malaria is one of the most prevalent infectious 
 diseases of the 
 developing world . in excess of 3 billion people
are at risk , and it 
 currently leads to the deaths of around 655,000 people each year , 
 with the majority of these occurring in sub - saharan africa among children 
 under five years of age . resistance to 
 existing antimalarial drugs is widespread , and therefore , new therapeutic approaches are urgently needed .
calcium - dependent 
 protein kinases ( cdpks ) are directly regulated by ca and 
 are found in plants and organisms in the alveolate lineage , but they are absent in humans .
they are present 
 in apicomplexan parasites including plasmodium 
 falciparum , the causative agent of the most severe form of 
 malaria .
cdpks in plasmodium are present as a multigene 
 family containing at least five members , and different cdpks are proposed to be functional at different stages 
 of the parasite life cycle .
falciparum calcium - dependent 
 protein kinase 1 ( pfcdpk1 ) , first identified by zhao 
 et al . , is expressed in the asexual blood 
 stages of the parasite responsible for disease pathology .
it has been 
 shown to be encoded by an important gene , and 
 it is implicated in parasite motility and host cell invasion , where 
 it is able to phosphorylate components of the molecular motor that 
 drives parasite invasion of red blood cells .
the 
 prevention of this invasion process could break the parasite lifecycle , 
 causing the parasites to die and the infection to be cleared .
pfcdpk1 therefore represents a novel target for the potential 
 treatment of malaria and offers promise for achieving selectivity 
 over the kinases of the human host .
more recently , its role in translational 
 regulation of motor complex transcripts in gametocytes and in schizont development has also been reported .
there has been interest in cdpks 
 as drug targets , although relatively 
 few inhibitors have been reported in the literature : kato et al . and
lemercier et al . have reported inhibitors of pfcdpk1 , and inhibitors 
 of the cdpk1 enzymes from the related apicomplexan protozoa toxoplasma gondii and cryptosporidium 
 parvum have also been described .
a high 
 throughput screen of our compound collection against the 
 isolated recombinant pfcdpk1 enzyme identified a 
 hit series containing an imidazopyridazine core as the primary series 
 of interest , and the initial development of the structure  activity 
 relationship ( sar ) in this series has been 
 described previously .
compounds with 
 potent enzyme inhibitory activity had been generated , which also showed 
 good kinase selectivity against a human kinase panel and promising in vitro adme profiles .
in particular , compound 1 ( figure 1 ) represented an early lead , with 
 low nanomolar inhibitory potency against pfcdpk1 , 
 sub-500 nm antiparasite activity , and modest in vivo efficacy in a p. berghei mouse model of malaria .
improvements were sought 
 in the in vitro antiparasite activity and pharmacokinetic 
 profile 
 of the series while maintaining a good selectivity profile against 
 human kinases to generate compounds with the potential to show improved in vivo efficacy .
a structure - guided 
 design approach using a homology model of pfcdpk1 
 ( based on tgcdpk1 , pdb ref : 3i7c ) was used in attempting to gain increased binding affinity 
 against the target and correspondingly increase the cellular potency 
 of the inhibitors .
the homology model had proved effective in explaining 
 the sar up to this point , and it was therefore used as a key component 
 in considering how additional potency could be gained . in particular , 
 it suggested that the binding pocket occupied by the isopentyl chain 
 of compound 1 was not optimally filled and that there 
 was potential to gain additional beneficial interactions with the 
 enzyme in this region .
virtual libraries were enumerated with a diverse 
 range of groups at this position and examined through docking using 
 glide sp . for enumeration purposes ,
the 
 basic amine group was set to be either n - methylpiperidine 
 or 1,4-diaminocyclohexane , which had been previously demonstrated 
 to be optimal for potency , and the heteroaryl 
 linker ring was set as either pyridine or pyrimidine .
analysis of 
 the docking results suggested that replacement of the isopentyl group 
 with an aromatic ring containing a suitably positioned hydrogen - bond 
 acceptor ( for example , a 2-pyridyl group , as in compound 2 ; figure 2 ) could 
 give increased binding affinity .
example of the 2-pyridyl variant with 
 superior predicted binding 
 affinity from docking studies . in comparison with the isopentyl group in this pocket ,
the 
 2-pyridyl 
 nitrogen atom could potentially form an additional hydrogen - bond interaction 
 with the backbone n - h of asp-212 ( depicted in figure 3b ) , and it also appeared to be an excellent spatial and electrostatic 
 fit into the rest of this pocket .
compound 2 was predicted 
 to be able to retain the other key interactions made by compound 1 : a hydrogen bond between the imidazopyridazine core and 
 the backbone n - h of tyr-148 at the hinge region , a hydrogen bond with 
 the side - chain carboxylic acid of asp-212 , and an additional hydrogen 
 bond between the basic amine group and glu-152 as it points out toward 
 the solvent . proposed binding modes from the docking of compound 1 ( a ) and compound 2 ( b ) illustrating the potential 
 to 
 gain an additional h - bond interaction with the backbone n - h of asp-212 .
the top scoring compounds identified 
 from the docking studies were 
 then synthesized according to the route in scheme 1 .
intermediate 3 was functionalized through nucleophilic 
 substitution on the chloro- at the 6-position to install the boc - protected 
 amine side chains in intermediates 4 and 17 .
these were elaborated through suzuki coupling with the appropriate 
 boronate reagents to give the 5-aminopyrimidine products 5 and 18 , the 5-thiomethylpyrimidine 13 or 
 5-aminopyridine 20 .
the final compounds were prepared 
 by either a buchwald coupling with an aryl halide or through oxidation 
 of the thiomethyl pyrimidine followed by nucleophilic displacement 
 with the appropriate amine .
reagents and conditions : ( a ) trans - cyclohexane-1,4-diamine , nmp , microwave , 180 c , 
 then di - tert - butyl dicarbonate , dmap , et3n , thf , reflux ; ( b ) 2-aminopyrimidine-5-boronic acid pinacol ester , 
 pd(dppf)cl2 , cs2co3 , dioxane / water , 
 90 c ; ( c ) aryl / heteroaryl halide , pd(oac)2 , cypf - bu or xantphos , naobu or cs2co3 , dme or dioxane , 80 c ; 
 ( d ) 4 m hcl / dioxane , meoh ; ( e ) 2-(thiomethyl)pyrimidine-5-boronic 
 acid , pd(dppf)cl2 , cs2co3 , dioxane / water , 
 90 c ; ( f ) m - chloroperoxybenzoic acid , ch2cl2 ; ( g ) 3-(aminomethyl)pyridine or 2-(aminomethyl)pyridine 
 or ( 1-methyl-1h - pyrazol-3-yl)methylamine , dioxane , 
 reflux ; ( h ) tert - butyl 4-aminopiperidine-1-carboxylate , 
 dipea , nmp , 130 c ; ( i ) 2-chloro-3-fluoropyridine , pd(oac)2 , xantphos , cs2co3 , dioxane , microwave , 
 130 c or thermal , 90 c ; ( j ) formaldehyde , acoh , sodium 
 triacetoxyborohydride , thf ; ( k ) 2-aminopyridine-5-boronic acid pinacol 
 ester , pd(dppf)cl2 , cs2co3 , dioxane / water , 
 90 c . the sar of the synthesized analogues 
 is shown in table 1 . as predicted by the docking 
 studies ,
variants 
 containing a 2-pyridyl or phenyl group at this position both showed 
 increased binding affinity to the enzyme versus compound 1 ( table 1 , examples 2 and 6 ) . as the potency of the most potent compounds was now below 
 the limit of detection of our primary kinase glo enzyme assay , a thermal 
 denaturation assay was used to quantify the differences and define 
 a rank order in binding affinity between the most potent compounds .
this revealed that the 2-pyridyl compound 2 resulted in a larger shift in the thermal denaturation temperature 
 of the protein ( tm ) than its phenyl 
 counterpart 6 , and both showed a higher thermal shift 
 than compound 1 in this assay , which displayed a tm of 15.7 k. gratifyingly , this difference in 
 enzyme affinity was reflected in the potency of the compounds against 
 the p. falciparum parasite , with compound 2 showing an ec50 of 80 nm compared with 180 nm for compound 6 .
the limit of detection of the pfcdpk1 kinase glo enzyme assay is 0.010 m ; nt = not tested .
alternative 
 heteroaryl groups were then explored : 2-pyrazine 7 showed 
 good potency , albeit weaker than those of 2 and 6 , but 3-pyridyl 8 and 2-pyrimidyl 9 lost potency against both the enzyme and parasite .
the addition 
 of substituents to the pyridyl ring was investigated : 3-fluoropyridyl 
 gave a boost in potency against both the enzyme and the parasite , 
 with compound 10 displaying a high thermal shift of 28.0 
 k and excellent ec50 of 12 nm against the parasite .
the 
 introduction of 5-position substituents to the pyridine ring such 
 as trifluoromethyl ( 11 ) and methyl ( 12 ) 
 led to excellent enzyme affinity and increased thermal shift values 
 relative to 10 , although their antiparasite potency decreased . 
 when a ch2 spacer group was introduced , the 3-pyridyl variant 14 was relatively weak against the enzyme , whereas the 2-pyridyl 
 variant 15 and the 3-pyrazole 16 showed 
 good enzyme inhibitory potency .
this was again consistent with the 
 predictions of the homology model , which suggested that 15 could form an h - bond with asp-212 , whereas 14 could 
 not .
switching to the n - methylpiperidine basic side chain 
 ( 19 ) gave good potency against the enzyme and the parasite , 
 while changing the pyrimidine linker ring to a pyridyl linker ring 
 ( 21 ) retained high enzyme potency but led to a loss in 
 potency against the parasite .
leading compounds were profiled 
 in in vitro adme 
 assays , and selected data are shown in table 2 .
in general , the compounds had low measured log d values and displayed good stability in both mouse and human microsomes 
 but poor pampa permeability .
kinase selectivity screening against 
 a human kinase panel revealed that they showed good selectivity , and 
 the selectivity profile of compound 10 is shown in figure 4 , in comparison with that of compound 1 .
compound 10 also showed ic50 > 25 m 
 against cyp - p450 isoforms 1a , 2c9 , 2c19 , 2d6 , and 3a4 .
however , when 10 was tested for in vivo efficacy in the 
 4-day peters test ( p. berghei murine model of malaria ) with a 50 mg / kg once daily oral dosing 
 regimen , it showed no significant reduction in parasitemia levels 
 ( 4% reduction ) .
this was thought to be a consequence of low plasma 
 exposure , consistent with poor absorption in accordance with its low 
 permeability .
kinase selectivity data on compounds 1 ( top ) and 10 ( bottom ) screened at 1 m inhibitor 
 concentration 
 against a 66-member human kinase panel ; green , < 50% inhibition ; 
 amber , 5080% inhibition ; and red , > 80% inhibition .
although the introduction of the 
 2-pyridyl group gave improved 
 enzyme and antiparasite potency , poor permeability was seemingly limiting 
 the bioavailability of the compounds when dosed in vivo .
efforts were therefore directed at increasing the permeability 
 to allow sufficient exposure while retaining high potency . in the 
 first case , the pyrimidine linker ring and distal 3-fluoro-2-aminopyridyl 
 group present in compound 10 were retained , and the basic 
 amine portion was modified .
the two amino substituents giving the 
 highest potency up to this point are both highly basic ( calculated 
 pka values for the conjugate acids of 10 and 19 are > 9 in both cases ) , and therefore , attenuating this basicity represents 
 one 
 approach to potentially gaining improved permeability .
compound 10 also contains four hydrogen bond donors ( hbds ) which may 
 be leading to poor permeability .
variations at this portion of the 
 molecule were explored , and selected variants are shown in table 3 . the requirement for a basic amine side chain in 
 order to achieve cell - based activity against the parasite was demonstrated 
 by example 22 , in which the basic side chain was replaced 
 by a methyl group .
although this compound is still relatively potent 
 against the enzyme with an ic50 of 44 nm , there is complete 
 loss of potency against the parasite ; this effect is consistent with 
 the observations of lemercier et al .
the 
 high permeability of 22 also confirms that the basic 
 center is making a significant contribution to the poor permeability 
 of the compounds .
piperazine 23 showed weaker enzyme , 
 and antiparasite potency and no improvement in permeability and although 
 the n - methylpiperazine 24 ( with just 
 one hbd and a calculated pka of 7.1 ) showed 
 improved permeability , it displayed weak antiparasite activity .
the more flexible variants 2729 showed lower enzyme affinity , and although 
 the weakly basic morpholine 27 showed improved permeability , 
 it was inactive against the parasite , whereas piperazine 28 and pyrrolidine 29 both showed low permeability . as 
 the diaminocyclohexane basic group seemed optimal for antiparasite 
 activity , cyclization of the free nh2 into a ring 

was explored as a way to potentially improve permeability , through 
 the attenuation of pka and/or the reduction 
 in the number of hbds .
the cyclized variants 30 and 31 showed good potency against both the enzyme and parasite ; 
 however , a modest improvement in permeability was only achieved with 
 morpholine variant 30 . compounds with oxygen - linked basic 
 amine groups such as piperidine 32 and pyrrolidine 33 were potent against the enzyme and showed improved permeability , 
 but they were inactive against the parasite .
carbon - linked piperidine 
 variant 34 showed good enzyme inhibitory potency but 
 a weak effect against the parasite , coupled with low permeability .
pka of 
 conjugate acid calculated according to ref ( 22 ) ; nt = not tested . in summary , although lowering the 
 pka and hbd count resulted in improved 
 permeability in some cases , this 
 tended to be accompanied by a significant reduction of potency against 
 the parasite .
the best combination was achieved with compound 30 , although it still showed only a weak effect ( 22% reduction 
 in parasitemia ) when tested for in vivo efficacy 
 in the p. berghei model under the same dosing regimen 
 as employed previously . despite complying with property criteria that 
 may normally be expected to be sufficient to allow permeability and 
 oral bioavailability , structure  property relationships suggested 
 that there were stricter requirements for this series and that the 
 desired balance in profile could not be obtained from modifying the 
 basic group alone .
it had been observed that the pyridine linker 
 ring had given higher 
 permeability than the pyrimidine ( comparison of examples 19 and 21 ) , so further variations at this linker ring 
 were then explored .
employing a phenyl ring in this position ( 35 ; see table 4 ) gave 
 a large increase in permeability versus its pyrimidine analogue ( 19 ) , although unfortunately this was accompanied by a significant 
 loss in antiparasite activity , with an ec50 of approximately 
 400 nm . in order to maintain the phenyl ring but mimic the dipole 
 moments of the pyridyl and pyrimidyl rings , fluorophenyl and difluorophenyl 
 variants 36 and 37 were investigated , although 
 this approach failed to restore potent antiparasite activity .
cyclization 
 of the nh2 of the diaminocyclohexane side chain 
 into a pyrrolidine ring with pyridyl or phenyl linker rings gave examples 38 and 39 ; these compounds showed good permeability 
 and marginally improved antiparasite activity relative to the n - methylpiperidines . reverting to the diaminocyclohexane 
 basic group with a free nh2 ( 40 ) gave 
 improved antiparasite activity but reduced permeability , and variants 
 with a pyrazole linker ring were also investigated , and these have 
 been described previously . in summary , although modifications of this linker ring led to significant 
 and steep improvements in pampa permeability , the variations that 
 gave improved permeability versus compound 10 while maintaining 
 good enzyme inhibitory potency were also accompanied by a significant 
 loss in antiparasite activity . although the desired balance in compound 
 profile had not yet been achieved through modification of this group , 
 the higher permeability of these compounds warranted pharmacokinetic 
 studies to confirm that they could achieve good in vivo exposure .
pharmacokinetic profiling in rats 
 revealed that there was a good correlation between in vitro adme and in vivo pk , with compound 35 showing the best pk profile of those tested ( data shown in table 5 ) .
compound 
 dosed as hydrochloride 
 salt , iv in aqueous vehicle containing 0.9% ( w / v ) nacl at 4.5 mg / kg ; 
 po in aqueous vehicle containing 0.5% ( w / v ) hydroxypropylmethylcellulose , 
 0.5% ( v / v ) benzyl alcohol , and 0.4% ( v / v ) tween-80 at 21 mg / kg .
compounds 35 , 38 , and 39 were then advanced to testing in the p. berghei in vivo mouse model , and despite their lower 
 antiparasite potency compared 
 to that of earlier examples such as compound 10 , they 
 showed superior efficacy .
the observed efficacy was still only modest , 
 with best results of 44% and 46% reduction in parasitemia achieved 
 with 35 and 38 , respectively , at a 50 mg / kg 
 oral dose ( table 6 ) .
percent remaining 
 at 30 min . measured in the p. berghei mouse model of malaria , 4-day peters test with 
 oral dosing once 
 daily at 50 mg / kg
; compounds were dosed as monohydrochloride salts , 
 dissolved in 30:70 etoh / tween-80 , and diluted 10-fold with water prior 
 to dosing . in order to evaluate 
 the exposure in the efficacy model ,
blood 
 samples were taken for compound 35 , which revealed that 
 it had achieved a good exposure with total plasma levels of 1660 ng / ml 
 at 1 h ( 10-fold over the antiparasite ec50 ) and 1460 ng / ml 
 at 4 h ( 8.5-fold over the antiparasite ec50 ) .
as the in vivo efficacy 
 model is performed with the rodent parasite p. 
 berghei rather than p. falciparum , there 
 remained a possibility that the low in vivo efficacy 
 achieved might be due to species differences .
significant efficacy 
 differences across the parasite species have been observed in the 
 development of other antimalarial compounds , and therefore , it was 
 desirable to address this possibility
. first , an in silico assessment of pfcdpk1 and pbcdpk1 
 revealed that across their entire protein sequences pbcdpk1 and pfcdpk1 have high homology , with 88% sequence 
 identity and 93% similarity .
furthermore , based on the residues within 
 10  of any atom of atp in the pbcdpk1 crystal 
 structure ( pdb i d : 3q5i ) , there is 100% identity .
docking of the inhibitors into the atp - binding 
 site of pbcdpk1 predicted that they would bind with 
 an affinity similar to that for pfcdpk1 .
this prediction 
 was confirmed through the expression of the recombinant pbcdpk1 enzyme and measurement of the ic50 values , which 
 showed that key compounds bound with similar affinity between the 
 two enzymes ( representative compounds 1 and 35 showed pbcdpk1 ic50 values of 22 and 
 15 nm , respectively ) .
finally , in order to probe in vivo differences , selected compounds were tested in a p. falciparum murine model , in which a severe combined immunodeficient ( scid ) 
 mouse can be injected with human erythrocytes infected with a suitable 
 strain of p. falciparum parasites .
compounds 1 and 35 were tested 
 in this efficacy model with the same dosing regimen as in the p. berghei model ( 50 mg / kg , oral , once daily ) , but disappointingly , 
 no efficacy was observed despite good compound exposure . in 

parallel with in vivo testing , efforts had also been 
 focused on achieving an improved compound profile with respect to 
 the combination of antiparasite potency and pharmacokinetics .
although 
 modification of the linker ring had led to improved pharmacokinetics , 
 this had been achieved at the expense of antiparasite potency , so 
 attention then turned to the distal pyridyl ring as a point of modification 
 to potentially restore potency .
the introduction of substituents around 
 this ring had previously produced compounds with the highest enzyme 
 affinities as determined by thermal denaturation experiments ( table 1 , compounds 11 and 12 ) , 
 so this approach offered the potential to yield increased antiparasite 
 potency .
molecular modeling suggested that there was sufficient space 
 in the binding pocket for small substituents to be accommodated , so 
 a number of variants were synthesized , and the resulting sar is shown 
 in table 7 . with n - methylpiperidine 
 as the basic group and a phenyl linker ring in place ,
the addition 
 of 5-chloro ( 41 ) , 6-methyl ( 42 ) , or 5-fluoro 
 ( 43 ) substituents was well tolerated , with sub-100 nm 
 antiparasite ec50 values for 41 and 42 and acceptable in vitro adme .
the 5- and 
 6-trifluoromethyl substituted pyridines 44 and 45 also showed excellent antiparasite ec50 values 
 of 50 and 30 nm , respectively , with good in vitro adme characteristics .
compounds 4648 , with the diaminocyclohexane basic group , also showed a good balance 
 between antiparasite activity and adme properties ; in particular , 
 compound 48 demonstrated the best profile with an antiparasite 
 ec50 of 80 nm combined with high stability in mouse and 
 human microsomes and acceptable permeability .
compounds 41 and 48 were advanced to 
 the p. berghei in vivo mouse model ; however , they 
 did not show significantly improved efficacy compared to that of previous 
 examples ( table 8) , and a maximum of 51% reduction 
 in parasitemia was achieved with compound 41 .
blood samples 
 revealed that compound 41 had achieved a good exposure 
 ( total plasma concentration 45-fold over in vitro antiparasite ec50 at 4 h and 35-fold at 24 h ) , whereas 48 showed lower exposure , consistent with its lower observed in vivo efficacy .
four - day peters test with once daily 
 oral dosing at 50 mg / kg ; compounds were dosed as monohydrochloride 
 salts , dissolved in 30:70 etoh / tween-80 , and diluted 10-fold with 
 water prior to dosing .
an imidazopyridazine series of pfcdpk1 inhibitors 
 was optimized employing a structure - guided design approach with a 
 homology model of pfcdpk1 .
initial 2-pyridyl variants 
 showed excellent potency in assays against the pfcdpk1 enzyme and p. falciparum parasite but did 
 not possess sufficient permeability to be effective in vivo .
modifications of the basic side chain and aryl linker rings allowed 
 permeability to be improved , but this led to a reduction in antiparasite 
 potency . finally , further optimization of the linker ring and distal 
 pyridyl ring led to molecules possessing well - balanced profiles with 
 respect to potency and in vitro adme and suitable 
 for in vivo dosing , with high resulting compound 
 exposures following oral administration . however , despite the 
 improvements made to the compound profiles 
 compared with those of the early lead compound 1 , the in vivo reduction in parasitemia in a murine p. 
 berghei model of malaria remained modest , with a maximum 
 reduction of 51% with an oral dose of 50 mg / kg for compound 41 .
the reason for this limited effect remains unclear : although 
 the pfcdpk1 enzyme has been shown to be encoded by 
 an important gene in the malaria parasite and high levels of in vivo compound exposure were achieved relative to in vitro antiparasite ec50 , it may be that a 
 higher multiple of this ec50 is required for a longer period 
 of time to produce higher efficacy or that low levels of residual 
 cdpk1 enzyme activity may be sufficient for parasite survival . there 
 may be a level of redundancy allowing a cdpk1-based inhibitory effect 
 to be circumvented , and indeed , a recent report has suggested that 
 cdpk1 may not in fact be required for host cell invasion in the erythrocytic 
 stage in plasmodium berghei but rather that it plays 
 an essential role in the mosquito sexual stages .
differences in parasite biology may also explain the limited 
 effect of the inhibitors in vivo : for example , p. berghei has only a 24-h multiplication cycle and develops 
 in reticulocytes in the mouse compared with a 48-h cycle in mature 
 red cells for p. falciparum , and differences in synchrony 
 of parasite development may also have contributed to the reduced efficacy 
 of the compounds .
further studies on these compounds including understanding 
 their mechanism of action , the identification of additional cdpk1 
 substrates , and the development of better read - outs of activity are 
 ongoing , and these will be reported in future publications .
silica gel chromatography was carried 
 out using a biotage sp4 or isolera mplc system with prepacked silica 
 gel cartridges .
the apparatus is constructed such that the chromatography 
 ( column : either a 19  100 mm ( 5 m ) c-18 waters xbridge 
 or a 19  100 mm ( 5 m ) c-6ph waters xbridge column , both 
 at a flow rate of 40 ml / min ) is monitored by a multiwavelength uv 
 detector ( g1365b manufactured by agilent ) and an mm - es + apci mass 
 spectrometer ( g-1956a , manufactured by agilent ) connected in series , 
 and if the appropriate criteria are met , the sample is collected by 
 an automated fraction collector ( g1364b manufactured by agilent ) . 

collection was triggered by a combination of uv or mass spectrometry 
 or based on time .
typical conditions for the separation process are 
 as follows : the gradient was run over a 7 min period ( gradient at 
 the start , 10% methanol and 90% water ; and gradient at the finish , 
 100% methanol and 0% water ; as buffer , 0.1% formic acid , 0.1% ammonium 
 hydroxide , or 0.1% trifluoroacetic acid was added to the water ) .
purity 
 of all final derivatives for biological testing was confirmed to be 
 > 95% as determined using the following conditions : an agilent hplc 
 instrument with a c-18 xbridge column ( 3.5 m , 4.6  30 
 mm , gradient at the start , 10% acetonitrile and 90% water ; gradient 
 at the finish , 100% acetonitrile and 0% water ; as buffer , either 0.1% 
 ammonium hydroxide or 0.1% trifluoroacetic acid was added to the water ) . 

a flow rate of 3 ml / min was used with uv detection at 254 and 210 
 nm .
the structure of the intermediates and final products was confirmed 
 by h nmr spectroscopy and mass spectrometry .
h nuclear magnetic resonance ( nmr ) spectroscopy was carried out using 
 a jeol ecx400 spectrometer in the stated solvent at around room temperature 
 unless otherwise stated .
characteristic chemical shifts (  ) 
 are given in parts - per - million using conventional abbreviations for 
 the designation of major peaks : e.g. , s , singlet ; d , doublet ; t , triplet ; 
 q , quartet ; dd , doublet of doublets ; and br , broad .
analytical mass 
 spectra were recorded using a mm - es + apci or es mass spectrometer 
 ( g-1956a or g-6120b , manufactured by agilent ) .
( a ) a solution of 3-bromo-6-chloroimidazo[1,2-b]pyridazine 3 ( 1.40 g , 6.02 mmol ) in nmp ( 8 
 ml ) was 
 treated with trans - cyclohexane-1,4-diamine ( 2.05 
 g , 18.0 mmol , 3.0 equiv ) and stirred with microwave heating at 180 
 c for 30 min .
it was then diluted with etoac ( 100 ml ) and washed 
 with water ( 2  100 ml ) .
chromatography on silica gel ( 2 m nh3 in meoh / etoac gradient ) gave trans - n-(3-bromo - imidazo[1,2-b]pyridazin-6-yl)-cyclohexane-1,4-diamine as a pale yellow 
 solid ( 981 mg , 52% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.66 ( d , j = 10.1 hz , 
 1h ) , 7.45 ( s , 1h ) , 6.95 ( br .
d , j = 7.3 hz , 1h ) , 
 6.65 ( d , j = 9.6 hz , 1h ) , 3.633.53 ( m , 1h ) , 
 2.602.52 ( m , 1h ) , 2.082.04 ( m , 2h ) , 1.821.78 
 ( m , 2h ) , 1.281.09 ( m , 4h ) .
m / z ( es + apci ) , 310/312 [ m + h ] .
( b ) a solution 
 of trans - n-(3-bromo - imidazo[1,2-b]pyridazin-6-yl)-cyclohexane-1,4-diamine ( 1.80 g , 5.79 mmol ) in thf 
 ( 20 ml ) was treated with et3n ( 1.21 ml , 8.70 mmol , 1.5 
 equiv ) , di - tert - butyl dicarbonate ( 1.90 g , 8.70 mmol , 
 1.5 equiv ) , and dmap ( 71 mg , 0.58 mmol , 0.1 equiv ) and stirred at 
 reflux for 18 h. concentration under reduced pressure and silica gel 
 chromatography ( 20% meoh / etoac ) gave 4 as a pale yellow 
 solid ( 1.60 g , 67% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.67 ( d , j = 9.6 hz , 1h ) , 
 7.46 ( s , 1h ) , 6.99 ( br .
d , j = 8.2 hz , 1h ) , 6.65 ( d , j = 9.6 hz , 1h ) , 3.593.52 ( m , 1h ) , 3.283.22 ( m , 1h ) , 
 2.112.08 ( m , 2h ) , 1.841.81 ( m , 2h ) , 1.38 ( s , 9h ) , 
 1.301.23 ( m , 4h ) .
m / z ( es 
 + apci ) : 410/412 [ m + h ] .
a mixture of 4 ( 1.20 g , 2.92 mmol , 1.0 equiv ) , 
 2-aminopyrimidine-5-boronic acid pinacol ester ( 970 mg , 4.39 mmol , 
 1.5 equiv ) , cs2co3 ( 3.81 g , 11.7 mmol , 4.0 equiv ) , 
 water ( 6 ml ) , and dioxane ( 12 ml ) was degassed with n2 , 
 then pd(dppf)cl2 ( 238 mg , 0.29 mmol , 0.1 equiv ) was added , 
 and the mixture heated at 90 c for 5 h. the mixture was allowed 
 to cool , then concentrated to dryness and purified by chromatography 
 on silica gel ( 220% meoh / etoac ) to give 5 as 
 a beige solid ( 1.07 g , 86% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.96 ( s , 2h ) , 7.78 ( s , 1h ) , 7.70 
 ( d , j = 9.6 hz , 1h ) , 6.96 ( d , j = 
 6.9 hz , 1h ) , 6.876.78 ( m , 3h ) , 6.62 ( d , j = 9.6 hz , 1h ) , 3.523.42 ( m , 1h ) , 3.303.21 ( m , 1h ) , 
 2.172.08 ( m , 2h ) , 1.901.79 ( m , 2h ) , 1.38 ( s , 9h ) , 
 1.321.21 ( m , 4h ) .
m / z ( es 
 + apci ) : 425 [ m + h ] . compound 5 ( 90 mg , 0.21 mmol , 1.1 equiv ) , 2-chloropyridine 
 ( 18 l , 22 mg , 0.19 mmol , 1.0 equiv ) , and sodium tert - butoxide ( 73 mg , 0.76 mmol , 4.0 equiv ) were added to a prestirred 
 solution of pd(oac)2 ( 4.3 mg , 0.1 equiv ) and cypf - bu ( 10.5 mg , 10 mol % ) in dme ( 1.5 ml ) , and 
 the mixture was heated at 80 c for 3 h. the mixture was allowed 
 to cool , diluted with ch2cl2 , and passed through 
 an isolute silica cartridge ( 1 g ) eluting with ch2cl2/meoh ( 7:3 ) .
the eluent was concentrated under reduced pressure , 
 then stirred in meoh ( 3 ml ) and 4 m hcl / dioxane ( 2 ml ) for 40 min , 
 concentrated under reduced pressure , and purified by prep - hplc to 
 give 2 as a white solid ( 5 mg , 6% ) .
h nmr 
 ( 400 mhz , cd3od )  ppm 9.24 ( s , 2h ) , 8.398.34 
 ( m , 1h ) , 8.298.24 ( m , 1h ) , 7.81 ( s , 1h ) , 7.807.76 
 ( m , 1h ) , 7.62 ( d , j = 10.1 hz , 1h ) , 7.057.00 
 ( m , 1h ) , 6.69 ( d , j = 9.6 hz , 1h ) , 3.733.63 
 ( m , 1h ) , 2.902.80 ( m , 1h ) , 2.312.22 ( m , 2h ) , 2.071.98 
 ( m , 2h ) , 1.501.30 ( m , 4h ) .
m / z ( es + apci ) : 402 [ m + h ] . compound 5 ( 90 mg , 0.21 mmol , 1.1 equiv ) , iodobenzene 
 ( 21 l , 39 mg , 0.19 mmol , 1.0 equiv ) , and sodium tert - butoxide ( 73 mg , 0.76 mmol , 4.0 equiv ) were added to a prestirred 
 solution of pd(oac)2 ( 4.3 mg , 0.1 equiv ) and cypf - bu ( 10.5 mg , 0.1 equiv ) in dioxane ( 1.5 ml ) , 
 and the mixture was heated at 80 c for 40 h. the mixture was 
 allowed to cool , and 4 m hcl / dioxane ( 1 ml ) was added and the mixture 
 stirred for 2 h , then concentrated to dryness , and purified by prep - hplc 
 to give 6 as an off - white solid ( 15 mg , 18% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 9.91 ( s , 1h ) , 9.24 ( s , 2h ) , 7.917.88 ( m , 1h ) , 7.837.77 
 ( m , 2h ) , 7.767.71 ( m , 1h ) , 7.337.26 ( m , 2h ) , 7.036.92 
 ( m , 2h ) , 6.67 ( d , j = 9.6 hz , 1h ) , 3.583.46 
 ( m , 1h ) , 2.662.56 ( m , 1h ) , 2.152.07 ( m , 2h ) , 1.871.78 
 ( m , 2h ) , 1.331.10 ( m , 4h ) .
m / z ( es + apci ) : 401 [ m + h ] . compound 5 ( 80 mg , 0.19 mmol , 1.0 equiv ) , 2-chloropyrazine 
 ( 22 mg , 0.19 mmol , 1.0 equiv ) , pd(oac)2 ( 4.3 mg , 0.1 equiv ) , 
 xantphos ( 11 mg , 0.1 equiv ) , and cs2co3 ( 248 
 mg , 0.76 mmol , 4.0 equiv ) in dioxane ( 1 ml ) under n2 were 
 heated at 90 c for 10 h. the mixture was allowed to cool , and 
 4 m hcl / dioxane ( 1 ml ) was added and the mixture stirred for 2 h then 
 concentrated to dryness .
purification by prep - hplc gave 7 as a white solid ( 8 mg , 10% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 10.52 ( br .
s , 1h ) , 9.52 ( d , j = 1.4 hz , 1h ) , 9.34 ( s , 2h ) , 8.368.34 ( m , 1h ) , 
 8.24 ( d , j = 2.3 hz , 1h ) , 7.97 ( s , 1h ) , 7.77 ( d , j = 9.6 hz , 1h ) , 7.04 ( d , j = 6.9 hz , 1h ) , 
 6.70 ( d , j = 9.6 hz , 1h ) , 3.583.49 ( m , 1h ) , 
 2.762.65 ( m , 1h ) , 2.172.09 ( m , 2h ) , 1.901.82 
 ( m , 2h ) , 1.341.16 ( m , 4h ) .
m / z ( es + apci ) : 403 [ m + h ] . following the method for 6 using 5 and 
 3-bromopyridine , we obtained 8 as an orange / brown solid 
 ( 28% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 10.1410.08 ( m , 1h ) , 9.29 ( s , 2h ) , 8.94 
 ( d , j = 1.8 hz , 1h ) , 8.288.24 ( m , 1h ) , 8.198.16 
 ( m , 1h ) , 7.93 ( s , 1h ) , 7.75 ( d , j = 9.6 hz , 1h ) , 
 7.367.32 ( m , 1h ) , 7.03 ( d , j = 6.9 hz , 1h ) , 
 6.67 ( d , j = 9.6 hz , 1h ) , 3.573.47 ( m , 1h ) , 
 2.662.58 ( m , 1h ) , 2.162.08 ( m , 2h ) , 1.881.80 
 ( m , 2h ) , 1.321.11 ( m , 4h ) .
m / z ( es + apci ) : 402 [ m + h ] . following the method for 7 using 5 and 
 2-chloropyrimidine , we obtained 9 as a white solid ( 10% 
 yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 10.45 ( br .
s , 1h ) , 9.30 ( s , 2h ) , 8.60 ( d , j = 5.0 hz , 2h ) , 7.97 ( s , 1h ) , 7.78 ( d , j = 9.6 hz , 
 1h ) , 7.087.03 ( m , 2h ) , 6.70 ( d , j = 9.6 hz , 
 1h ) , 3.603.50 ( m , 1h ) , 2.822.73 ( m , 1h ) , 2.182.09 
 ( m , 2h ) , 1.921.84 ( m , 2h ) , 1.341.20 ( m , 4h ) .
m / z ( es + apci ) : 403 [ m + h ] . following the method for 7 using 5 and 2-chloro-3-fluoropyridine , we obtained 10 as a 
 yellow solid ( 3% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 10.04 ( br .
s , 1h ) , 9.16 ( s , 2h ) , 
 8.258.19 ( m , 1h ) , 7.87 ( s , 1h ) , 7.777.69 ( m , 2h ) , 
 7.327.23 ( m , 1h ) , 6.99 ( d , j = 6.9 hz , 1h ) , 
 6.67 ( d , j = 10.5 hz , 1h ) , 3.573.44 ( m , 1h ) , 
 2.652.56 ( m , 1h ) , 2.142.03 ( m , 2h ) , 1.861.76 
 ( m , 2h ) , 1.311.08 ( m , 4h ) .
m / z ( es + apci ) : 420 [ m + h ] . following the method for 7 using 2-bromo-3-fluoro-5-(trifluoromethyl)pyridine
h nmr ( 400 mhz , dmso - d6 )  ppm 
 9.26 ( s , 2h ) , 8.61 ( s , 1h ) , 8.25 ( dd , j = 10.5 , 1.8 
 hz , 1h ) , 7.94 ( s , 1h ) , 7.75 ( d , j = 9.6 hz , 1h ) , 
 7.03 ( d , j = 6.9 hz , 1h ) , 6.68 ( d , j = 9.6 hz , 1h ) , 3.563.46 ( m , 1h ) , 2.652.57 ( m , 1h ) , 
 2.132.05 ( m , 2h ) , 1.861.78 ( m , 2h ) , 1.311.09 
 ( m , 4h ) .
m / z ( es + apci ) : 488 [ m + h ] . following the method for 7 using 2-chloro-5-methylpyridine , 
 we obtained 12 as an off - white solid ( 5% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 9.92 ( s , 1h ) , 9.26 ( s , 2h ) , 8.17 ( d , j = 8.7 
 hz , 1h ) , 8.148.11 ( m , 1h ) , 7.92 ( s , 1h ) , 7.75 ( d , j = 9.6 hz , 1h ) , 7.60 ( dd , j = 2.3 , 8.7 
 hz , 1h ) , 7.00 ( d , j = 6.9 hz , 1h ) , 6.67 ( d , j = 9.6 hz , 1h ) , 3.573.47 ( m , 1h ) , 2.652.56 
 ( m , 1h ) , 2.25 ( s , 3h ) , 2.142.07 ( m , 2h ) , 1.861.79 
 ( m , 2h ) , 1.321.13 ( m , 4h ) .
m / z ( es + apci ) : 416 [ m + h ] . following the method for 5 using 2-(thiomethyl)pyrimidine-5-boronic 
 acid , we obtained 13 as a pale yellow solid ( 90% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 9.38 ( s , 2h ) , 8.03 ( s , 1h ) , 7.77 ( d , j = 9.6 
 hz , 1h ) , 7.09 ( br .
d , j = 8.2 hz , 1h ) , 6.71 ( d , j = 
 9.6 hz , 1h ) , 3.533.47 ( m , 1h ) , 3.313.26 ( m , 1h ) , 2.58 
 ( s , 3h ) , 2.152.11 ( m , 2h ) , 1.871.82 ( m , 2h ) , 1.39 
 ( s , 9h ) , 1.351.23 ( m , 4h ) .
m / z ( es + apci ) : 456 [ m + h ] .
compound 13 ( 2.00 g , 4.39 mmol , 1.0 equiv ) 
 in ch2cl2 ( 60 ml ) was treated with mcpba ( 2.38 
 g , 9.66 mmol , 2.2 equiv ) and stirred at rt for 2 h. the mixture was 
 diluted with saturated aqueous na2so3 ( 20 ml ) 
 and ch2cl2 ( 50 ml ) and washed with saturated 
 aqueous nahco3 ( 80 ml ) .
the aqueous layer was extracted 
 with ch2cl2 ( 50 ml ) , then the combined organic 
 layers were washed with brine ( 60 ml ) , dried , and concentrated to 
 give an orange solid ( 2.1 g , 98% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.80 ( s , 2h ) , 8.30 ( s , 1h ) , 7.85 
 ( d , j = 9.6 hz , 1h ) , 7.24 ( br .
d , j = 8.2 hz , 1h ) , 6.82 
 ( d , j = 10.3 hz , 1h ) , 3.603.52 ( m , 1h ) , 3.44 
 ( s , 3h ) , 3.333.25 ( m , 1h ) , 2.172.13 ( m , 2h ) , 1.881.83 
 ( m , 2h ) , 1.39 ( s , 9h ) , 1.371.23 ( m , 4h ) .
a solution of the 
 intermediate ( 60 mg , 0.12 mmol , 1.0 equiv ) in dioxane ( 2 ml ) was treated 
 with 1-(pyridin-3-yl)methanamine ( 54 mg , 0.49 mmol , 4.0 equiv ) and 
 stirred at reflux for 4 h. concentration under reduced pressure , purification 
 by prep - hplc , and treatment with 4 m hcl / dioxane ( 1 ml ) followed by 
 elution through an isolute aminopropyl cartridge gave 14 as an off - white solid ( 12 mg , 24% ) .
h nmr ( 400 mhz , 
 dmso - d6 )  ppm 9.02 ( br .
s , 2h ) , 
 8.58 ( d , j = 1.8 hz , 1h ) , 8.488.38 ( m , 1h ) , 
 8.08 ( t , j = 6.4 hz , 1h ) , 7.787.68 ( m , 3h ) , 
 7.437.27 ( m , 1h ) , 6.94 ( d , j = 6.9 hz , 1h ) , 
 6.62 ( d , j = 9.6 hz , 1h ) , 4.55 ( d , j = 6.4 hz , 2h ) , 3.533.41 ( m , 1h ) , 2.642.55 ( m , 1h ) , 
 2.132.01 ( m , 2h ) , 1.881.75 ( m , 2h ) , 1.331.08 
 ( m , 4h ) .
m / z ( es + apci ) : 416 [ m + h ] . following the method for 14 using 1-(pyridin-2-yl)methanamine , 
 we obtained 15 as an off - white solid ( 27% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 9.00 ( br .
s , 2h ) , 8.50 ( dt , j = 0.9 , 2.5 hz , 
 1h ) , 8.047.96 ( m , 1h ) , 7.787.67 ( m , 3h ) , 7.32 ( d , j = 7.8 hz , 1h ) , 7.24 ( ddd , j = 0.9 , 4.8 , 
 7.6 hz , 1h ) , 6.93 ( d , j = 6.9 hz , 1h ) , 6.62 ( d , j = 9.6 hz , 1h ) , 4.64 ( d , j = 6.4 hz , 2h ) , 
 3.553.40 ( m , 1h ) , 2.662.56 ( m , 1h ) , 2.182.02 
 ( m , 2h ) , 1.891.76 ( m , 2h ) , 1.311.06 ( m , 4h ) .
m / z ( es + apci ) : 416 [ m + h ] . following the method for 14 using 1-(1-methyl-1h - pyrazol-3-yl)methanamine
, we obtained 16 as 
 an off - white solid ( 11% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.00 ( br .
s , 2h ) , 7.817.65 
 ( m , 3h ) , 7.55 ( d , j = 2.3 hz , 1h ) , 6.93 ( d , j = 6.9 hz , 1h ) , 6.62 ( d , j = 9.6 hz , 1h ) , 
 6.13 ( d , j = 2.3 hz , 1h ) , 4.47 ( d , j = 6.0 hz , 2h ) , 3.77 ( s , 3h ) , 3.553.40 ( m , 1h ) , 2.642.54 
 ( m , 1h ) , 2.152.03 ( m , 2h ) , 1.891.74 ( m , 2h ) , 1.371.08 
 ( m , 4h ) .
m / z ( es + apci ) : 419 [ m + h ] . a solution of 3 ( 3.00 g , 12.9 mmol 
 1.0 equiv ) in nmp 
 ( 15 ml ) was treated with tert - butyl 4-aminopiperidine-1-carboxylate 
 ( 5.10 g , 25.8 mmol , 2.0 equiv ) , dipea ( 5.60 ml , 32.3 mmol , 2.5 equiv ) , 
 and heated at 130 c for 5 h. the reaction mixture was diluted 
 with ch2cl2 ( 100 ml ) and washed with deionized 
 water ( 3  150 ml ) .
the separated organic layer was concentrated 
 under reduced pressure and column chromatography 
 ( 1080% etoac / pet ether ) to give 17 as a brown 
 solid ( 3.01 g , 59% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.71 ( d , j = 9.6 hz , 1h ) , 
 7.48 ( s , 1h ) , 7.10 ( br .
d , j = 7.3 hz , 1h ) , 6.68 
 ( d , j = 10.1 hz , 1h ) , 3.873.82 ( m , 3h ) , 3.022.93 
 ( m , 2h ) , 2.021.98 ( m , 2h ) , 1.41 ( s , 9h ) , 1.401.31 
 ( m , 2h ) .
m / z ( es + apci ) : 396/398 [ m + h ] . following the procedure for 5 using 17 , we obtained 18 as a beige solid ( 100% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 8.95 ( s , 2h ) , 7.79 ( s , 1h ) , 7.74 ( d , j = 9.6 
 hz , 1h ) , 7.06 ( d , j = 6.4 hz , 1h ) , 6.85 ( s , 2h ) , 
 6.65 ( d , j = 9.6 hz , 1h ) , 3.933.83 ( m , 2h ) , 
 3.803.71 ( m , 1h ) , 3.012.88 ( m , 2h ) , 2.062.00 
 ( m , 2h ) , 1.42 ( s , 9h ) , 1.391.31 ( m , 2h ) .
m / z ( es + apci ) : 411 [ m + h ] . ( a ) to a mixture of 18 ( 100 mg , 0.24 
 mmol , 1.0 equiv ) , 2-chloro-3-fluoropyridine ( 28 mg , 0.30 mmol , 1.2 
 equiv ) and cs2co3 ( 316 mg , 0.97 mmol , 4.0 equiv ) 
 in dioxane ( 3.0 ml ) was added pd(oac)2 ( 6 mg , 0.1 equiv ) 
 and xantphos ( 16 mg , 0.1 equiv ) .
the reaction mixture was heated in 
 the microwave at 130 c for 50 min , diluted with ch2cl2 ( 20 ml ) , and washed with deionized water ( 20 ml ) . 
 the organic was separated and treated with 4 m hcl / dioxane ( 2 ml ) 
 and methanol ( 5 ml ) .
the product was purified by prep - hplc give a 
 white solid ( 24 mg , 24% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.99 ( s , 1h ) , 9.13 ( s , 2h ) , 8.23 
 ( td , j = 4.6 , 1.4 hz , 1h ) , 7.87 ( s , 1h ) , 7.77 ( d , j = 9.6 hz , 1h ) , 7.767.70 ( m , 1h ) , 7.29 ( ddd , j = 8.4 , 4.9 , 3.7 hz , 1h ) , 7.10 ( d , j = 
 6.9 hz , 1h ) , 6.69 ( d , j = 9.6 hz , 1h ) , 3.833.61 
 ( m , 1h ) , 3.133.03 ( m , 2h ) , 2.782.63 ( m , 2h ) , 2.141.94 
 ( m , 2h ) , 1.591.31 ( m , 2h ) .
m / z ( es + apci ) : 406 [ m + h ] .
( b ) to a solution 
 of 3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-yl}-n-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine 
 ( 30 mg , 0.07 mmol , 1.0 equiv ) in thf ( 0.5 ml ) was added formaldehyde 
 ( 37% aq .
, 6 l , 0.07 mmol , 1.0 equiv ) , acoh ( 8 l , 0.148 
 mmol , 2.0 equiv ) , and sodium triacetoxyborohydride ( 31 mg , 0.015 mmol , 
 2.0 equiv ) .
the reaction mixture was stirred for 2 h at rt and concentrated 
 under reduced pressure .
purification by prep - hplc gave 19 as an off - white solid ( 12 mg , 39% ) .
h nmr ( 400 mhz , 
 dmso - d6 )  ppm 10.01 ( d , j = 4.1 hz , 1h ) , 9.159.12 ( m , 2h ) , 8.248.21 
 ( m , 1h ) , 7.87 ( s , 1h ) , 7.777.69 ( m , 2h ) , 7.29 ( ddd , j = 8.1 , 4.7 , 3.7 hz , 1h ) , 7.097.03 ( m , 1h ) , 6.69 
 ( d , j = 9.6 hz , 1h ) , 3.643.50 ( m , 1h ) , 2.822.70 
 ( m , 2h ) , 2.18 ( s , 3h ) , 2.091.90 ( m , 4h ) , 1.541.31 
 ( m , 2h ) .
m / z ( es + apci ) : 420 [ m + h ] . following the method for 5 using 17 and 2-aminopyridine-5-boronic acid pinacol ester , we obtained 20 as an off - white solid ( 91% yield ) .
h nmr ( 400 
 mhz , dmso - d6 )  ppm 8.69 ( d , j = 1.8 hz , 1h ) , 8.07 ( dd , j = 2.3 , 8.7 
 hz , 1h ) , 7.747.65 ( m , 2h ) , 7.00 ( d , j = 6.4 
 hz , 1h ) , 6.61 ( d , j = 10.1 hz , 1h ) , 6.54 ( dd , j = 0.9 , 8.7 hz , 1h ) , 6.12 ( s , 2h ) , 3.963.83 ( m , 
 2h ) , 3.823.68 ( m , 1h ) , 3.042.84 ( m , 2h ) , 2.111.96 
 ( m , 2h ) , 1.471.27 ( m , 11h ) .
m / z ( es + apci ) : 410 [ m + h ] . following the method for 7 using 20 , we obtained an off - white solid ( 33% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 9.30 ( br .
s , 1h ) , 9.139.01 ( m , 1h ) , 8.42 ( dd , j = 2.5 , 8.9 hz , 1h ) , 8.198.09 ( m , 1h ) , 8.098.04 ( m , 
 1h ) , 7.87 ( s , 1h ) , 7.74 ( d , j = 9.6 hz , 1h ) , 7.65 
 ( ddd , j = 1.4 , 8.0 , 11.2 hz , 1h ) , 7.096.94 
 ( m , 2h ) , 6.68 ( d , j = 10.1 hz , 1h ) , 3.773.59 
 ( m , 1h ) , 3.062.89 ( m , 2h ) , 2.632.52 ( m , 2h ) , 2.071.87 
 ( m , 2h ) , 1.431.20 ( m , 2h ) .
m / z ( es + apci ) : 405 [ m + h ] .
this was then 
 treated following the method for 19b , which gave 21 as a yellow solid ( 22 mg , 76% ) .
h nmr ( 400 
 mhz , dmso - d6 )  ppm 9.31 ( s , 1h ) , 
 9.07 ( d , j = 2.3 hz , 1h ) , 8.458.36 ( m , 1h ) , 
 8.158.09 ( m , 1h ) , 8.06 ( d , j = 8.2 hz , 1h ) , 
 7.87 ( s , 1h ) , 7.75 ( d , j = 9.6 hz , 1h ) , 7.65 ( ddd , j = 1.4 , 8.0 , 11.2 hz , 1h ) , 7.076.94 ( m , 2h ) , 6.69 
 ( d , j = 9.6 hz , 1h ) , 3.60 ( d , j = 
 6.4 hz , 1h ) , 2.812.70 ( m , 2h ) , 2.19 ( s , 3h ) , 2.121.97 
 ( m , 4h ) , 1.601.38 ( m , 2h ) .
m / z ( es + apci ) : 419 [ m + h ] . ( a ) to a solution of 3-amino-6-methylpyridazine ( 5.0 g , 45.8 mmol , 
 1.0 equiv ) in butan-1-ol ( 10 ml ) was added 50% chloroacetaldehyde 
 in water ( 6.4 ml , 50.4 mmol , 1.1 equiv ) .
the reaction mixture was 
 heated at reflux for 4 h , then concentrated under reduced pressure , 
 and purification by column chromatography ( 010% meoh in ch2cl2 ) gave 6-methylimidazo[1,2-b]pyridazine as a beige solid ( 4.2 g , 69% ) .
h nmr ( 400 
 mhz , dmso - d6 )  ppm 8.62 ( d , j = 2.3 hz , 1h ) , 8.41 ( d , j = 9.2 hz , 1h ) , 
 8.32 ( d , j = 2.3 hz , 1h ) , 7.75 ( d , j = 9.2 hz , 1h ) , 2.65 ( s , 3h ) .
b ) to a solution of 6-methylimidazo[1,2-b]pyridazine ( 4.0 g , 30.0 mmol , 1.0 equiv ) in mecn ( 40 ml ) 
 was added n - bromosuccinimide ( 5.9 g , 33.0 mmol , 1.1 
 equiv ) portionwise .
the reaction mixture was stirred overnight at 
 rt , then concentrated under reduced pressure , dissolved in ch2cl2 ( 200 ml ) , and washed with deionized water ( 3 
  200 ml ) .
the organic phase was separated and dried ( mgso4 ) , and purification by column chromatography ( 010% 
 meoh in ch2cl2 ) gave 3-bromo-6-methylimidazo[1,2-b]pyridazine as a red solid ( 2.0 g , 31% ) .
h 
 nmr ( 400 mhz , dmso - d6 )  ppm 8.05 
 ( d , j = 9.2 hz , 1h ) , 7.83 ( s , 1h ) , 7.21 ( d , j = 9.2 hz , 1h ) , 2.58 ( s , 3h ) .
( c ) to a solution of 3-bromo-6-methylimidazo[1,2-b]pyridazine ( 2.0 g , 9.43 mmol , 1.0 equiv ) in dioxane ( 10 
 ml ) was added 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine 
 ( 2.5 g , 11.3 mmol , 1.2 equiv ) , bis(di - tert- butyl(4- 
 dimethylaminophenyl ) phosphine ) dichloropalladium(ii ) ( 332 mg , 0.47 
 mmol , 0.05 equiv ) , and 2 m sodium carbonate ( 18.8 ml , 37.7 mmol , 4.0 
 equiv ) .
the reaction mixture was heated at 80 c for 6 h , concentrated 
 under reduced pressure , and the residue taken up in ch2cl2 ( 100 ml ) .
the organic phase was washed with deionized 
 water ( 3  100 ml ) and dried ( mgso4 ) , and purification 
 by column chromatography ( 010% meoh in ch2cl2 ) gave 5-(6-methylimidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine 
 as an off - white solid ( 205 mg , 10% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.92 ( s , 2h ) , 8.128.00 
 ( m , 2h ) , 7.16 ( d , j = 9.2 hz , 1h ) , 6.93 ( s , 2h ) , 
 2.57 ( s , 3h ) .
m / z ( es + apci ) : 227 [ m + h ] .
( d ) a mixture of 5-(6-methylimidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine ( 95 mg , 0.42 mmol , 1.0 
 equiv ) , 2-chloro-3-fluoropyridine ( 82 mg , 0.63 mmol , 1.5 equiv ) , pd(oac)2 ( 19 mg , 0.08 mmol , 0.2 equiv ) , xantphos ( 49 mg , 0.08 mmol , 
 0.2 equiv ) , and cs2co3 ( 547 mg , 1.68 mmol , 4.0 
 equiv ) in dioxane ( 3 ml ) was heated at 120 c for 2 h. the reaction 
 mixture was diluted with ch2cl2 ( 100 ml ) and 
 washed with deionized water ( 3  100 ml ) .
purification by column 
 chromatography ( 220% meoh in ch2cl2 ) 
 gave 22 as a yellow solid ( 36 mg , 28% ) .
h 
 nmr ( 400 mhz , dmso - d6 )  ppm 10.06 
 ( s , 1h ) , 9.16 ( s , 2h ) , 8.25 ( td , j = 1.3 , 4.7 hz , 
 1h ) , 8.19 ( s , 1h ) , 8.10 ( d , j = 9.2 hz , 1h ) , 7.76 
 ( ddd , j = 10.5 , 8.2 , 1.4 hz , 1h ) , 7.31 ( ddd , j = 3.9 , 4.8 , 8.2 hz , 1h ) , 7.20 ( d , j = 
 9.6 hz , 1h ) , 2.58 ( s , 3h ) .
m / z ( es 
 + apci ) : 322 [ m + h ] . ( a ) following the method for 17 using tert - butyl piperazine-1-carboxylate , we obtained tert - butyl 4-(3-bromoimidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate 
 as a yellow solid ( 33% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.91 ( d , j = 
 10.1 hz , 1h ) , 7.62 ( s , 1h ) , 7.24 ( d , j = 10.1 hz , 
 1h ) , 3.623.42 ( m , 8h ) , 1.43 ( s , 9h ) .
m / z ( es + apci ) : 382/384 [ m + h ] . 

( b ) following the method for 5 using tert - butyl 4-(3-bromoimidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate , 
 we obtained tert - butyl 4-[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]piperazine-1-carboxylate as a brown solid 
 ( 902 mg , 77% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.91 ( s , 2h ) , 7.93 ( d , j = 9.6 hz , 1h ) , 7.90 ( s , 1h ) , 7.20 ( d , j = 10.1 
 hz , 1h ) , 6.86 ( s , 2h ) , 3.49 ( br .
m / z ( es + apci ) : 397 [ m + h ] .
( c ) to a solution of tert - butyl 4-[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]piperazine-1-carboxylate ( 300 mg , 0.76 
 mmol , 1.0 equiv ) , 2-chloro-3-fluoropyridine ( 15 mg , 1.14 mmol , 1.5 
 equiv ) , and cs2co3 ( 990 mg , 3.03 mmol , 4.0 equiv ) 
 in dioxane ( 10 ml ) was added pd(oac)2 ( 50 mg , 0.23 mmol , 
 0.3 equiv ) and xantphos ( 130 mg , 0.23 mmol , 0.3 equiv ) .
the reaction 
 mixture was heated at reflux for 2 h , diluted with ch2cl2 ( 100 ml ) , and washed with deionized water ( 3  100 ml ) . 

purification by column chromatography ( 220% meoh / dcm ) gave 
 112 mg of boc - protected intermediate , which was treated with 4 m hcl 
 in dioxane ( 2 ml ) .
purification by column chromatography 
 ( 530% meoh / dcm ) gave 23 as an off - white solid 
 ( 45 mg , 15% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 10.00 ( s , 1h ) , 9.14 ( s , 2h ) , 8.24 ( td , j = 4.6 , 1.4 hz , 1h ) , 8.00 ( s , 1h ) , 7.91 ( d , j = 9.6 hz , 1h ) , 7.75 ( ddd , j = 10.5 , 8.2 , 1.4 hz , 
 1h ) , 7.30 ( ddd , j = 8.2 , 4.6 , 3.7 hz , 1h ) , 7.22 ( d , j = 10.1 hz , 1h ) , 3.473.37 ( m , 4h ) , 2.902.78 
 ( m , 4h ) .
m / z ( es + apci ) : 392 [ m + h ] . following the method for 19b using 23 , 
 we obtained 24 as an off - white solid ( 41% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 10.00 ( s , 1h ) , 9.13 ( s , 2h ) , 8.24 ( td , j = 4.9 , 
 1.2 hz , 1h ) , 8.00 ( s , 1h ) , 7.93 ( d , j = 10.1 hz , 
 1h ) , 7.75 ( ddd , j = 10.5 , 8.0 , 1.6 hz , 1h ) , 7.30 
 ( ddd , j = 8.1 , 4.7 , 3.7 hz , 1h ) , 7.25 ( d , j = 10.1 hz , 1h ) , 3.593.44 ( m , 4h ) , 2.482.39 
 ( m , 4h ) , 2.22 ( s , 3h ) .
m / z ( es + 
 apci ) : 406 [ m + h ] . ( a ) compound 3 ( 1.00 g , 4.30 mmol , 
 1.0 equiv ) , ( r , s)-3-amino-1-n - boc - pyrrolidine ( 2.00 g , 2.5 equiv ) , and nmp ( 4 ml ) were 
 heated at 140 c for 16 h. the mixture was diluted with etoac 
 ( 80 ml ) and washed with water ( 60 ml ) .
the aqueous layer was extracted 
 with etoac ( 30 ml ) , and the combined organic layers were dried and 
 concentrated under reduced pressure .
purification by silica gel chromatography 
 ( 0.23.5% meoh / etoac ) gave ( r , s)-tert - butyl 3-[(3-bromoimidazo[1,2-b]pyridazin-6-yl)amino]pyrrolidine-1-carboxylate as a brown - yellow 
 solid ( 588 mg , 36% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.73 ( d , j = 9.6 hz , 1h ) , 
 7.50 ( s , 1h ) , 7.457.38 ( m , 1h ) , 6.71 ( d , j = 9.6 hz , 1h ) , 4.314.20 ( m , 1h ) , 3.733.58 ( m , 1h ) , 
 3.423.13 ( m , 3h ) , 2.212.12 ( m , 1h ) , 1.951.83 
 ( m , 1h ) , 1.431.36 ( m , 9h ) .
( b ) following the method for 5 using ( r , s)-tert - butyl 3-[(3-bromoimidazo[1,2-b]pyridazin-6-yl)amino]pyrrolidine-1-carboxylate , 
 we obtained ( r , s)-tert - butyl 3-{[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]amino}pyrrolidine-1-carboxylate as a pale yellow 
 solid ( 272 mg , 45% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.94 ( d , j = 7.8 hz , 2h ) , 
 7.817.73 ( m , 2h ) , 7.357.28 ( m , 1h ) , 6.84 ( br .
s , 2h ) , 
 6.66 ( d , j = 9.6 hz , 1h ) , 4.264.19 ( m , 1h ) , 
 3.683.46 ( m , 1h ) , 3.413.34 ( m , 2h ) , 3.303.18 
 ( m , 1h ) , 2.212.06 ( m , 1h ) , 2.041.87 ( m , 1h ) , 1.39 
 ( d , j = 12.8 hz , 9h ) .
( c ) ( r , s)-tert - butyl 3-{[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]amino}pyrrolidine-1-carboxylate ( 100 mg , 
 0.25 mmol , 1.0 equiv ) , 2-chloro-3-fluoropyridine ( 35 l , 46 
 mg , 0.35 mmol , 1.4 equiv ) , pd(oac)2 ( 11 mg , 0.2 equiv ) , 
 xantphos ( 0.2 equiv ) , and cs2co3 ( 326 mg , 1.00 
 mmol , 4.0 equiv ) in dioxane ( 1.5 ml ) were heated at 100 c for 
 8 h. the mixture was allowed to cool and concentrated under reduced 
 pressure , and purification by silica chromatography ( 110% 
 meoh / etoac ) gave ( r , s)-tert - butyl 3-[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-yl}imidazo[1,2-b]pyridazin-6-yl)amino]pyrrolidine-1-carboxylate as a pale 
 yellow solid ( 68 mg , 55% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.99 ( br .
s , 1h ) , 9.179.08 
 ( m , 2h ) , 8.258.20 ( m , 1h ) , 7.90 ( s , 1h ) , 7.81 ( d , j = 10.1 hz , 1h ) , 7.777.69 ( m , 1h ) , 7.417.34 
 ( m , 1h ) , 7.337.24 ( m , 1h ) , 6.72 ( d , j = 10.1 
 hz , 1h ) , 4.304.20 ( m , 1h ) , 3.713.46 ( m , 1h ) , 3.403.35 
 ( m , 2h ) , 3.303.15 ( m , 1h ) , 2.202.08 ( m , 1h ) , 2.021.92 
 ( m , 1h ) , 1.431.31 ( m , 9h ) .
( d ) ( r , s)-tert - butyl 3-[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-yl}imidazo[1,2-b]pyridazin-6-yl)amino]pyrrolidine-1-carboxylate ( 67 mg , 
 0.14 mmol , 1.0 equiv ) was stirred with 4 m hcl / dioxane ( 1.5 ml ) and 
 meoh ( 1.5 ml ) for 5 h. the mixture was concentrated under reduced 
 pressure , then dissolved in meoh / ch2cl2 and 
 eluted through an isolute aminopropyl cartridge ( 1 g ) , which gave 25 as a beige solid ( 52 mg , 98% ) .
h nmr ( 400 mhz , 
 dmso - d6 )  ppm 9.99 ( br .
s , 1h ) , 
 9.209.12 ( m , 2h ) , 8.268.20 ( m , 1h ) , 7.89 ( s , 1h ) , 
 7.787.71 ( m , 2h ) , 7.327.26 ( m , 1h ) , 7.23 ( d , j = 6.0 hz , 1h ) , 6.69 ( d , j = 9.6 hz , 1h ) , 
 4.164.08 ( m , 1h ) , 3.10 ( dd , j = 11.4 , 6.4 
 hz , 1h ) , 2.952.72 ( m , 3h ) , 2.102.00 ( m , 1h ) , 1.731.63 
 ( m , 1h ) .
m / z ( es ) : 392 
 [ m + h ] . following the method 
 for 19b using 25
h nmr ( 400 mhz , cd3od )  ppm 9.15 ( s , 2h ) , 8.19 ( d , j = 
 5.0 hz , 1h ) , 7.82 ( s , 1h ) , 7.717.59 ( m , 2h ) , 7.267.19 
 ( m , 1h ) , 6.76 ( d , j = 9.6 hz , 1h ) , 4.464.37 
 ( m , 1h ) , 3.273.20 ( m , 1h ) , 3.143.03 ( m , 1h ) , 3.012.93 
 ( m , 1h ) , 2.922.83 ( m , 1h ) , 2.59 ( s , 3h ) , 2.552.42 
 ( m , 1h ) , 2.031.91 ( m , 1h ) .
m / z ( es ) : 406 [ m + h ] . ( a ) following the method 
 for 17 using 
 2-(morpholin-4-yl)ethanamine , we obtained 3-bromo - n-[2-(morpholin-4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine 
 as a yellow oil ( 41% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.69 ( d , j = 
 9.6 hz , 1h ) , 7.47 ( s , 1h ) , 7.09 ( t , j = 5.5 hz , 1h ) , 
 6.74 ( d , j = 9.6 hz , 1h ) , 3.643.49 ( m , 4h ) , 
 3.453.36 ( m , 2h ) , 2.54 ( t , j = 6.6 hz , 2h ) , 
 2.482.39 ( m , 4h ) .
m / z ( es 
 + apci ) : 326/328 [ m + h ] .
( b ) following the 
 method for 5 using 3-bromo - n-[2-(morpholin-4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine , we obtained 3-(2-aminopyrimidin-5-yl)-n-[2-(morpholin-4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine as an off - white solid ( 91% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 8.93 ( s , 2h ) , 7.76 ( s , 1h ) , 7.71 ( d , j = 9.6 hz , 
 1h ) , 7.03 ( t , j = 5.3 hz , 1h ) , 6.83 ( s , 2h ) , 6.70 
 ( d , j = 9.6 hz , 1h ) , 3.58 ( t , j = 
 4.4 hz , 4h ) , 3.433.31 ( m , 2h ) , 2.55 ( t , j = 6.6 hz , 2h ) , 2.472.34 ( m , 4h ) .
m / z ( es + apci ) : 341 [ m + h ] .
c ) following 
 the method for 25c using 3-(2-aminopyrimidin-5-yl)-n-[2-(morpholin-4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine 
 with purification by prep - hplc , we obtained 27 as an 
 off - white solid ( 32% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.97 ( s , 1h ) , 9.16 ( s , 2h ) , 8.23 
 ( td , j = 4.6 , 1.4 hz , 1h ) , 7.88 ( s , 1h ) , 7.797.69 
 ( m , 2h ) , 7.30 ( ddd , j = 8.2 , 4.6 , 3.7 hz , 1h ) , 7.09 
 ( t , j = 5.5 hz , 1h ) , 6.74 ( d , j = 
 9.6 hz , 1h ) , 3.593.49 ( m , 4h ) , 3.443.34 ( m , 2h ) , 2.54 
 ( t , j = 6.6 hz , 2h ) , 2.472.35 ( m , 4h ) .
m / z ( es + apci ) : 436 [ m + h ] . ( a ) following the method 
 for 17 using 
 1-(2-aminoethyl)-4-methylpiperazine , we obtained 3-bromo - n-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine 
 as a brown solid ( 40% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.68 ( d , j = 
 9.6 hz , 1h ) , 7.47 ( s , 1h ) , 7.05 ( t , j = 5.3 hz , 1h ) , 
 6.74 ( d , j = 9.6 hz , 1h ) , 3.423.34 ( m , 2h ) , 
 2.572.51 ( m , 2h ) , 2.502.21 ( m , 8h ) , 2.14 ( s , 3h ) .
m / z ( es + apci ) : 339/341 [ m 
 + h ] . ( b ) following the method for 5 using 
 3-bromo - n-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine , we obtained an off - white solid ( 68% 
 yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.93 ( s , 2h ) , 7.76 ( s , 1h ) , 7.72 ( d , j = 10.1 hz , 1h ) , 7.297.05 ( m , 1h ) , 6.83 ( s , 2h ) , 6.71 ( d , j = 9.6 hz , 1h ) , 3.453.35 ( m , 2h ) , 3.022.52 
 ( m , 13h ) .
m / z ( es + apci ) : 354 [ m + h ] .
( c ) following the method for 25c using 3-(2-aminopyrimidin-5-yl)-n-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine , we obtained 28 after purification 
 by prep - hplc as an off - white solid ( 11% yield ) .
h nmr 
 ( 400 mhz , cd3od )  ppm 9.16 ( s , 2h ) , 8.19 ( d , j = 4.6 hz , 1h ) , 7.79 ( s , 1h ) , 7.687.59 ( m , 2h ) , 
 7.23 ( ddd , j = 8.1 , 4.7 , 3.7 hz , 1h ) , 6.73 ( d , j = 9.6 hz , 1h ) , 3.51 ( t , j = 6.9 hz , 2h ) , 
 2.692.63 ( m , 2h ) , 2.722.36 ( m , 8h ) , 2.29 ( s , 3h ) .
m / z ( es + apci ) : 449 [ m + h ] . ( a ) 3-bromo-6-chloro - imidazo[1,2-b]pyridazine ( 500 mg , 2.15 mmol , 1.0 equiv ) , 1,2-aminoethylpyrrolidine 
 ( 682 l , 614 mg , 5.38 mmol , 2.5 equiv ) , and nmp ( 3 ml ) were 
 heated at 170 c under microwave irradiation for 60 min .
the 
 mixture was diluted with etoac ( 60 ml ) and washed with water ( 50 ml ) . 

the aqueous layer was basified to ph 10 with 2 m naoh ( aq . ) , extracted 
 with etoac ( 40 ml ) , and the combined organic layers were dried ( mgso4 ) and concentrated under reduced pressure .
aqueous 
 ammonia ) gave 3-bromo - n-[2-(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine as a yellow solid ( 450 mg , 68% ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 7.67 ( d , j = 9.6 hz , 1h ) , 7.46 ( s , 1h ) , 7.11 
 ( t , j = 5.3 hz , 1h ) , 6.75 ( d , j = 
 9.6 hz , 1h ) , 3.423.35 ( m , 2h ) , 2.66 ( t , j = 6.6 hz , 2h ) , 2.542.49 ( m , 4h ) , 1.721.65 ( m , 4h ) .
m / z ( es + apci ) : 310/312 [ m 
 + h ] . ( b ) following the procedure for 5 using 
 3-bromo - n-[2-(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine , we obtained 3-(2-aminopyrimidin-5-yl)-n-[2-(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine as an off - white solid ( 91% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 8.93 ( s , 2h ) , 7.787.72 ( m , 2h ) , 7.15 ( br .
s , 1h ) , 6.83 ( s , 
 2h ) , 6.70 ( d , j = 9.6 hz , 1h ) , 3.45 ( br .
s , 2h ) , 
 3.092.61 ( m , 6h ) , 1.851.70 ( m , 4h ) .
m / z ( es + apci ) : 325 [ m + h ] .
( c ) following the procedure for 25c using 3-(2-aminopyrimidin-5-yl)-n-[2-(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine , we obtained 29 as a pale yellow 
 solid ( 60% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.97 ( s , 1 h ) , 9.17 ( s , 2h ) , 8.24 ( td , j = 1.4 , 5.0 hz , 1h ) , 7.89 ( s , 1h ) , 7.777.70 ( m , 
 2h ) , 7.327.27 ( m , 1h ) , 7.10 ( t , j = 5.5 hz , 
 1h ) , 6.76 ( d , j = 9.6 hz , 1h ) , 3.413.33 ( m , 
 2h ) , 2.67 ( t , j = 6.9 hz , 2h ) , 2.482.45 ( m , 
 4h ) , 1.701.63 ( m , 4h ) .
m / z ( es + apci ) : 420 [ m + h ] . ( a ) to a stirred 
 solution of trans - n-(3-bromo - imidazo[1,2-b]pyridazin-6-yl)-cyclohexane-1,4-diamine ( 1.00 g , 3.24 
 mmol , 1.0 equiv ) in n - buoh ( 15 ml ) was added k2co3 ( 2.20 g , 15.94 mmol , 5.0 equiv ) and potassium 
 iodide ( 1.18 g , 7.11 mmol , 2.2 equiv ) at 0 c .
after 15 min , 
 1-chloro-2-(2-chloroethoxy)ethane ( 925 mg , 6.47 mmol , 2.0 equiv ) was 
 added , and the reaction mixture was heated at 100 c for 16 h. 
 the reaction mixture was cooled to rt and filtered .
the filtrate was 
 diluted with dichloromethane ( 50 ml ) and washed with h2o ( 2  20 ml ) and a brine solution ( 20 ml ) .
the organic layer 
 was dried ( na2so4 ) and concentrated , and purification 
 by silica gel chromatography ( 5% meoh / chcl3 ) gave 3-bromo - n-(4-morpholino - trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine as a yellow solid ( 600 mg , 49% ) .
h nmr ( 400 mhz , cdcl3 )  ppm 7.58 ( d , j = 10.0 hz , 1h ) , 7.47 ( s , 1h ) , 6.40 ( d , j = 9.6 hz , 1h ) , 4.27 ( d , j = 6.8 hz , 1h ) , 3.753.69 
 ( m , 5h ) , 2.602.55 ( m , 4h ) , 2.402.20 ( m , 3h ) , 2.052.01 
 ( m , 2h ) , 1.451.40 ( m , 2h ) , 1.151.10 ( m , 2h ) .
( b ) following the method for 5 using 3-bromo - n-(4-morpholino - trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine ( 1.30 g , 3.42 mmol , 1.0 equiv ) , we obtained 
 3-(2-aminopyridin-5-yl)-n-(4-morpholino - trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine as an 
 off - white solid ( 950 mg , 70% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.98 ( s , 2h ) , 7.79 ( s , 1h ) , 7.70 
 ( d , j = 10.0 hz , 1h ) , 6.97 ( d , j = 6.8 hz , 1h ) , 6.86 ( s , 2h ) , 6.62 ( d , j = 10.0 
 hz , 1h ) , 3.573.49 ( m , 4h ) , 2.502.40 ( m , 4h ) , 2.302.10 
 ( m , 4h ) , 2.001.90 ( m , 2h ) , 1.401.20 ( m , 4h ) .
( c ) a mixture of 3-(2-aminopyridin-5-yl)-n-(4-morpholino - trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine 
 ( 150 mg , 0.38 mmol , 1.0 equiv ) , 2-chloro-3-fluoropyridine ( 100 mg , 
 0.76 mmol , 2.0 equiv ) , xantphos ( 22 mg , 0.04 mmol , 0.1 equiv ) , and 
 cs2co3 ( 496 mg , 1.52 mmol , 4.0 equiv ) in 1,4-dioxane 
 ( 8 ml ) was degassed using argon for 45 min .
pd(pph3)4 ( 44 mg , 0.04 mmol , 0.1 equiv ) was added and the mixture further 
 degassed for 45 min .
the reaction mixture was heated at 130 c 
 in a sealed tube for 6 h then cooled to rt , h2o was added 
 ( 30 ml ) and extracted with 20% meoh / chcl3 ( 2  20 
 ml ) .
the combined organic extracts were washed with brine solution 
 ( 20 ml ) , dried ( na2so4 ) , and concentrated .
the 
 crude compound was purified by neutral alumina chromatography ( 2% 
 meoh / chcl3 ) to give 30 as a pale yellow solid 
 ( 120 mg , 64% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 10.02 ( s , 1h ) , 9.16 ( s , 2h ) , 8.22 ( d , j = 4.4 hz , 1h ) , 7.88 ( s , 1 h ) , 7.737.67 ( m , 2h ) , 
 7.317.27 ( m , 1h ) , 7.067.04 ( m , 1h ) , 6.66 ( d , j = 10.0 hz , 1h ) , 3.723.40 ( m , 5h ) , 2.502.40 
 ( m , 4h ) , 2.302.10 ( m , 3h ) , 2.011.80 ( m , 2h ) , 1.401.20 
 ( m , 4h ) .
m / z ( es ) : 490 
 [ m + h ] . ( a )
to a solution of 3 ( 500 mg , 1.61 mmol 1.0 equiv ) 
 and 1,4-dibromobutane ( 291 l , 524 mg , 1.5 equiv ) in anhydrous 
 dmf ( 4 ml ) was added k2co3 ( 670 mg , 4.85 mmol , 
 3.0 equiv ) , and the reaction mixture was heated at 70 c for 
 4 h. the reaction mixture was cooled to rt and concentrated , and purification 
 by neutral alumina chromatography ( 10% meoh / chcl3 ) gave 
 3-bromo - n-4-(pyrrolidin-1-yl)-trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine as an 
 off - white solid ( 150 mg , 25% ) .
h nmr ( 400 mhz , cdcl3 )  ppm 7.58 ( d , j = 9.6 hz , 1h ) , 7.47 
 ( s , 1h ) , 6.41 ( d , j = 10.0 hz , 1h ) , 4.29 ( d , j = 6.8 hz , 1h ) , 3.773.71 ( m , 1h ) , 2.62 ( s , 4h ) , 
 2.272.24 ( m , 2h ) , 2.122.08 ( m , 3h ) , 1.8 ( s , 4h ) , 1.521.46 
 ( m , 2h ) , 1.291.23 ( m , 2h ) .
( b ) a mixture of 3-bromo - n-(4-(pyrrolidin-1-yl)-trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine ( 150 mg , 0.41 mmol , 1.0 equiv ) , 2-aminopyrimidine-5-boronic 
 acid pinacol ester ( 136 mg , 0.61 mmol , 1.5 equiv ) , na2co3 ( 175 mg , 1.65 mmol , 4.0 equiv ) in dmf ( 5 ml ) , and water ( 1 
 ml ) was degassed using argon for 30 min .
( aphos)2pdcl2 ( 30 mg , 5 mol % ) was added , and the reaction mixture was 
 heated at 100 c for 4 h. the mixture was cooled to rt , concentrated , 
 and purified by neutral alumina chromatography ( 1040% meoh / chcl3 ) to give 3-(2-aminopyrimidin-5-yl)-n-(4-(pyrrolidin-1-yl)-trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine 
 as an off - white solid ( 100 mg , 64% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.99 ( s , 2h ) , 7.80 ( s , 1h ) , 6.99 
 ( d , j = 3.2 hz , 1h ) , 6.86 ( s , 2h ) , 6.71 ( s , 1 h ) , 
 6.62 ( d , j = 9.6 hz , 1 h ) , 3.54 ( s , 1h ) , 2.112.01 
 ( m , 5h ) , 1.67 ( s , 4h ) , 1.331.23 ( m , 4h ) , 1.06 ( s , 4h ) .
( c ) a mixture of 3-(2-aminopyrimidin-5-yl)-n-(4-(pyrrolidin-1-yl)-trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine ( 100 mg , 0.26 mmol , 1.0 equiv ) , 2-chloro-3-fluoropyridine 
 ( 52 l , 69 mg , 0.52 mmol , 2.0 equiv ) , xantphos ( 15 mg , 0.02 
 mmol , 0.1 equiv ) , and cs2co3 ( 338 mg , 1.04 mmol , 
 4.0 equiv ) in 1,4-dioxane ( 5 ml ) was degassed using argon for 30 min
. 
 pd(pph3)4 ( 30 mg , 0.02 mmol , 0.1 equiv ) was 
 then added and the mixture further degassed for 15 min .
the reaction 
 mixture was heated at 130 c in a sealed tube for 6 h , then cooled 
 to rt , concentrated , and purified by prep - hplc to give 31 as an off - white solid ( 50 mg , 40% ) .
h nmr ( 400 mhz , 
 cdcl3 )  ppm 12.45 ( s , 1h ) , 9.14 ( s , 2h ) , 8.29 ( d , j = 5.2 hz , 1h ) , 7.777.70 ( m , 2h ) , 7.497.46 
 ( m , 1h ) , 7.087.03 ( m , 1h ) , 6.48 ( d , j = 10.4 
 hz , 1h ) , 4.40 ( d , j=6.8 hz , 1h ) , 3.72 ( m , 2h ) , 3.082.91 
 ( m , 3h ) , 2.422.26 ( m , 6h ) , 2.011.85 ( m , 4h ) , 1.431.31 
 ( m , 3h ) .
m / z ( es ) : 474 
 [ m + h ] . ( a )
to a solution of 1-methylpiperidin-4-ol ( 375 mg , 3.2 mmol , 1.5 
 equiv ) in anhydrous thf ( 15 ml ) was added nah ( 60% in mineral oil , 
 130 mg , 3.2 mmol , 1.5 equiv ) at 0 c .
the mixture was allowed 
 to stir at rt for 30 min , then 3-bromo-6-chloro - imidazo[1,2-b]pyridazine ( 500 mg , 2.14 mmol , 1.0 equiv ) was added and 
 heated to 65 c for 4 h. the mixture was allowed to cool , diluted 
 with etoac ( 100 ml ) , and washed with water ( 50 ml ) . the organic layer 
 was dried ( na2so4 ) and concentrated under reduced 
 pressure , and purification by silica gel chromatography ( 510% 
 meoh / chcl3 ) gave 3-bromo-6-(1-methylpiperidin-4-yloxy)imidazo[1,2-b]pyridazine as a pale yellow solid ( 100 mg , 15% ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 8.04 ( d , j = 10.0 hz , 1h ) , 7.74 ( s , 1h ) , 6.93 
 ( d , j = 9.6 hz , 1h ) , 5.045.00 ( m , 1h ) , 2.752.65 
 ( m , 2h ) , 2.322.24 ( m , 5h ) , 2.152.05 ( m , 2h ) , 1.851.75 
 ( m , 2h ) .
( b ) following the method for 5 using 3-bromo-6-(1-methylpiperidin-4-yloxy)imidazo[1,2-b]pyridazine , we obtained 5-(6-(1-methylpiperidin-4-yloxy)imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine as a pale yellow solid 
 ( 44% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.93 ( s , 2h ) , 8.068.01 ( m , 2h ) , 6.99 
 ( s , 2h ) , 6.86 ( d , j = 9.6 hz , 1h ) , 4.954.85 
 ( m , 1h ) , 2.752.65 ( m , 2h ) , 2.192.08 ( m , 7h ) , 1.791.74 
 ( m , 2h ) .
( c ) following the method for 30c using 5-(6-(1-methylpiperidin-4-yloxy)imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine , we obtained 32 as 
 a pale yellow solid ( 42% yield ) .
h nmr ( 400 mhz , cdcl3 )  ppm 9.16 ( s , 2h ) , 8.30 ( d , j = 
 4.4 hz , 1h ) , 7.87 ( d , j = 8.2 hz , 2h ) , 7.72 ( s , 1h ) , 
 7.497.44 ( m , 1h ) , 7.087.04 ( m , 1h ) , 6.75 ( d , j = 10.0 hz , 1h ) , 5.105.00 ( m , 1h ) , 2.702.65 
 ( m , 2h ) , 2.452.35 ( m , 2h ) , 2.33 ( s , 3h ) , 2.132.10 
 ( m , 2h ) , 1.941.92 ( m , 2h ) .
m / z ( apci ) : 421 [ m + h ] . ( a ) following the method for 32a using ( r , s)-1-methylpyrrolidin-3-ol , we obtained ( r , s)-3-bromo-6-[(1-methylpyrrolidin-3-yl)oxy]imidazo[1,2-b]pyridazine as a pale yellow solid ( 1.4 g , crude ) .
h nmr ( 400 mhz , cdcl3 )  ppm 7.74 ( d , j = 9.6 hz , 1h ) , 7.59 ( s , 1h ) , 6.73 ( d , j = 8.0 hz , 1h ) , 5.485.44 ( m , 1h ) , 2.972.89 ( m , 2h ) , 
 2.822.78 ( m , 1h ) , 2.522.45 ( m , 1h ) , 2.42 ( s , 3h ) , 
 2.402.34 ( m , 1h ) , 2.102.04 ( m , 1h ) .
( b ) following the method for 5 using ( r , s)-3-bromo-6-[(1-methylpyrrolidin-3-yl)oxy]imidazo[1,2-b]pyridazine , we obtained ( r , s)-5-(6-(1-methylpyrrolidin-3-yloxy)imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine as a pale yellow solid ( 39% ) .
h nmr ( 400 mhz , cdcl3 )  ppm 8.93 ( s , 2h ) , 
 7.84 ( d , j = 9.2 hz , 1h ) , 7.81 ( s , 1h ) , 6.75 ( d , j = 8.0 hz , 1h ) , 5.405.35 ( m , 1h ) , 5.22 ( br .
s , 
 2h ) , 3.052.97 ( m , 2h ) , 2.702.65 ( m , 1h ) , 2.42 ( s , 
 3h ) , 2.352.30 ( m , 1h ) , 2.112.03 ( m , 2h ) .
( c ) following the method for 30c using ( r , s)-5-(6-(1-methylpyrrolidin-3-yloxy)imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine , we obtained 33 as a pale yellow solid ( 90 mg , 53% ) .
h nmr ( 400 mhz , 
 cdcl3 )  ppm 9.18 ( s , 2h ) , 8.30 ( d , j = 4.4 hz , 1h ) , 7.88 ( s , 1h ) , 7.86 ( d , j = 9.6 hz , 
 1h ) , 7.74 ( s , 1h ) , 7.47 ( t , j = 8.0 hz , 1h ) , 7.07 
 ( m , 1h ) , 6.78 ( d , j = 8.0 hz , 1h ) , 5.455.40 
 ( m , 1h ) , 3.042.94 ( m , 2h ) , 2.702.65 ( m , 1h ) , 2.512.43 
 ( m , 1h ) , 2.41 ( s , 3h ) , 2.362.27 ( m , 1h ) , 2.112.04 
 ( m , 1h ) .
m / z ( apci ) : 
 325 [ m + h ] . ( a ) to a solution of 1-boc-4-methylene - piperidine ( 1.29 g , 6.5 mmol , 
 2.0 equiv ) in dry thf ( 10 ml ) under nitrogen was added 9-bbn ( 0.5 
 m in thf , 16.3 ml , 8.16 mmol , 2.5 equiv ) .
the reaction mixture was 
 heated at 75 c for 3 h. after cooling , the resulting solution 
 was added to a mixture of 6-chloroimidazo[1,2-b]pyridazine 
 ( 0.5 g , 3.2 mmol ) , pd(dppf)cl2 ( 133 mg , 0.16 mmol , 0.05 
 equiv ) , and k2co3 ( 1.35 g , 9.8 mmol , 3.0 equiv ) 
 in dmf ( 5 ml ) and water ( 1 ml ) .
the reaction mixture was heated at 
 75 c for 16 h then concentrated under reduced pressure .
the 
 residue was dissolved in etoac and washed with water ( 50 ml ) and brine 
 ( 20 ml ) .
purification by chromatography on silica gel 
 gave tert - butyl 4-(imidazo[1,2-b]pyridazin-6-ylmethyl)piperidine-1-carboxylate as a solid ( 0.406 
 g , 40% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.21 ( s , 1h ) , 8.03 ( d , j = 9.1 hz , 
 1h ) , 7.71 ( s , 1h ) , 7.16 ( d , j = 9.1 hz , 1h ) , 3.91 
 ( m , 2h ) , 2.792.55 ( m , 4h ) , 1.92 ( m , 1h ) , 1.651.52 
 ( m , 2h ) , 1.38 ( s , 9h ) , 1.220.99 ( m , 2h ) .
m / z ( es + apci ) : 317 [ m + h ] .
( b ) to a solution of tert - butyl 4-(imidazo[1,2-b]pyridazin-6-ylmethyl)piperidine-1-carboxylate ( 0.40 g , 
 1.26 mmol ) in dry ch2cl2 ( 6 ml ) was added dropwise 
 a solution of nbs ( 0.246 g , 1.39 mmol , 1.1 equiv ) in dry acetonitrile 
 ( 6 ml ) .
the reaction mixture was stirred at room temperature for 1 
 h. nbs ( 24 mg , 0.13 mmol ) was added , and the reaction mixture was 
 stirred for a further hour .
the residue was dissolved in ch2cl2 ( 100 ml ) and washed with water ( 2  30 ml ) .
purification by chromatography on silica gel gave tert - butyl 4-[(3-bromoimidazo[1,2-b]pyridazin-6-yl)methyl]piperidine-1-carboxylate 
 as a solid ( 0.42 g , 83% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.09 ( d , j = 
 9.1 hz , 1h ) , 7.86 ( s , 1h ) , 7.26 ( d , j = 9.1 hz , 1h ) , 
 3.963.82 ( m , 2h ) , 2.80 ( d , j = 6.9 hz , 2h ) , 
 2.772.59 ( m , 2h ) , 1.981.87 ( m , 1h ) , 1.641.55 
 ( m , 2h ) , 1.38 ( s , 9h ) , 1.241.01 ( m , 2h ) .
( c ) following the 
 method for 5 , we obtained tert - butyl 
 4-{[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]methyl}piperidine-1-carboxylate 
 as a yellow solid ( 0.170 g , 70% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.93 ( s , 2h ) , 8.10 ( s , 1h ) , 8.09 
 ( d , j = 9.1 hz , 1h ) 7.18 ( d , j = 
 9.1 hz , 1 h ) , 6.93 ( br . s , 2h ) , 3.953.85 ( m , 2h ) , 2.79 ( d , j = 7.3 hz , 2h ) , 2.752.55 ( m , 2h ) , 2.031.89 
 ( m , 1h ) , 1.701.58 ( m , 2h ) , 1.38 ( s , 9h ) , 1.211.03 
 ( m , 2h ) .
m / z ( es + apci ) : 410 [ m + h ] .
( d ) following the method for 25c , we obtained tert - butyl 4-[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-yl}imidazo[1,2-b]pyridazin-6-yl)methyl]piperidine-1-carboxylate as a yellow 
 solid ( 0.10 g , 48% ) .
h nmr ( 400 mhz , cd3od ) 
  ppm 9.21 ( s , 2h ) .
8.248.20 ( m , 1h ) , 8.12 ( s , 1h ) , 
 8.01 ( d , j = 9.6 hz , 1h ) , 7.67 ( ddd , j = 10.2 , 8.4 , 1.6 hz , 1h ) , 7.317.18 ( m , 2h ) , 4.134.01 
 ( m , 2h ) , 2.87 ( d , j = 7.3 hz , 2h ) , 2.852.65 
 ( m , 2h ) , 2.152.05 ( m , 1h ) , 1.801.65 ( m , 2h ) , 1.44 
 ( s , 9h ) , 1.321.15 ( m , 2h ) .
m / z ( es + apci ) : 505 [ m + h ] .
( e ) to a solution 
 of tert - butyl 4-[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-yl}imidazo[1,2-b]pyridazin-6-yl)methyl]piperidine-1-carboxylate ( 0.10 g , 
 0.19 mmol ) in meoh ( 2 ml ) was added 4 m hcl ( 2 ml ) , and the reaction 
 mixture was stirred at room temperature for 2 h. the reaction mixture 
 was concentrated under reduced pressure .
the residue was purified 
 by prep - lcms to give the product as a tfa salt , which was eluted through 
 an isolute aminopropyl cartridge to give n-(3-fluoropyridin-2-yl)-5-[6-(piperidin-4-ylmethyl)imidazo[1,2-b]pyridazin-3-yl]pyrimidin-2-amine as a solid ( 75 mg , 94% ) .
h nmr ( 400 mhz , cd3od )  ppm 9.20 ( s , 2h ) , 
 8.248.19 ( m , 1h ) , 8.12 ( s , 1h ) , 8.01 ( d , j = 9.1 hz , 1h ) , 7.67 ( ddd , j = 10.5 , 8.2 , 1.3 hz , 
 1h ) , 7.317.21 ( m , 2h ) , 3.133.04 ( m , 2h ) , 2.88 ( d , j = 7.3 hz , 2h ) , 2.67 ( td , j = 12.5 , 2.7 
 hz , 2h ) , 2.302.07 ( m , 1h ) , 1.77 ( m , 2h ) , 1.34 ( qd , j = 12.5 , 4.1 hz , 2h ) .
m / z ( es + apci ) : 405 [ m + h ] .
( f ) following 
 the procedure for 19b , we obtained 34 as 
 a yellow solid ( 34 mg , 65% ) .
h nmr ( 400 mhz , cd3od )  ppm 9.16 ( s , 2h ) , 8.258.17 ( m , 1h ) , 8.10 ( s , 
 1h ) , 8.01 ( d , j = 9.1 hz , 1h ) , 7.66 ( ddd , j = 10.1 , 8.2 , 1.0 hz , 1h ) , 7.307.21 ( m , 2h ) , 3.48 
 ( m , 2h ) 3.092.97 ( m , 2h ) , 2.94 ( d , j = 6.8 
 hz , 2h ) , 2.84 ( s , 3h ) , 2.312.13 ( m , 1h ) , 2.02 ( m , 2h ) , 1.711.53 
 ( m , 2h ) .
m / z ( es + apci ) : 419 [ m + h ] . ( a ) following the method 
 for 5 using 17 and 4-aminophenylboronic 
 acid pinacol ester , we obtained tert - butyl 4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-6-yl]amino}piperidine-1-carboxylate as a yellow 
 solid 
 ( 84% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.887.78 ( m , 2h ) , 7.68 ( d , j = 9.6 hz , 1h ) , 7.63 ( s , 1h ) , 6.94 ( d , j = 6.4 hz , 
 1h ) , 6.676.60 ( m , 2h ) , 6.58 ( d , j = 9.6 hz , 
 1h ) , 5.27 ( s , 2h ) , 4.013.85 ( m , 2h ) , 3.853.70 ( m , 
 1h ) , 3.052.87 ( m , 2h ) , 2.132.00 ( m , 2h ) , 1.481.27 
 ( m , 11h ) .
m / z ( es + apci ) : 409 [ m + h ] .
( b ) following the method for 7 using tert - butyl 4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-6-yl]amino}piperidine-1-carboxylate , we obtained 
 3-{4-[(3-fluoropyridin-2-yl)amino]phenyl}-n-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine as an off - white solid ( 25% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 8.97 ( d , j = 1.8 hz , 1h ) , 8.158.06 ( m , j = 8.7 hz , 2h ) , 8.057.98 ( m , 1h ) , 7.927.84 
 ( m , j = 8.7 hz , 2h ) , 7.80 ( s , 1h ) , 7.72 ( d , j = 9.6 hz , 1h ) , 7.57 ( ddd , j = 1.4 , 7.9 , 
 11.8 hz , 1h ) , 6.96 ( d , j = 6.9 hz , 1h ) , 6.83 ( ddd , j = 3.2 , 4.8 , 8.0 hz , 1h ) , 6.65 ( d , j = 
 9.6 hz , 1h ) , 3.793.62 ( m , 1h ) , 3.092.96 ( m , 2h ) , 2.662.56 
 ( m , 2h ) , 2.121.98 ( m , 2h ) , 1.441.25 ( m , 2h ) .
m / z ( es + apci ) : 404 [ m + h ] .
( c ) following the method for 19b using 3-{4-[(3-fluoropyridin-2-yl)amino]phenyl}-n-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine , 
 we obtained 35 as a yellow solid ( 70% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 9.00 ( d , j = 1.8 hz , 1h ) , 8.148.06 ( m , 2h ) , 
 8.047.97 ( m , 1h ) , 7.947.85 ( m , 2h ) , 7.80 ( s , 1h ) , 
 7.72 ( d , j = 9.6 hz , 1h ) , 7.627.51 ( m , 1h ) , 
 6.97 ( d , j = 6.4 hz , 1h ) , 6.84 ( ddd , j = 3.4 , 4.7 , 7.9 hz , 1h ) , 6.65 ( d , j = 9.6 hz , 1h ) , 
 3.683.47 ( m , 1h ) , 2.892.74 ( m , 2h ) , 2.20 ( s , 3h ) , 
 2.121.99 ( m , 4h ) , 1.601.42 ( m , 2h ) .
m / z ( es + apci ) : 418 [ m + h ] . ( a ) following 
 the method for 5 using 17 and 4-amino-3-fluorophenylboronic 
 acid pinacol ester , we obtained tert - butyl 4-{[3-(4-amino-3-fluorophenyl)imidazo[1,2-b]pyridazin-6-yl]amino}piperidine-1-carboxylate as an off - white 
 solid ( 56% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.98 ( dd , j = 2.1 , 14.0 
 hz , 1h ) , 7.78 ( s , 1h ) , 7.73 ( d , j = 9.6 hz , 1h ) , 
 7.64 ( dd , j = 1.8 , 8.2 hz , 1h ) , 7.08 ( d , j = 6.4 hz , 1h ) , 6.83 ( dd , j = 8.2 , 9.6 
 hz , 1h ) , 6.64 ( d , j = 9.6 hz , 1h ) , 5.35 ( br .
s , 2h ) , 
 4.043.88 ( m , 2h ) , 3.843.69 ( m , 1h ) , 3.052.85 
 ( m , 2h ) , 2.142.04 ( m , 2h ) , 1.451.28 ( m , 11h ) .
m / z ( es + apci ) : 427 [ m + h ] .
( b ) following the method for 7 using tert - butyl 4-{[3-(4-amino-3-fluorophenyl)imidazo[1,2-b]pyridazin-6-yl]amino}piperidine-1-carboxylate and 2-chloropyridine , 
 we obtained 3-[3-fluoro-4-(pyridin-2-ylamino)phenyl]-n-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine 
 as a yellow solid ( 28% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.898.75 ( m , 1h ) , 8.368.25 
 ( m , 2h ) , 8.16 ( dd , j = 1.4 , 5.0 hz , 1h ) , 7.90 ( s , 
 1h ) , 7.84 ( dd , j = 1.8 , 8.7 hz , 1h ) , 7.74 ( d , j = 10.1 hz , 1h ) , 7.59 ( ddd , j = 1.8 , 7.0 , 
 8.6 hz , 1h ) , 7.086.98 ( m , 2h ) , 6.79 ( ddd , j = 0.9 , 5.5 , 6.4 hz , 1h ) , 6.67 ( d , j = 9.6 hz , 1h ) , 
 3.773.62 ( m , 1h ) , 3.082.94 ( m , 2h ) , 2.652.54 
 ( m , 2h ) , 2.161.97 ( m , 2h ) , 1.421.25 ( m , 2h ) .
m / z ( es + apci ) : 404 [ m + h ] .
( c ) following the method for 19b using 3-[3-fluoro-4-(pyridin-2-ylamino)phenyl]-n-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine , 
 we obtained 36 as a yellow solid ( 62% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 8.84 ( s , 1h ) , 8.398.27 ( m , 2h ) , 8.16 ( td , j = 1.4 , 5.0 hz , 1h ) , 7.91 ( s , 1h ) , 7.82 ( dd , j = 
 1.8 , 8.7 hz , 1h ) , 7.75 ( d , j = 9.6 hz , 1h ) , 7.637.54 
 ( m , 1h ) , 7.116.99 ( m , 2h ) , 6.79 ( ddd , j = 
 0.9 , 5.5 , 6.4 hz , 1h ) , 6.68 ( d , j = 9.6 hz , 1h ) , 
 3.703.51 ( m , 1h ) , 2.892.75 ( m , 2h ) , 2.19 ( s , 3h ) , 
 2.161.99 ( m , 4h ) , 1.601.40 ( m , 2h ) .
m / z ( es + apci ) : 418 [ m + h ] . ( a ) a solution of 4-bromo-2,6-difluoroaniline 
 ( 2.50 g , 12.0 mmol , 1.0 equiv ) , bis(pinacolato)diboron ( 3.36 g , 13.2 
 mmol , 1.1 equiv ) , pd(dppf)cl2 ( 150 mg , 0.18 mmol , 15 mol 
 % ) , and potassium acetate ( 3.54 g , 36.06 mmol , 3.0 equiv ) in dmso 
 was heated at 80 c under n2 for 90 min .
the reaction 
 mixture was partitioned between etoac ( 100 ml ) and saturated aqueous 
 bicarbonate ( 100 ml ) .
the organic layer was washed with brine ( 3  
 100 ml ) , dried over mgso4 , and concentrated under reduced 
 pressure to give 2,6-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline 
 as a brown solid ( 3.0 g , 97% ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.076.94 ( m , 2h ) , 5.66 
 ( s , 2h ) , 1.281.17 ( m , 12h ) .
m / z ( es + apci ) : 256 [ m + h ] .
( b ) to a solution 
 of 1-methylpiperidine-4-amine ( 11.8 g , 103 mmol , 3.0 equiv ) in nmp 
 ( 32 ml ) was added 3-bromo-6-chloroimidazo[1,2-b]pyridazine 
 ( 8.0 g , 34.0 mmol , 1.0 equiv ) .
the reaction mixture was heated in 
 a microwave at 180 c for 35 min .
the reaction mixture was diluted 
 with ethyl acetate ( 200 ml ) and washed with deionized water ( 3  
 250 ml ) .
the organic layer was separated , dried ( mgso4 ) , 
 filtered , and concentrated under reduced pressure .
purification by 
 chromatography on silica gel ( 2.5%25% 2 m nh3 in 
 meoh / etoac ) gave 3-bromo - n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine as a pale yellow solid ( 4.8 g , 45% ) .
h nmr ( 400 mhz , cd3od )  ppm 7.52 ( d , j = 9.6 hz , 1h ) , 7.37 ( s , 1h ) , 6.67 ( d , j = 9.6 hz , 1h ) , 3.913.66 ( m , 1h ) , 2.972.78 ( m , 2h ) , 
 2.30 ( s , 3h ) , 2.262.08 ( m , 4h ) , 1.791.72 ( m , 2h ) .
m / z ( es + apci ) : 310 [ m + h ] .
( c ) following the method for 5 using 3-bromo - n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine and 2,6-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline , 
 we obtained 3-(4-amino-3,5-difluorophenyl)-n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine as a yellow solid ( 521 mg , 45% ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 7.967.80 ( m , 3h ) , 7.71 ( d , j = 9.6 hz , 
 1h ) , 7.05 ( d , j = 6.9 hz , 1h ) , 6.64 ( d , j = 9.6 hz , 1h ) , 5.40 ( s , 2h ) , 3.673.42 ( m , 1h ) , 2.912.76 
 ( m , 2h ) , 2.19 ( s , 3h ) , 2.151.94 ( m , 4h ) , 1.591.34 
 ( m , 2h ) .
m / z ( es + apci ) : 359 [ m + h ] .
( d ) following the method for 25c using 3-(4-amino-3,5-difluorophenyl)-n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine and 2-chloropyridine , we obtained 37 as a yellow solid ( 9% yield ) .
h nmr ( 400 mhz , 
 dmso - d6 )  ppm 8.54 ( s , 1h ) , 8.078.00 
 ( m , 3h ) , 7.98 ( dd , j = 1.4 , 5.0 hz , 1h ) , 7.75 ( d , j = 9.6 hz , 1h ) , 7.52 ( ddd , j = 1.8 , 7.0 , 
 8.6 hz , 1h ) , 7.13 ( d , j = 6.4 hz , 1h ) , 6.776.60 
 ( m , 3h ) , 3.673.43 ( m , 1h ) , 2.932.69 ( m , 2h ) , 2.16 
 ( br . s , 3h ) , 2.121.92 ( m , 4h ) , 1.611.32 ( m , 2h ) .
m / z ( es + apci ) : 436 [ m + h ] . ( a ) following 
 the procedure for 5 using 3-bromo - n-4-(pyrrolidin-1-yl)-trans - cyclohexyl)imidazo[1,2-b]pyridazin-6-amine 
 ( 500 mg , 1.37 mmol , 1.0 equiv ) and 2-aminopyridine-5-boronic acid 
 pinacol ester ( 452 mg , 2.06 mmol , 1.5 equiv ) , we obtained 3-(6-aminopyridin-3-yl)-n-[trans-4-(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine as a beige solid ( 468 mg , 90% ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 8.768.73 ( m , 1h ) , 8.08 ( dd , j = 2.3 , 
 8.7 hz , 1h ) , 7.707.66 ( m , 2h ) , 6.93 ( d , j = 6.9 hz , 1h ) , 6.59 ( d , j = 9.6 hz , 1h ) , 6.53 ( d , j = 8.7 hz , 1h ) , 6.11 ( s , 2h ) , 3.643.46 ( m , 1h ) , 
 2.882.61 ( m , 3h ) , 2.212.00 ( m , 5h ) , 1.841.63 
 ( m , 4h ) , 1.441.19 ( m , 5h ) .
m / z ( es + apci ) : 378 [ m + h ] .
( b ) following 
 the method for 25c using 3-(6-aminopyridin-3-yl)-n-[trans-4-(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine , we obtained 38 as a pale 
 yellow solid ( 24% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.379.32 ( m , 1h ) , 9.11 
 ( d , j = 1.8 hz , 1h ) , 8.42 ( dd , j = 2.3 , 8.7 hz , 1h ) , 8.118.06 ( m , 2h ) , 7.88 ( s , 1h ) , 7.73 
 ( d , j = 9.6 hz , 1h ) , 7.677.61 ( m , 1h ) , 7.046.96 
 ( m , 2h ) , 6.66 ( d , j = 9.6 hz , 1h ) , 3.58 ( br .
s , 1h ) , 
 2.742.51 ( m , 3h ) , 2.192.08 ( m , j = 
 8.7 hz , 2h ) , 2.071.96 ( m , 3h ) , 1.721.63 ( m , 4h ) , 1.371.20 
 ( m , 5h ) .
m / z ( es + apci ) : 473 [ m + h ] . ( a ) following 
 the method for 5 using 3-bromo - n-[trans-4-(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine and 4-aminophenylboronic acid pinacol 
 ester , we obtained 3-(4-aminophenyl)-n-[trans-4-(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine 
 as an off - white solid ( 37% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.907.81 ( m , 2h ) , 7.697.60 
 ( m , 2h ) , 6.84 ( d , j = 6.9 hz , 1h ) , 6.676.59 
 ( m , 2h ) , 6.596.50 ( m , 2h ) , 5.27 ( s , 2h ) , 2.612.52 
 ( m , 3h ) , 2.232.09 ( m , 2h ) , 2.091.94 ( m , 3h ) , 1.741.62 
 ( m , 4h ) , 1.361.13 ( m , 5h ) .
m / z ( es + apci ) : 377 [ m + h ] .
( b ) following 
 the method for 25c using 3-(4-aminophenyl)-n-[trans-4-(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine , we obtained 39 as an off - white 
 solid ( 27% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.99 ( d , j = 1.8 hz , 1h ) , 
 8.188.06 ( m , 2h ) , 8.00 ( d , j = 5.0 hz , 1h ) , 
 7.947.85 ( m , j = 9.2 hz , 2h ) , 7.80 ( s , 1h ) , 
 7.71 ( d , j = 9.6 hz , 1h ) , 7.57 ( ddd , j = 1.6 , 8.1 , 11.8 hz , 1h ) , 6.94 ( d , j = 6.9 hz , 
 1h ) , 6.84 ( ddd , j = 3.2 , 4.8 , 8.0 hz , 1h ) , 6.63 ( d , j = 9.6 hz , 1h ) , 3.663.51 ( m , 1h ) , 2.582.52 
 ( m , 3h ) , 2.242.11 ( m , 2h ) , 2.101.95 ( m , 3h ) , 1.741.61 
 ( m , 4h ) , 1.401.20 ( m , 5h ) .
m / z ( es + apci ) : 472 [ m + h ] . ( a ) following the method for 5 using 4 and 4-aminophenylboronic acid pinacol ester , we obtained tert - butyl ( trans-4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-6-yl]amino}cyclohexyl)carbamate as a yellow 
 solid ( 66% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 7.957.76 ( m , 2h ) , 7.65 ( d , j = 9.6 hz , 1h ) , 7.637.58 ( m , 1h ) , 6.84 ( d , j = 6.9 hz , 1h ) , 6.79 ( s , 1h ) , 6.706.59 ( m , 2h ) , 
 6.56 ( d , j = 10.1 hz , 1h ) , 5.26 ( s , 2h ) , 3.643.42 
 ( m , 1h ) , 3.393.13 ( m , 1h ) , 2.242.10 ( m , 2h ) , 1.931.76 
 ( m , 2h ) , 1.39 ( s , 9h ) , 1.351.17 ( m , 4h ) .
m / z ( es + apci ) : 423 [ m + h ] .
( b ) following the method for 7 using tert - butyl ( trans-4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-6-yl]amino}cyclohexyl)carbamate , we obtained 40 as a yellow solid ( 17% yield ) .
h nmr ( 400 mhz , 
 dmso - d6 )  ppm 9.00 ( d , j = 1.8 hz , 1h ) , 8.158.07 ( m , 2h ) , 8.057.97 
 ( m , 1h ) , 7.937.85 ( m , 2h ) , 7.79 ( s , 1h ) , 7.70 ( d , j = 9.6 hz , 1h ) , 7.57 ( ddd , j = 1.6 , 8.1 , 
 11.8 hz , 1h ) , 6.89 ( d , j = 6.9 hz , 1h ) , 6.83 ( ddd , j = 3.2 , 4.8 , 8.0 hz , 1h ) , 6.63 ( d , j = 
 9.6 hz , 1h ) , 3.613.46 ( m , 1h ) , 2.692.55 ( m , 1h ) , 2.192.09 
 ( m , 2h ) , 1.901.79 ( m , 2h ) , 1.341.10 ( m , 4h ) .
m / z ( es + apci ) : 418 [ m + h ] . ( a ) following the method 
 for 5 using 
 3-bromo - n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine and 4-aminophenylboronic acid pinacol 
 ester , we obtained 3-(4-aminophenyl)-n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine as a yellow solid ( 42% yield ) .
h nmr ( 400 mhz , dmso - d6 )  
 ppm 7.937.79 ( m , 2h ) , 7.66 ( d , j = 9.6 hz , 
 1h ) , 7.63 ( s , 1h ) , 6.89 ( d , j = 6.4 hz , 1h ) , 6.666.60 
 ( m , 2h ) , 6.58 ( d , j = 9.6 hz , 1h ) , 5.28 ( s , 2h ) , 
 3.703.47 ( m , 1h ) , 2.882.77 ( m , 2h ) , 2.21 ( s , 3h ) , 
 2.111.93 ( m , 4h ) , 1.591.35 ( m , 2h ) .
m / z ( es + apci ) : 323 [ m + h ] .
( b ) following the method for 25c using 3-(4-aminophenyl)-n-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine , we obtained 41 as a yellow solid 
 ( 32% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.18 ( d , j = 1.8 hz , 1h ) , 8.228.02 
 ( m , 3h ) , 7.977.77 ( m , 4h ) , 7.72 ( d , j = 9.6 
 hz , 1h ) , 6.97 ( d , j = 6.4 hz , 1h ) , 6.65 ( d , j = 9.6 hz , 1h ) , 3.733.51 ( m , 1h ) , 2.912.72 
 ( m , 2h ) , 2.21 ( s , 3h ) , 2.131.95 ( m , 4h ) , 1.611.35 
 ( m , 2h ) .
m / z ( es + apci ) : 452 [ m + h ] . following the method 
 for 41 using 
 2-bromo-3-fluoro-6-methylpyridine , we obtained 42 as 
 a yellow solid ( 15% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 8.91 ( d , j = 
 2.3 hz , 1h ) , 8.218.05 ( m , 2h ) , 8.037.89 ( m , 2h ) , 7.82 
 ( s , 1h ) , 7.72 ( d , j = 9.6 hz , 1h ) , 7.44 ( dd , j = 8.0 , 11.7 hz , 1h ) , 6.98 ( d , j = 6.4 
 hz , 1h ) , 6.766.54 ( m , 2h ) , 3.703.48 ( m , 1h ) , 2.922.77 
 ( m , 2h ) , 2.39 ( s , 3h ) , 2.20 ( s , 3h ) , 2.151.94 ( m , 4h ) , 1.581.39 
 ( m , 2h ) .
m / z ( es + apci ) : 432 [ m + h ] . following the method 
 for 41 using 
 2-bromo-3,5-difluoropyridine , we obtained 43 as a yellow 
 solid ( 27% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 9.02 ( d , j = 1.8 hz , 1h ) , 
 8.158.04 ( m , 3h ) , 7.897.78 ( m , 4h ) , 7.72 ( d , j = 9.6 hz , 1h ) , 6.97 ( d , j = 6.4 hz , 1h ) , 
 6.65 ( d , j = 9.6 hz , 1h ) , 3.683.51 ( m , 1h ) , 
 2.852.74 ( m , 2h ) , 2.20 ( s , 3h ) , 2.121.98 ( m , 4h ) , 
 1.561.43 ( m , 2h ) .
m / z ( es 
 + apci ) : 436 [ m + h ] . following the 
 method for 41 using 2-chloro-5-trifluoromethylpyridine
, 
 we obtained 44 as an off - white solid ( 7% yield ) .
h nmr ( 400 mhz , cd3od )  ppm 8.458.40 
 ( m , 1h ) , 8.138.06 ( m , 2h ) , 7.797.68 ( m , 4h ) , 7.61 
 ( d , j = 9.6 hz , 1h ) , 6.92 ( d , j = 
 8.7 hz , 1h ) , 6.68 ( d , j = 9.6 hz , 1h ) , 3.843.74 
 ( m , 1h ) , 2.992.90 ( m , 2h ) , 2.36 ( s , 3h ) , 2.332.16 
 ( m , 4h ) , 1.721.57 ( m , 2h ) .
m / z ( es + apci ) : 468 [ m + h ] . following the procedure 
 for 41 using 2-chloro-6-trifluoromethylpyridine
h nmr ( 400 
 mhz , cd3od )  ppm 8.068.01 ( m , 2h ) , 7.917.85 
 ( m , 2h ) , 7.767.70 ( m , 2h ) , 7.67 ( d , j = 9.6 
 hz , 1h ) , 7.14 ( d , j = 7.3 hz , 1h ) , 7.03 ( d , j = 8.7 hz , 1h ) , 6.70 ( d , j = 9.6 hz , 1h ) , 
 4.053.95 ( m , 1h ) , 3.573.40 ( m , 2h ) , 3.213.01 
 ( m , 2h ) , 2.83 ( s , 3h ) , 2.532.38 ( m , 2h ) , 1.991.69 
 ( m , 2h ) m / z ( es + apci ) : 468 [ m + h ] . following the method for 40 using 2-bromo-3,5-difluoropyridine , 
 we obtained 46 as a yellow solid ( 31% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 9.03 ( d , j = 1.8 hz , 1h ) , 8.168.04 ( m , 3h ) , 
 7.897.78 ( m , 4h ) , 7.70 ( d , j = 9.6 hz , 1h ) , 
 6.90 ( d , j = 6.9 hz , 1h ) , 6.62 ( d , j = 9.6 hz , 1h ) , 3.603.48 ( m , 1h ) , 2.682.57 ( m , 1h ) , 
 2.192.10 ( m , 2h ) , 1.901.80 ( m , 2h ) , 1.321.13 
 ( m , 4h ) .
m / z ( es + apci ) : 436 [ m + h ] . following the method for 40 using 2,5-dichloro-3-fluoropyridine , 
 we obtained 47 as a yellow solid ( 13% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 9.19 ( d , j = 1.8 hz , 1h ) , 8.188.05 ( m , 3h ) , 
 7.907.77 ( m , 4h ) , 7.71 ( d , j = 10.1 hz , 1h ) , 
 6.91 ( d , j = 6.9 hz , 1h ) , 6.63 ( d , j = 9.6 hz , 1h ) , 3.623.43 ( m , 1h ) , 2.722.57 ( m , 1h ) , 
 2.212.08 ( m , 2h ) , 1.911.76 ( m , 3h ) , 1.331.13 
 ( m , 4h ) .
m / z ( es + apci ) : 452 [ m + h ] . following the method for 40 using 2-bromo-3-fluoro-6-methylpyridine , 
 we obtained 48 as a yellow solid ( 16% yield ) .
h nmr ( 400 mhz , dmso - d6 )  ppm 
 8.91 ( d , j = 1.8 hz , 1h ) , 8.198.03 ( m , 2h ) , 
 8.037.86 ( m , 2h ) , 7.81 ( s , 1h ) , 7.70 ( d , j = 9.6 hz , 1h ) , 7.43 ( dd , j = 7.8 , 11.5 hz , 1h ) , 
 6.91 ( d , j = 6.4 hz , 1h ) , 6.726.55 ( m , 2h ) , 
 3.633.46 ( m , 1h ) , 2.702.57 ( m , 1h ) , 2.38 ( s , 3h ) , 
 2.252.11 ( m , 2h ) , 1.921.81 ( m , 2h ) , 1.391.14 
 ( m , 4h ) .
m / z ( es + apci ) : 432 [ m + h ] .
ic50 determinations were performed 
 using kinase glo ( promega ) to measure 
 atp depletion resulting from the kinase reaction .
compounds were added 
 to 22 l reaction mixtures in white 384-well plates containing 
 10 nm full length recombinant pfcdpk1 and 8 m 
 myoa - tail domain interacting protein ( mtip ) in assay buffer ( tris - hcl 
 buffer at ph 8.0 containing 0.1 mm egta , 0.2 mm cacl2 , 
 1 mm dtt , and 0.01% triton x-100 ) and incubated for 30 min at rt prior 
 to initiating the reaction with 10 m atp ( km ) and 20 mm mgcl2 at final concentrations .
reactions were allowed to proceed 
 for 120 min at ambient temperature and stopped by the addition of 
 22 l of kinase glo plus detection reagent .
luminescence proportional 
 to the remaining atp at the end of the reaction was measured using 
 a pherastar plate reader ( bmg labtech ) .
response 
 curves were obtained from half - log dilutions of the test compound 
 diluted in assay buffer at a constant final dmso concentration of 
 1% .
purified recombinant pfcdpk1 was diluted to 1 m in 10 mm hepes buffer 
 at ph 7.5 containing 150 mm nacl , 1 mm ca , and 1/1000 
 sypro orange dye ( invitrogen ) .
test compounds were prediluted to 400 
 m in 40% v / v dmso in water , and 1 l of diluted compound 
 was added to 39 l of enzyme / dye mix in white 96-well quantitative 
 pcr plates ( thermo scientific ) to give a final compound concentration 
 of 10 m and preincubated for 30 min at rt .
reference melting 
 temperatures were obtained in parallel by the addition of 1 l 
 of 40% v / v dmso to 39 l of diluted pfcdpk1 . 

the plates were sealed with transparent adhesive covers ( biorad ) and 
 subjected to a temperature gradient from 25 to 95 k at a rate of 1 
 k / min using a quantitative pcr machine ( mx3005p , stratagene ) .
fluorescence 
 data were acquired at 1 min intervals using the fam / rox filter set 
 ( ex 492 nm / em 610 nm ) and the raw data exported to excel ( microsoft ) 
 for analysis .
data were processed to identify the fluorescence maxima 
 and minima , and the midpoints of melting curves were determined by 
 fitting to the boltzmann equation ( xlfit add - in , idbs software ) .
results 
 were expressed as tm values relative 
 to dmso controls where tm = tm ( inhibitor )  tm ( dmso controls ) .
falciparum ec50 values were measured using an in vitro model of 
 malaria parasite growth which measures merozoite invasion of red blood 
 cells .
test cultures were set up at 0.5% parasitemia and 2% hematocrit 
 from a synchronized stock culture of 3d7 p. falciparum .
compounds were diluted into 2% dmso and added to parasites 24 h 
 postinvasion using a 95 l parasite culture in a 96-well plate 
 and incubated under static conditions .
cells were recovered 48 h later and processed for facs 
 analysis : 50 l of parasite culture was transferred into a facs 
 tube and mixed with 500 l of 500 g / ml hydroethidine 
 in pbs to stain parasite dna .
the parasites were incubated for 20 
 min at 37 c , then diluted with 1 ml of pbs , and stored on ice 
 prior to facs analysis .
the data were acquired using cellquest pro 
 software on a facscalibur ( becton dickinson ) .
growth inhibition was 
 calculated using the following formula : % growth inhibition = ( 1  
 [ parasitemia of culture / parasitemia of control culture ] )  100 . to establish 
 activity of the compounds against recombinant p. berghei cdpk1 enzyme
, atpase activity was measured using a biosensor sensitive 
 to adp ( rhodamine - labeled parm , gift of m. webb , nimr ) .
the progress 
 of the reactions was monitored by an increase in fluorescence corresponding 
 to the accumulation of adp using a pherastar plate reader ( bmg labtech ) .
kinase selectivity profiling was 
 carried out at the national centre for protein kinase profiling in 
 the mrc protein phosphorylation unit at the university of dundee .
compounds were solubilized or suspended 
 in a solution consisting of 70% tween-80 and 30% ethanol , followed 
 by a 10-fold dilution in h2o .
chloroquine ( sigma c6628 ) 
 was used as a control drug . test procedure : day 0 , from a donor mouse 
 with approximately 30% parasitemia , heparinized blood ( containing 
 50 l of 200 u / ml heparin ) is taken and diluted in physiological 
 saline to 10 parasitized erythrocytes per ml . of this 
 suspension , 0.2 ml was injected intravenously ( i.v . ) into experimental 
 groups of 3 mice and a control group of 3 mice .
four hours postinfection , 
 the experimental groups were treated with a single dose of compound 
 by the oral ( p.o . ) route .
days 13 : 24 , 48 , and 72 h postinfection , 
 the experimental groups were treated with a single daily dose of compound 
 p.o . at 50 mg / kg .
day 4 : 24 h after the last drug treatment , 1 l 
 of tail blood was taken and suspended in 1 ml of pbs buffer .
parasitemia 
 was determined with a facscan ( becton dickinson ) by counting 100000 
 red blood cells .
the difference of the mean infection rate of the 
 control group (= 100% ) to the test group was calculated and expressed 
 as percent reduction .
the results are expressed as the reduction of 
 parasitemia on day 4 in % as compared to the untreated control group . 
 as an example , activity determination with a mean of , e.g. , 2% parasitemia 
 in treated mice and a mean of , e.g. , 40% parasitemia in the control 
 animals is calculated as follows : ( 40%  2%)/40% * 100 = 95% 
 reduction in parasitemia .
blast was used with 
 the pfcdpk1 sequence ( uniprot : p62344 ) to search 
 the pdb to identify suitable templates for homology modeling .
sequence 
 alignments were generated and structures inspected using the schrodinger 
 molecular modeling suite , 2010 version .
the structure of tgcdpk1 ( pdb : 3i7c ) was selected as 
 the most suitable template for a variety of reasons including good 
 crystallographic quality and high sequence identity , particularly 
 around the atp site ; of the 49 residues within 6  of the crystal 
 ligand , 69% are identical , and 92% are homologous .
homology modeling 
 was carried out using prime , and further 
 refinements were not required due to the excellent correspondence 
 between the target and template .
the resultant structure was prepared 
 for docking studies involving solvent removal , valence and charge 
 assignment , addition of hydrogen atoms , orientation of ambiguous groups 
 ( e.g. , amide groups of asn and gln ) , and restrained minimization to 
 relieve residual strain .
a docking grid was generated with size and 
 location based on the 3i7c crystal ligand , including optional constraints 
 to prominent h - bonding groups including tyr148 at the hinge , glu152 
 at the entrance to the pocket , and asp212 of the dfg - loop .
virtual 
 libraries of compounds were enumerated and prepared for docking using 
 ligprep then docked flexibly using glidesp with a h - bonding constraint to tyr148 n - h , outputting 
 up to three diverse poses per ligand .
high scoring candidate molecules 
 were prioritized for synthesis following manual inspection and consideration 
 of additional factors such as favorable / unfavorable contacts and ligand 
 strain .
 a structure - guided 
 design approach using a homology model of plasmodium falciparum calcium - dependent protein kinase 1 
 ( pfcdpk1 ) was used to improve the potency of a series 
 of imidazopyridazine inhibitors as potential antimalarial agents . 
 
 this resulted in high affinity compounds with pfcdpk1 
 enzyme ic50 values less than 10 nm and in vitrop . 
 falciparum antiparasite ec50 values 
 down to 12 nm , although these compounds did not have suitable adme 
 properties to show in vivo efficacy in a mouse model . 
 
 structural modifications designed to address the adme issues , in particular 
 permeability , were initially accompanied by losses in antiparasite 
 potency , but further optimization allowed a good balance in the compound 
 profile to be achieved . upon testing in vivo in a 
 murine model of efficacy against malaria , 
 high levels of compound 
 exposure relative to their in vitro activities were 
 achieved , and the modest efficacy that resulted raises questions about 
 the level of effect that is achievable through the targeting of pfcdpk1 . 
Introduction
Results and Discussion
Conclusions
Experimental Section