hz , 2h , arh ) , 7.95 ( d , j = 8.8 
 hz , 2h , arh ) , 8.46 ( s , 1h , pyridine 6-h ) , 10.24 ( s , 1h , conh ) ; lc / ms 
 ( esi , m / z ) tr = 2.50 min , 385 , 387 [ ( m + h ) , cl isotopic pattern ] . to a mixture 
 of 4-((2-amino-5-chloro-3-nitropyridin-4-yl)oxy)-n - phenylbenzamide ( 0.077 g , 0.20 mmol ) and dmf ( 4.2 ml )
was added 
 1,3-dimethyl-1h - pyrazole-4-carbaldehyde ( 0.030 g , 
 0.24 mmol ) followed by a freshly prepared aqueous solution of na2s2o4 ( 1 m , 1.0 ml , 1.0 mmol ) .
the reaction 
 mixture was stirred at 84 c for 20 h , then allowed to cool to 
 room temperature , and partitioned between etoac ( 180 ml ) and water 
 ( 20 ml ) .
the organic layer washed with more h2o ( 15 ml ) , 
 and brine ( 15 ml ) , dried ( na2so4 ) , and concentrated 
 in vacuo .
the resulting residue was triturated with diethyl ether 
 ( 15 ml ) ; the precipitate was collected by filtration and washed with 
 diethyl ether .
this solid was absorbed on silica gel ( 1.4 g ) , and 
 the free - running powder was placed on a 10 g isolute silica column 
 which was eluted with dichloromethane , dichloromethane / ethyl acetate 
 ( 1:1 , v / v ) , 2.5% methanol in dichloromethane / ethyl acetate ( 1:1 , v / v ) , 
 and finally 4% methanol in dichloromethane / ethyl acetate ( 1:1 , v / v ) . 

the obtained residue was triturated with diethyl ether ; the precipitate 
 was collected by filtration and washed with diethyl ether to give 
 the title compound as an off - white solid ( 0.010 g , 11% ) : h nmr ( 500 mhz , dmso - d6 ) 2.21 ( s , 3h , 
 pyrazole 3-me ) , 3.82 ( s , 3h , pyrazole n - me ) , 7.09 
 ( t , j = 7.4 hz , 1h , p - phh ) , 7.18 
 ( br d , j = 7.5 hz , 2h , arh ) , 7.34 ( t , j = 7.8 hz , 2h , m - phh ) , 7.75 ( d , j = 8.0 hz , 2h , arh ) , 7.98 ( d , j = 8.8 hz , 2h , arh ) , 
 8.21 ( br s , 1h , pyrazole 5-h or imidazo[4,5-b]pyridine 
 5-h ) , 8.38 ( br s , 1h , pyrazole 5-h or imidazo[4,5-b]pyridine 5-h ) , 10.16 ( s , 1h , conh ) , 13.35 ( s , 1h , imidazo[4,5-b]pyridine nh ) ; lc / ms ( esi , m / z ) tr = 2.56 min , 459 , 461 [ ( m + h ) , cl isotopic pattern ] ; hrms m / z found 459.1348 , calcd for c24h20cln6o2 ( m + h ) 459.1331 . to a round - bottom flask containing 
 4-acetoxybenzoic acid ( 0.306 g , 
 1.70 mmol ) was slowly added a solution of oxalyl chloride ( 0.430 g , 
 3.40 mmol ) in anhydrous dichloromethane ( 2.0 ml ) under argon . to this 
 mixture
was slowly added anhydrous dmf ( 0.030 ml ) , and a clear solution 
 was obtained .
the solution was stirred at room temperature for 45 
 min under argon and then concentrated in vacuo , and the residue was 
 dissolved in anhydrous dichloromethane ( 3.0 ml ) .
cyclobutylamine ( 0.363 
 g , 5.10 mmol ) was slowly added ; a precipitate had formed by completion 
 of the amine addition .
then the reaction mixture was diluted with anhydrous dichloromethane 
 ( 2.0 ml ) , and the precipitate was removed by filtration and washed 
 with dichloromethane ( 4  3 ml ) .
the filtrate was concentrated 
 in vacuo and absorbed on silica gel ( 1.6 g ) , and the free - running 
 powder was placed on a 20 g isolute silica column which eluted with 
 dichloromethane and then 5% , 10% , and 15% etoac in dichloromethane . 

the obtained product was dissolved in etoac ( 150 ml ) , and the solution 
 was washed with saturated aqueous nahco3 ( 3  20 ml ) , 
 dried ( na2so4 ) , and concentrated in vacuo to 
 give the title compound as a white solid ( 0.150 g , 38% ) : h nmr ( 500 mhz , dmso - d6 ) 1.66 ( m , 2h , 
 cyclobutylamine ch ) , 2.06 ( m , 2h , cyclobutylamine ch ) , 2.20 ( m , 2h , 
 cyclobutylamine ch ) , 2.28 ( s , 3h , coch3 ) , 4.39 ( m , 1h , 
 nhch ) , 7.20 ( d , j = 8.6 hz , 2h , 
 arh ) , 7.87 ( d , j = 8.6 hz , 2h , arh ) , 8.57 ( d , j = 7.4 hz , 1h , conh ) ; lc / ms ( esi , m / z ) tr = 1.91 min , 234 ( m + h ) . to a mixture of 4-(cyclobutylcarbamoyl)phenyl 
 acetate 
 ( 0.145 g , 0.62 mmol ) in methanol ( 4.0 ml )
was added an aqueous solution 
 of naoh ( 1 m , 1.6 ml , 1.6 mmol ) followed by water ( 0.4 ml ) ; a clear 
 solution was obtained .
the reaction mixture was stirred at room temperature 
 for 1 h and 20 min .
approximately half of the solvent was then removed 
 under reduced pressure , and the remaining solution was diluted with 
 water ( 2.0 ml ) .
the ph was adjusted to about 7 with 1 m hcl ; a white 
 suspension was obtained which was diluted with water ( 5 ml ) .
this 
 was extracted with etoac ( 3  50 ml ) , and the combined extracts 
 were dried ( na2so4 ) and concentrated in vacuo 
 to give the title compound as a white solid ( 0.105 g , 89% ) : h nmr ( 500 mhz , dmso - d6 ) 1.67 ( m , 2h , 
 cyclobutylamine ch ) , 2.05 ( m , 2h , cyclobutylamine ch ) , 2.18 ( m , 2h , 
 cyclobutylamine ch ) , 4.37 ( m , 1h , nhch ) , 6.77 ( d , j = 8.7 hz , 2h , arh ) , 7.70 ( d , j = 8.7 
 hz , 2h , arh ) , 8.28 ( d , j = 7.5 hz , 1h , conh ) , 9.87 
 ( s , 1h , oh ) ; lc / ms ( esi , m / z ) tr = 1.66 min,192 ( m + h ) . to a solution 
 of n - cyclobutyl-4-hydroxybenzamide ( 0.100 g , 0.52 
 mmol ) in 
 anhydrous dmf ( 3.3 ml )
then 4,5-dichloro-3-nitropyridin-2-amine ( 0.102 
 g , 0.49 mmol ) was added portionwise over a 5 min period .
stirring 
 was continued at room temperature for 1 h and 30 min under argon ; 
 the reaction mixture was then partitioned between ethyl acetate ( 200 
 ml)/dichloromethane ( 5 ml ) and h2o ( 30 ml ) .
the organic 
 layer was washed with h2o ( 20 ml ) , brine ( 30 ml ) , saturated 
 aqueous nahco3 ( 20 ml ) , and brine ( 20 ml ) , dried ( na2so4 ) , and concentrated in vacuo to give a yellow 
 solid .
this solid was triturated with etoac / dichloromethane ( 1:1 , 
 v / v ; approximately 10 ml ) ; the precipitate was collected by filtration 
 and washed with dichloromethane to afford the title compound as a 
 yellow solid ( 0.120 g , 67% ) : h nmr ( 500 mhz , dmso - d6 ) 1.66 ( m , 2h , cyclobutylamine ch ) , 2.05 ( m , 
 2h , cyclobutylamine ch ) , 2.20 ( m , 2h , cyclobutylamine ch ) , 4.38 ( m , 
 1h , nhch ) , 7.04 ( d , j = 8.9 hz , 
 2h , arh ) , 7.54 ( s , 2h , nh2 ) , 7.83 ( d , j = 8.9 hz , 2h , arh ) , 8.43 ( s , 1h , pyridine 6-h ) , 8.53 ( d , j = 7.5 hz , 1h , conh ) ; lc / ms ( esi , m / z ) tr = 2.31 min , 363 , 365 [ ( m 
 + h ) , cl isotopic pattern ] . to a mixture 
 of 4-((2-amino-5-chloro-3-nitropyridin-4-yl)oxy)-n - cyclobutylbenzamide ( 0.094 g , 0.26 mmol ) and dmf ( 5.9 ml )
was added 
 1,3-dimethyl-1h - pyrazole-4-carbaldehyde ( 0.038 g , 
 0.31 mmol ) followed by a freshly prepared aqueous solution of na2s2o4 ( 1 m , 1.3 ml,1.30 mmol ) .
the reaction 
 mixture was stirred at 85 c for 24 h , then allowed to cool to 
 room temperature , and partitioned between etoac ( 220 ml ) and water 
 ( 30 ml ) .
the organic layer was washed with more h2o ( 20 
 ml ) and brine ( 20 ml ) , dried ( na2so4 ) , and concentrated 
 in vacuo .
the residue was treated with diethyl ether ( approximately 
 15 ml ) ; a brown precipitate was obtained which was collected by filtration 
 and washed with diethyl ether . this solid was absorbed on silica gel 
 ( 1.2 g ) , and the free - running powder was placed on a 10 g isolute 
 silica column which was eluted with dichloromethane , dichloromethane / ethyl 
 acetate ( 1:1 , v / v ) , 2.5% methanol in dichloromethane / ethyl acetate 
 ( 1:1 , v / v ) , and finally 4% methanol in dichloromethane / ethyl acetate 
 ( 1:1 , v / v ) .
the obtained residue was triturated with diethyl ether ; 
 the precipitate was collected by filtration and washed with diethyl 
 ether to give the title compound as an off - white solid ( 0.016 g , 14% ) : h nmr ( 500 mhz , dmso - d6 ) 1.67 
 ( m , 2h , cyclobutylamine ch ) , 2.06 ( m , 2h , cyclobutylamine ch ) , 2.19 
 ( m , 5h , cyclobutylamine ch and pyrazole 3-me ) , 3.82 ( s , 3h , pyrazole 
 n - me ) , 4.40 ( m , 1h , nhch ) , 7.10 ( d , j = 8.1 hz , 2h , arh ) , 7.85 ( d , j = 8.7 hz , 2h , arh ) , 
 8.19 ( s , 1h ) and 8.34 ( s , 1h ) ( pyrazole 5-h and imidazo[4,5-b]pyridine 5-h ) , 8.51 ( d , j = 7.2 hz , 1h , 
 conh ) , 13.31 ( s , 1h , imidazo[4,5-b]pyridine nh ) ; 
 lc / ms ( esi , m / z ) tr = 2.44 min , 437 , 439 [ ( m + h ) , cl isotopic 
 pattern ] ; hrms m / z found 437.1492 , 
 calcd for c22h22cln6o2 ( m + h ) 437.1487 .
 aurora - a differs from aurora - b / c 
 at three positions in the atp - binding 
 pocket ( l215 , t217 , and r220 ) . exploiting these differences , crystal 
 structures of ligand  
 aurora protein interactions formed the 
 basis of a design principle for imidazo[4,5-b]pyridine - derived 
 aurora - a - selective inhibitors . 
 guided by a computational modeling 
 approach , appropriate c7-imidazo[4,5-b]pyridine derivatization 
 led to the discovery of highly selective inhibitors , such as compound 28c , of aurora - a over aurora - b . in hct116 human colon carcinoma 
 cells , 28c and 40f 
 inhibited the aurora - a 
 l215r and r220k mutants with ic50 values similar to those 
 seen for the aurora - a wild type . 
 however , the aurora - a t217e mutant 
 was significantly less sensitive to inhibition by 28c and 40f compared to the aurora - a wild type , suggesting 
 that the t217 residue plays a critical role in governing the observed 
 isoform selectivity for aurora - a inhibition . these compounds are useful 
 small - molecule chemical tools to further explore the function of aurora - a 
 in cells . 
Introduction
Chemistry
Results and Discussion
Experimental Section