human african trypanosomiasis 
 ( hat ) or african sleeping sickness 
 is a serious life threatening disease . around 60 million people in 36 african countries are currently in 
 constant threat of infection .
although the reported number of cases 
 has dropped over recent years , the actual number of unreported cases 
 is estimated to be around 7000080000 .
hat is caused by infection with trypanosoma brucei , a vector - borne parasite , which is transmitted by the bite of tsetse 
 flies .
the symptoms of the disease occur in two main stages . in the 
 first stage , known as the hemolymphatic phase , the parasites multiply 
 in blood , subcutaneous tissues , and lymph , causing headaches , fever , 
 itching , joint pains , and swelling of lymph nodes . in the second stage , 
 or neurological phase ,
this phase entails 
 confusion , change of behavior , reduced coordination , sensory disturbances , 
 disturbance of sleep cycle , and finally death .
most available drugs 
 for hat display severe toxic side effects , require long periods of 
 administration , and/or are expensive due to the logistics to reach 
 rural african areas .
further , resistance 
 to all in use drugs has been observed in the laboratory and/or in 
 the field , resulting in an urgent requirement 
 for better , safer , and inexpensive therapeutic alternatives to the 
 current treatments .
genetic knockdown studies have identified 
 several proteins that 
 are essential for the survival of the parasite , including members 
 of the protein kinase ( pk ) family . in trypanosoma brucei pks
are essential in many fundamental cellular processes , e.g. , 
 proliferation , differentiation , and cell cycle control , and can therefore 
 be considered as potential drug targets for the treatment of hat . in the t. brucei genome
there 
 are 
 two kinases that are highly homologous to human glycogen synthase 
 kinase 3 ( hsgsk3 ) : tbgsk3 short 
 and tbgsk3 long . rna 
 interference ( rnai ) knockdown of tbgsk3 has shown 
 that tbgsk3 short is critical for cell growth , with 
 a role in the control of mitosis and/or cytokinesis . the ability to selectively inhibit tbgsk3 
 over 
 the off - target hsgsk3 is highly desirable because 
 mouse knockout studies revealed that the disruption of the murine 
 gsk3 gene causes embryonic lethality ; consequently , nonselective 
 inhibitors are not applicable for use in infants and women of child 
 bearing age . from a homology perspective ,
tbgsk3 is not only 
 very closely related to hsgsk3 but also to 
 other human pks such as cyclin dependent kinase 1 ( hscdk1 ) and cyclin dependent kinase 2 ( hscdk2).hscdk2 and hscdk1 are essential for g1/g2 progression and s / m - phase entry of the 
 cell cycle .
off - target inhibition of these human kinases will therefore 
 result in cell cycle arrest and reduction of cellular proliferation 
 and as such potentially lead to severe side effects . over the 
 past decade
, various groups and pharmaceutical companies 
 have identified multiple series of hsgsk3 
 inhibitors .
recently , astex therapeutics 
 and researchers at the university of osaka have developed a series 
 of aminopyrazoles that are potent inhibitors of hsgsk3. co - crystal structures of this series with hsgsk3 are not available to date ; however , complex 
 structures with the closely related hscdk2 have been 
 determined . in all structures , the pyrazole 
 scaffold forms two hydrogen bonds to the hinge region of hscdk2 ( figure 1 ) .
further , the nh group of 
 the 3-position amide forms an additional hydrogen - bond interaction 
 to the backbone of leu83 . a water - mediated hydrogen bond from the 
 amide carbonyl oxygen atom to the backbone
the r residues ( figure 2 ) access 
 the gatekeeper region between the gatekeeper residue phe80 and the 
 catalytic asp145 ( figure 1 ) .
the r substituents occupy the hydrophobic pocket ii , formed by the backbone 
 of the linker region , leu83 , phe82 , and side chains of ile10 , asp86 , 
 and leu134 .
finally , an intramolecular hydrogen bond between the r - nh and r - carbonyl group is present .
the similarity 
 of hscdk2 , hsgsk3 , and tbgsk3 indicates that aminopyrazoles will also bind into 
 the atp - binding site of the latter enzyme .
co - crystal 
 structure ( 2vu3 ) of at7519 ( carbon atoms 
 in gray ) bound to cdk2 .
key : red sphere , water molecule ; black dashed 
 lines , protein  ligand and water  ligand hydrogen bonds ; 
 yellow stick , hydrogen atom .. generic binding mode of the r and r substituted 
 aminopyrazole scaffold ( carbon atoms in gray ) .
herein , we describe the design , synthesis , and biological 
 evaluation 
 of aminopyrazole inhibitors which bind to tbgsk3 
 short .
the inhibitors were also tested against the closely related 
 off - targets hsgsk3 and hscdk2 and evaluated against a panel of mammalian protein kinases . 

the most potent compound has nanomolar affinity for tbgsk3 short , is selective over hsgsk3 and hscdk2 , and clean in the kinase panel . by using computer - aided 
 molecular modeling
enzyme affinity correlated with inhibition of t. b. brucei proliferation , albeit a 100-fold offset 
 in potency , was found . in light of these results ,
the aminopyrazole derivatives developed 
 by astex therapeutics and yumiko uno et al . for inhibition of hscdk2 and hsgsk3 enzymes were chosen 
 as a starting point for the investigation of tbgsk3 
 short inhibitors .
aminopyrazoles analogues were generated by substituting 
 at either r or r position ( figure 2 ) using two synthetic routes ( scheme 1a , b ) .
reagents and conditions 
 used 
 in routes a and b : ( a ) trans-4-methoxycyclohexylamine , 
 edc , hobt , dmf , rt ; ( b ) 10% pd / c , h2 , dmf , rt ; ( c ) rcooh , edc , hobt , dipea , rt ; ( d ) socl2 , meoh , 0c , 
 rt ; ( e ) 10% pd / c , h2 , etoh , rt ; ( f ) 2,6-dimethoxybenzoylchloride , 
 et3n , dioxane , rt ; ( g ) naoh , dioxane , h2o , rt ; 
 ( h ) r2nh2 , polystyrene - bound carbodiimide , hobt , 
 acetonitrile , mw , 100 c . a structural model 
 of tbgsk3 was built to assess 
 the differences in the binding sites of tbgsk3 short , hsgsk3 , and hscdk2 and to guide 
 ligand design .
an overlay of 42 hsgsk3 crystal structures 
 showed that there is low flexibility in the atp binding site . only 
 the regions including phe67 and arg141 showed some mobility .
arg141 also spans 
 a number of distinct conformations , including examples where it occupies 
 space in the binding site ( 1j1b , 1j1c , 1o9u , 2o5k ) and therefore could 
 influence docking results .
however , to allow for ligands of a significant 
 size , we have used examples with arg141 pointing out of the binding 
 site .
therefore , two homology models for tbgsk3 were 
 generated representing both states of phe67 . as we were mainly interested 
 in aminopyrazoles with less extended r groups , the crystal 
 structure with phe67 pointing toward the hinge ( with structure 1r0e as a representative ) 

the selection of 1r0e instead of other 
 members of this group ( with phe67 pointing toward the hinge ) was arbitrary . 

for this analysis , all residues that are located within 6  of 
 the ligand bound to the template structure ( 1r0e ) were considered . the binding pockets of tbgsk3 and hsgsk3 differ by nine amino acid residues ( table 1 , figure 3a ) . of the amino acid side 
 chains that point toward the ligand , the most significant differences 
 are the replacement of tyr134 in hsgsk3 with 
 phe103 , leu132 with met101 , gln72 with leu36 , and tyr140 with his109 . 

in total 16 out of 26 amino acids were 
 found to be different ( table 1 , figure 3b ) , the most important of these differences being 
 the replacement of lys20 in cdk2 with leu36 , phe80 with met101 , his84 
 with pro105 , lys89 with arg110 , and ala144 with cys170 .
interestingly , 
 most of the amino acid differences occurred in the hydrophobic pocket 
 ii and the gatekeeper region .
therefore , we decided to direct the 
 optimization of the lead scaffold toward suitable interactions with 
 amino acids which are located in these subpockets of the atp binding 
 site .
amino acids of hgsk3 or hscdk2 which differ in tbgsk3 short are 
 shown in boldface .
superposition of the 
 binding sites of the homology model of tbgsk3 
 short ( blue carbon atoms ) with ( a ) the hsgsk3 crystal structure 
 ( pdb code 1r0e ) and
the solvent accessible 
 surface of tbgsk3 short is shown in light blue . only residues that 
 differ between the binding pockets are shown .
the synthesis of r and r substituted aminopyrazole derivatives started from 4-nitro - pyrazole-3-carboxylic 
 acid 1 and is described by two different routes ( scheme 1 ) based on previous work from wyatt et al . in route
reduction of the subsequent intermediate 2 by 
 hydrogenation in the presence of palladium on carbon generated amino 
 pyrazole 3 .
the conversion to compounds 4a  z was accomplished by coupling of 3 with a suitable selection of carboxylic acids . in route
b , after 
 esterification of the carboxyl group of 1 , the nitro 
 group of intermediate 5 was reduced to afford amine 6 .
treatment of 6 with 2,6-dimethoxybenzoyl chloride 
 under standard conditions , followed by base hydrolysis of the ester , 
 provided acid 8 . in the final step ,
8 was 
 coupled with appropriate amines in a microwave reaction using polystyrene - bound 
 carbodiimide to yield final compounds 9a  x .
twenty - six r substituted 
 aminopyrazole analogues ( table 2 ) were made 
 according to the synthetic route shown 
 in scheme 1a .
a range of r groups 
 varying in size and polarity was chosen to probe whether the differences 
 in the gate keeper region between tbgsk3 , hsgsk3 , and hscdk2 could be exploited to 
 derive selective and potent tbgsk3 inhibitors ( figure 3 ) .
concentration 
 required to inhibit 
 the growth of t. b. brucei in culture 
 by 50% over 72 h. concentration 
 required to inhibit 
 the growth of mrc5 cells in culture by 50% over 72 h. all compounds showed 
 good potency against tbgsk3 ( < 1 m ) .
an 
 unsubstituted phenyl ring ( 4f ) provided on average a 
 20-fold improvement of inhibition 
 potency relative to saturated six - membered ring systems ( 4a and 4b ) and benzyl groups ( 4c , 4d , and 4e ) .
in general , a variety of different aryl and 
 heteroaryl rings ( 4g4y compared 
 to 4a and 4b ) in the r position 
 led to significantly improved potency against tbgsk3 . 

additionally , a wide variety of substituents were tolerated on the 
 phenyl ring . in general
, ortho - substituted phenyl 
 rings gave the best improvement in activity compared to the unsubstituted 
 phenyl group ( 4j , 4k , 4 m , and 4n ) .
the methoxyphenyl moieties in 4j , 4 m , and 4n , which had tbgsk3 
 ic50 values of 4 , 2 , and 3 nm , respectively , were the most 
 favorable substituents .
interestingly , this was 
 also the most selective compound for hscdk2 ( > 1000-fold ) . to rationalize the observed selectivity ,
all analogues were docked 
 into the binding sites of tbgsk3 , hsgsk3 , and hscdk2 and their poses were visually 
 analyzed . for most compounds ,
a binding mode similar to that observed 
 for at7519 in hscdk2 
 ( figure 1 ) was predicted in tbgsk3 , hsgsk3 , and hscdk2 . 

one important difference between hscdk2 , tbgsk3 , and hsgsk3 is the gatekeeper 
 residue ( table 1 , figure 3a , b ) . while hsgsk3 and tbgsk3 enzymes 
 have leu or met , respectively , in this position , in hscdk2 phe is present . as a consequence ,
the gatekeeper region of hscdk2 ( located between phe80 and asp145 ) is more restricted 
 compared to the other two enzymes .
this resulted in a higher energy , 
 out of plane conformation of the amide group of 4f when 
 binding into this pocket ( figure 4 ) , while 
 a low energy conformation was found when binding into t. brucei and human gsk3 ( not shown ) .
further , without 
 induced fit adaptations , the bulky r - substituents such 
 as the 2,6-dimethoxybenzamide group of 4 m , the 2,4,6-trimethoxybenzamide 
 groups 4n , and the phenylaminobenzamide groups of 4k and 4l can only be accommodated by the gatekeeper 
 region of tbgsk3 and hsgsk3 
 but not the narrower hscdk2 gatekeeper region .
these 
 observations might explain the reduced binding affinity of 4f for hscdk2 compared to hsgsk3 . 
 of note ,
this explanation is further supported by the report by wyatt 
 et al . , which found that the aryl groups 
 which are located in the same position need to twist in order to provide 
 potent cdk2 activity
docking results suggested that 
 the phenyl ring of compound 4f needs to be significantly 
 twisted out of plane by approximately 60 compared to the amide 
 in order to fit into the gatekeeper region ..
all r substituted 
 compounds were tested for their ability to inhibit the proliferation 
 of bloodstream form ( bsf ) t. b. brucei in culture .
as an initial indication of potential toxicity , compounds 4a4z were additionally tested against 
 proliferating human fetal lung fibroblast cells ( mrc5 cell line ) . 

four compounds ( 4 g , 4j , 4 m , 
 and 4y ) had ec50 values < 1 m and 
 a further 11 compounds had ec50 values <3 m against 
 bsf t. b. brucei ( table 2 ) .
the ec50 values correlated well with enzyme 
 activity ( r = 0.73 , figure 5 ) . however , a 100-fold drop from enzyme to cellular activity 
 was observed .
selectivity over the mrc5 cells was achieved with compounds 4 m ( 60-fold ) , 4s ( > 19-fold ) , 4x ( 12-fold ) , 
 and 4n ( > 9-fold ) , however , the majority of compounds 
 showed a poor selectivity over mrc5 cells .
correlation between the 
 inhibition of recombinant tbgsk3 and bloodstream 
 form t. b. brucei proliferation by 
 r substituted aminopyrazole derivatives ( 4a  z ) . as 4
m was the most potent inhibitor of tbgsk3 and proliferation of t. b. brucei cells , we retained the 2,6-dimethoxybenzamide group at position 
 r for optimization of the r substituent .
r substituted aminopyrazole analogues ( 9a9x ) were made according to the synthetic route shown in scheme 1b to explore the structural requirements for improvement 
 of antiparasitic activity and selectivity over the closely related 
 human kinases .
the majority of 
 variations led to potent tbgsk3 inhibitors , indicating 
 that chemical diversity at 
 this position was well tolerated ( table 3 ) . 

one of the sar trends observed was that six - membered saturated rings 
 ( 9c ) and seven - membered saturated rings ( 9d ) were favored over their three- and four - membered equivalents ( 9a and 9b ) .
further , it was noted that the replacement 
 of the cyclohexane of 9c with a phenyl ring or 4-pyridine , 
 to give 9k or 9l , gave a 6-fold decrease 
 in potency against tbgsk3 . the 2-pyridine analogue 
 ( 9 m ) , on the other hand , was much less active ( 50-fold ) 
 against tbgsk3 .
homologation of aromatic ( 9 g ) and saturated six - membered ( 9j ) rings by one carbon 
 atom produced inhibitors with 1 nm activity for tbgsk3 .
for aliphatic side chain derivatives 9r9w , the pentanyl and 1-isopropoxypropanyl analogues had ic50 values of 1 nm .
the impact of replacing the amide group 
 ( 3-position ) with carboxylic acid and ester groups was investigated 
 with compounds 8 and 7 .
compound 8 containing a carboxylate group in the 4-position showed a dramatic 
 loss in activity ( ic50 > 50 m ) .
the ester group 
 of 
 compound 7 on the other hand was better tolerated ( ic50 0.5 m ) .
interestingly , compared with 4a4z , a majority of r substituted 
 analogues ( 9a9x ) showed selectivity 
 over hscdk2 and hsgsk3. these 
 are the most selective tbgsk3 inhibitors described 
 to date .
concentration 
 required to inhibit 
 the growth of t. b. brucei in culture 
 by 50% over 72 h. concentration 
 required to inhibit 
 the growth of mrc5 cells in culture by 50% over 72 h. the predicted binding mode of 9 g in tbgsk3 offers an explanation for the 
 observed selectivity ( figure 6 ) . in the highest 
 scoring docking pose , the core 
 scaffold adopts a similar binding mode as observed for at7519 in hscdk2 ( figure 1 ) .
in addition , the docking results suggested that the hydrophobic 
 pocket ii of tbgsk3 was occupied by the n - benzylamide group of 9 g in such a way that its phenyl 
 moiety formed t - shaped edge - to - face interactions with the side chain 
 of phe103 and hydrophobic interactions with leu36 and ala26 . in hgsk3 , phe103 is replaced with a tyr ( table 1 , figure 3a ) , resulting in 
 steric clash and electrostatic repulsion toward the benzyl moiety 
 of 9 g .
further , leu36 is substituted with gln72 in hgsk3 and lys20 in hscdk2 , diminishing 
 hydrophobic interactions between the benzyl moiety of 9 g and these residues .
overall , these changes together with differences 
 in the gatekeeper region of hscdk2 ( see above ) are 
 likely to be responsible for the high selectivity of 9 g for tbgsk3 over hgsk3 and hscdk2 .
similar observations regarding the r group placement and selectivity were also made for compound 9h supporting this model .
proposed binding mode of 9 g in the homology model 
 of tbgsk3 ( blue carbon atoms ) overlaid on the hsgsk3 crystal 
 structure ( pink carbon atoms ) .
both ligand and protein are represented 
 as sticks and color coded by atom types .
ligand carbon atoms are shown 
 in gray , protein carbon atoms of tbgsk3 are shown in blue , and hsgsk3 
 carbon atoms in salmon .
hydrogen bonds and hydrophobic interactions are shown as black dotted 
 lines , with interaction distances in angstroms .
tbgsk3 amino acids 
 which are involved in hydrophobic interactions with the benzyl group 
 are marked in bold .
the gold sphere represents the center of the phenyl 
 ring .. the r substituted 
 compounds were tested against bsf t. b. brucei and mrc5 cells . as for the r - substituted analogues , 
 a good correlation between the ec50 and ic50 values and a 100-fold drop in activity between the biochemical and 
 cell assay was observed ( figure 7 ) .
compound 9c had an ec50 for t. b. brucei of 4 m ( table 3 ) .
limited selectivity 
 ( > 7-fold ) over mrc5 cells was achieved with compounds 9c , 9d , 9 g , 9s , and 9 t .
it was found that the compounds showed selective inhibition of tbgsk3 over hsgsk3 ( > 20-fold ) and hscdk2 ( > 1200-fold ) .
correlation between the inhibition of 
 recombinant tbgsk3 and bloodstream 
 from t. b. brucei proliferation using 
 r substituted aminopyrazole derivatives ( 9a  x ) .
pk inhibitors frequently 
 inhibit multiple kinases , often leading to off - target toxic effects . 
 to assess the selectivity of the aminopyrazole inhibitors , remaining 
 activity at 10 m concentration was measured for compounds 4f , 4 m , and 4y against a panel of 
 80 human pks and for compound 9 g against 124 human pks . 

compounds 4 m and 9 g were found to be highly 
 specific ( table 4 ) .
compound 4 m inhibited only two pks , namely gsk3 and cdk2 , at more than 
 80% .
9 g showed activity against three pks : gsk3 , 
 mapkap - k2 , and mink1 at more than 80% .
compound 4f was 
 found to inhibit seven pks and compound 4y 15 pks by 
 greater than 80% at 10 m .
numbers represent average percentage 
 of activity compared to the control at 10 m . in this table , 
 only kinases with activity values < 20% are shown ( for full table 
 see supporting information ) .
in this work , we exploited 
 the knowledge of the previously described 
 aminopyrazoles inhibitors of hscdk2 and hsgsk3 to identify selective inhibitors 
 of the tbgsk3 short isoform .
this kinase has been 
 shown using genetic manipulation studies to be essential for the survival 
 of the t. b. brucei parasite . however , we wanted to confirm if antiparasitic 
 activity could be gained using selective , small molecule inhibitors 
 of tbgsk3 .
the ability to selectively inhibit tbgsk3 over hsgsk3 and hscdk2 is essential to avoid potential side effects .
therefore , more 
 than 50 aminopyrazole derivatives were synthesized and screened against tbgsk3 , hsgsk3 , hscdk2 , and proliferating t. b. brucei and human cells in culture . the results ( table 2 and table 3 ) showed that almost all 
 compounds were highly potent tbgsk3 inhibitors .
the 
 activity could be rationalized using 
 the homology models and subsequent molecular docking studies ( figure 4 and figure 6 ) .
the aminopyrazole 
 derivatives make three h - bond interactions with the kinase hinge region , 
 driving much of the potency of the compounds against the three kinases 
 studied .
selectivity could be derived from substitution at both r and r positions . from a homology perspective
, hscdk2 is the most 
 closely related kinase to hsgsk3. although , the enzymes only share approximately 
 33% amino acid identity , their atp binding pockets are highly conserved , resulting in the majority of known hscdk2 inhibitors also potently inhibiting hsgsk3. 
 the results demonstrated that the required profile could be achieved , 
 with several compounds with high affinity ( < 18 nm ) for tbgsk3 showing high selectivity ( > 500-fold ) over hscdk2 .
docking studies provided a number of important insights 
 into the binding modes and the selectivity profile of aminopyrazole 
 derivatives .
first , the docking results suggested that if the phenyl 
 ring of compound 4f is planar with the amide group at 
 r position it can not bind into the truncated gate keeper 
 region of hscdk2 , defined by phe80 in hscdk2 , compared to met101 in tbgsk3 . to fit into 
 this region of hscdk2 , the phenyl ring needs to significantly 
 twist out of plane of the amide , with a torsion angle of approximately 
 60 , resulting in reduced binding affinity ( figure 4 ) . to stabilize this twist , di - ortho - substituents 
 on the r phenyl group
are required to cause a steric / electronic 
 clash with the carbonyl of the amide bond . however , this region of 
 the pocket in hscdk2 is narrow ( in a plane perpendicular 
 to the hinge backbone and the pyrazole core ) , only allowing small ortho - substituents ( such as in compound 9 g ) 
 on the phenyl group .
in contrast , the wider gatekeeper regions of hgsk3 and tbgsk3 can tolerate large substituents 
 such as the ortho - dimethoxy groups of compound 4 m or 4n and the ortho - phenylaminobenzamide 
 groups of 4k or 4l .
second , the highest 
 increase in selectivity ( > 10000-fold ) over hscdk2 
 was achieved by accessing the hydrophobic pocket ii . exploitation 
 of hydrophobic interactions in these two pockets not only reliably 
 increased ligand - binding affinity but also impacted on the selectivity 
 profile of these compounds .
on the basis of the biological results 
 of compound 9 g and structural modeling studies , we have 
 shown that selectivity over hsgsk3 can be achieved 
 by exploiting the phe103tyr , leu36gln , and ala26ile active site differences 
 in the hydrophobic pocket ii of tbgsk3 enzyme ( figure 6 ) . taken together ,
the region between the gate keeper 
 residue and the catalytic aspartate of the dfg loop , together with 
 the hydrophobic pocket ii , are the key areas to exploit to achieve 
 high selectivity over hscdk2 . the tbgsk3 short 
 enzyme ic50 values correlated well 
 with the t. b. brucei antiproliferative 
 ec50 activities of the described substituted aminopyrazole 
 inhibitors ( figures 5 and 7 ) , indicating that the compounds act on target .
however , a 
 100-fold drop in cell activity was observed , compared to that in the tbgsk3 assay ( 1 m ) .
the calculated physical properties 
 ( mw < 473 ; log p 0.43.6 ; psa < 
 130  ) of the series of compounds suggest this loss of activity 
 was not driven by lack of cellular penetration .
in addition , the compound 
 series was observed to be highly chemically stable under the range 
 of synthetic conditions used during the chemistry campaign , suggesting 
 that chemical degradation was not responsible for the loss of activity 
 in the proliferation assay . although metabolism by the parasite can not 
 be ruled out , the high degree of correlation between enzyme inhibition 
 and antiparasitic activity suggests this is not the case , as it would 
 be not expected that all compounds be metabolized to a constant extent . 

therefore , the drop of activity was probably due to the high atp concentration 
 ( millimolar range ) in the cell compared to the kinase assay conditions .
furthermore , chemical proteomic profiling conducted 
 in parasite cell extracts confirmed that compound 4 m binds 
 the endogenous tbgsk3 short with nanomolar affinity 
 and very few other kinase targets with much lower affinity in the 
 micromolar range .
in 
 this study , we have developed a series of substituted aminopyrazole 
 amides as tbgsk3 short inhibitors starting from a 
 compound series initially designed by astex therapeutics to inhibit hscdk2 and hsgsk3. sar investigation 
 and optimization successfully provided 18 low nanomolar ( ic50 < 10 nm ) inhibitors of tbgsk3 with high selectivity 
 ( > 10000-fold ) over hscdk2 . with compound 9 g
, we have shown that good ( 330-fold ) selectivity over hsgsk3 can be achieved by targeting the hydrophobic pocket 
 ii .
in addition , 9 g proved to be highly selective against a panel of 124 human 
 pks , showing > 90% inhibition at 10 m against only one pk , hsgsk3. molecular modeling has also shown that despite 
 overall conservation in sequence and conformation between the three 
 pks ( hsgsk3 , hscdk2 , and tbgsk3 ) , the binding pockets have distinct features that 
 determine their specificity for particular compounds .
further , we 
 have shown that enzymatic inhibition correlates well with cell efficacy 
 over a wide range of concentrations and a representative member of 
 this series binds the endogenous tbgsk3 with nanomolar 
 potency , indicating that compounds definitely act on target . however , a general 100-fold drop in activity 
 between target and cellular activities resulted at best in compounds 
 with low micromolar antiparasitic activity . taken together , this data 
 suggests that specific atp competitive hinge binders of tbgsk3 short require low picomolar potency to obtain nanomolar antiproliferative 
 activity against t. brucei .
first , 
 non - atp competitive approaches to inhibition of tbgsk3 , through irreversible hinge binders or allosteric inhibitors , 
 could be pursued .
however , these approaches have potential downsides , 
 through the introduction of a reactive functionality or an increased 
 chance of resistance causing mutations , respectively .
second , a polypharmacology 
 approach through the inhibition of a number of essential t. brucei kinases in addition to tbgsk3 could be investigated , although obtaining selectivity over human 
 kinases would be more problematical .
however , the aminopyrazole compounds 
 ( 4a4z and 9a9x ) reported here represent an excellent start for chemistry 
 optimization of selective tbgsk3 short inhibitors 
 and an outstanding probe for studying the physiological functions 
 of tbgsk3 short in t. brucei parasites .
subsequently , 
 modeler 9.2 was used to build homology 
 models of tbgsk3 short , whereas the hsgsk3 crystal structure ( pdb code 1r0e ) served as template .
modeler was run 
 with default settings , and only the highest - scoring structure was 
 used for further analysis and modeling .
flexx 2.0.1 ( biosolveit gmbh ) was used 
 to dock ligands flexible into protein binding sites .
the active sites were defined as the areas within 7  
 of the co - crystallized ligands of hscdk2 ( pdb code 2vu3 ) and hsgsk3 ( pdb code 1r0e ) or the equivalent residues in the homology model of tbgsk3 . in all three structures , protonation states of amino 
 acids and the orientations of the protons of hydroxyl and amine groups 
 of active - site residues
a highly conserved water molecule ( h2o 82 in 1r0e or h2o 2134 in 2vu3 ) was kept in all three protein structures used for docking .
docking 
 was carried out using default settings , and only the highest scoring 
 binding modes were visually analyzed .
all figures of protein 
 binding sites were prepared using pymol . for compound potency determinations , 

compounds were solubilized in dmso at a top concentration of 3 mm 
 and serially diluted to achieve 10-point titration of final assay 
 concentrations from 30 m to 0.3 nm with a final dmso concentration 
 of 1% ( v / v ) .
the reaction mixtures contained 1 m biotinylated 
 gsp2 substrate , 1 m atp , 3.7 kbq / well [ -33p]-atp and 
 2.5 nm tbgsk3 in the tbgsk3 kinase 
 assay buffer .
gsk3 inhibitors were screened for selectivity assessment 
 also against hsgsk3. for hsgsk3 assay , the reaction mixes contained 1 m biotinylated 
 gsp2 substrate , 2 m atp , 7.4 kbq / well [ -33p]-atp and 
 15 nm hsgsk3 in the tbgsk3 
 kinase assay buffer ( 25 mm tris - hcl , ph 7.5 , 10 mm mgcl2 , 5 mm dtt , 0.02% chaps , 2 u / ml heparin ) .
for hscdk2/cyclin a assay , the reaction mixtures contained 1 mm cdk5 biotinylated 
 peptide substrate ( biotin - c6-pktpkkakkl ) , 1 m atp , 
 7.4 kbq / well [ -p]-atp and 2 nm hscdk2/cyclin a in the kinase assay buffer ( 50 mm tris - hcl , ph 7.5 , 
 10 mm mgcl2 , 2 mm dtt , 100 mm nacl , 0.2 mm egta , 0.02% 
 ( v / v ) brij35 ) .
the 
 statistical significance of the compound potency ( ic50 ) 
 was based on the performance of standard molecules which have been 
 tested to a high replication . in the case of tbgsk3 
 short assay
the average pic50 value was 8.26 with 
 a sd ( standard deviation ) of 0.23 .
the minimum significant ratio ( msr ) 
 of 0.4 was evaluated considering the following formula : where sd 
 is the standard deviation and n the number of replicate 
 values routinely used for the 
 assay ( 2 in our case ) .
this implies that 
 a difference of > 0.4 in pic50 can be considered statistically 
 significant for this assay . in the case of hsgsk3 assay ,
the standard compound gw8510 was tested 47 times across 
 5 independent runs , with an average pic50 value of 8.10 
 and a sd of 0.21 .
this implies that a difference of > 0.3 in pic50 can be considered statistically significant for this assay . for the hscdk2 assay ,
the analysis was performed 
 using two different standards ( gw8510 and staurosporine ) tested respectively 
 five times in a single run and 19 times in 2 independent runs .
this 
 implies that a difference of > 0.3 in pic50 can be considered 
 statistically significant for this assay .
selected compounds were 
 screened against a panel of mammalian kinases routinely run by the 
 division of signal transduction therapy ( dstt ) at the university of 
 dundee in duplicate at 10 m .
enzymes 
 included in the panel and assay conditions are reported in the literature . 

all biochemical assays are run below the km for the atp for each enzyme , allowing comparison of 
 inhibition across the panel .
measurement 
 of inhibition of the proliferation of mrc5 ( human lung fibroblast ) 
 cells and t. b. brucei bloodstream 
 stage cells was performed using a modification of the cell viability 
 assay previously described .
compounds 
 ( 50 m to 0.5 nm ) were incubated with 2  10 cells / well in 0.2 ml of the appropriate culture medium ( mem with 
 10% fetal bovine serum for mrc5 cells ) in clear 96-well plates .
plates 
 were incubated at 37 c in the presence of 5% co2 for 
 69 h. resazurin was then added to a final concentration of 50 m , 
 and plates were incubated as above for a further 4 h before being 
 read on a biotek flx800 fluorescent plate reader .
h and c nmr 
 spectra were recorded on either a bruker avance dpx 
 300 or 500 mhz spectrometer .
chemical shifts (  ) are expressed 
 in parts per million ( ppm ) and coupling constants ( j ) are in hertz ( hz ) .
signal splitting patterns are described as singlet 
 ( s ) , broad singlet ( br s ) , doublet ( d ) , triplet ( t ) , quartet ( q ) , 
 quintuplet ( quin ) , sextuplet ( sex ) , septet ( sept ) , multiplet ( m ) , 
 or combinations thereof .
lcms ( liquid chromatography mass spectrometry ) 
 analyses were performed with either an agilent hplc 1100 series connected 
 to a bruker daltonics microtof or an agilent technologies 1200 series 
 hplc connected to an agilent technologies 6130 quadrupole lcms , and 
 both instruments were connected to an agilent diode array detector . 

lcms chromatographic separations were conducted with a phenomenex 
 gemini c18 column , 50 mm  3.0 mm , 5 m particle size ; 
 mobile phase / acetonitrile + 0.1% hcooh 80:20 to 5:95 over 3.5 min , 
 and then held for 1.5 min ; flow rate 0.5 ml min .
high resolution electrospray measurements ( hrms ) were performed 
 with a bruker daltonics microtof mass spectrometer .
thin layer chromatography 
 ( tlc ) was carried out on merck silica gel 60 f254 plates using uv 
 light and/or kmno4 for visualization .
when 
 applicable , all glassware was oven - dried overnight and all reactions 
 were carried out under dry and inert conditions ( argon atmosphere ) .
all in this work synthesized compounds had a measured purity of 
 greater than 95% ( measured on analytical hplc - ms system ) .
m data are given below to substantiate the purity and integrity of 
 the compounds .
h nmr , c nmr , and hrms experiments 
 were also used to confirm compound identity and purity .
a mixture of 4-nitro-3-pyrazolecarboxylic 
 acid ( 1 ) ( 2.33 g , 14.8 mmol ) , trans-4-methoxy - cyclohexylamine 
 ( 2.39 g , 18.5 mmol ) , edc ( 3.55 g , 18.5 mmol ) , and hobt ( 2.50 g , 18.5 
 mmol ) in dmf ( 75 ml ) was stirred at ambient temperature for 16 h. 
 the mixture was reduced in vacuo and partitioned between saturated 
 aqueous sodium bicarbonate and etoac .
the organic layer was washed 
 ( water , brine ) , dried ( mgso4 ) , and reduced in vacuo to 
 give a yellow oil , which was purified by column chromatography , eluting 
 0100% etoac in petroleum ether to give 2 .
h nmr ( dmso - d6 dmso - d6 )  ( ppm ) 14.02 
 ( s , 1h ) , 8.73 ( s , 1h ) , 8.57 ( d , j = 7.81 hz , 1h ) , 
 3.74 ( m , 1h ) , 3.24 ( s , 3h ) , 3.11 ( m , 1h ) , 1.95 ( dd , j = 55.8 , 10.9 hz , 4h ) , 1.27 ( m , 4h ) .
c nmr ( dmso - d6 )  ( ppm ) 159.04 , 141.49 , 132.17 , 131.44 , 
 77.40 , 55.01 , 47.58 , 29.67 , 29.30 . lrms ( es ) :
a solution of 2 ( 1.13 g , 4.2 
 mmol ) in dmf ( 100 ml ) was treated with 10% 
 palladium on carbon then shaken under hydrogen at room temperature 
 and atmospheric pressure for 5 h. the reaction mixture was diluted 
 with etoac , filtered through celite , washing with further etoac , and 
 the filtrate reduced in vacuo to give crude 3 as brown 
 oil . yield : 982 mg , 98% .
h nmr ( cd3od )  
 ( ppm ) 7.23 ( s , 1h ) , 3.84 ( m , 1h ) , 3.37 ( s , 3h ) , 3.23 ( m , 1h ) , 2.07 
 ( dd , j = 45.8 , 11.2 hz , 4h ) , 1.38 ( m , 4h ) .
c nmr ( meod - d4 )  ( ppm ) 
 165.58 , 134.15 , 133.13 , 118.22 , 79.72 , 56.15 , 48.58 , 31.37
. lrms ( es ) : m / z 239 [ m + h ] . a mixture of benzoic 
 acid ( 0.051 g , 0.42 mmol ) , 3 ( 0.1 g , 0.42 mmol ) ,
edc 
 ( 0.096 g , 0.5 mmol ) , and hobt ( 0.068 g , 0.5 mmol ) in dmf ( 10 ml ) was 
 stirred at ambient temperature for 16 h. the mixture was reduced in 
 vacuo and partitioned between saturated aqueous sodium bicarbonate 
 and etoac . the organic layer was washed ( water , brine ) , dried ( mgso4 ) , and reduced in vacuo to give a creamy solid 4f , which was purified by column chromatography .
evaporation of the 
 appropriate fraction yielded the desired compound as an amorphous 
 solid .
h nmr ( cdcl3 )  
 ( ppm ) 10.65 ( s , 1h ) , 8.52 ( s , 1h ) , 8.01 ( d , j = 
 7.2 hz , 2h ) , 7.57 ( t , j = 7.2 hz , 1h ) , 7.51 ( m , 
 2h ) , 6.86 ( d , j = 8.3 hz , 1h ) , 4.01 ( m , 1h ) , 3.39 
 ( s , 3h ) , 3.22 ( m , 1h ) , 2.16 ( m , 4h ) , 1.42 ( m , 4h ) . c 
 nmr ( cdcl3 )  ( ppm ) 164.36 , 163.21 , 133.54 , 133.28 , 
 132.03 , 128.83 , 127.24 , 123.74 , 120.82 , 78.13 , 55.91 , 47.48 , 30.69 , 
 30.11 .
hrms ( es ) : calcd for c18h23n4o3 [ m + h ] 343.1765 , found 343.1751 .
h nmr ( cdcl3 )  ( ppm ) 
 9.86 ( s , 1h ) , 8.35 ( s , 1h ) , 6.83 ( d , j = 8.3 hz , 
 1h ) , 3.95 ( m , 1h ) , 3.39 ( s , 3h ) , 3.21 ( m , 1h ) , 2.44 ( m , 1h ) , 2.22 
 ( m , 2h ) , 2.11 ( m , 6h ) , 1.94 ( m , 2h ) , 1.81 ( m , 2h ) , 1.40 ( m , 4h ) .
c nmr ( cdcl3 )  ( ppm ) 171.61 , 163.21 , 133.28 , 
 123.22 , 122.55 ( t , j = 239.4 hz ) , 120.71 , 78.07 , 
 55.91 , 47.50 , 42.74 , 32.85 ( t , j = 23.5 hz ) , 30.35 , 
 25.73 .
hrms ( es ) : calcd for c18h27f2n4o3 [ m 
 + h ] 385.2046 , found 385.2036 .
h nmr ( cdcl3 )  ( ppm ) 9.86 ( s , 1h ) , 8.34 ( s , 1h ) , 
 6.81 ( d , j = 8.1 hz , 1h ) , 4.07 ( m , 2h ) , 3.95 ( m , 
 1h ) , 3.47 ( m , 2h ) , 3.39 ( s , 3h ) , 3.20 ( m , 1h ) , 2.59 ( m , 1h ) , 2.13 
 ( d , j = 11.0 hz , 4h ) , 1.91 ( m , 4h ) , 1.40 ( m , 4h ) .
c nmr ( cdcl3 )  ( ppm ) 171.97 , 163.30 , 133.22 , 
 123.23 , 120.71 , 78.12 , 67.26 , 55.92 , 47.50 , 42.20 , 30.64 , 30.12 , 29.10 . 

lrms ( es ) : m / z 351 [ m 
 + h ] .
hrms ( es ) : calcd for c17h27n4o4 [ m + h ] 351.2027 , 
 found 351.2011 .
h nmr ( cd3od )  ( ppm ) 
 8.20 ( s , 1h ) , 7.38 ( t , 7.7 hz , 1h ) , 7.347.26 ( m , 1h ) , 7.187.05 
 ( m , 2h ) , 3.83 ( m , 1h ) , 3.80 ( s , 2h ) , 3.33 ( s , 3h ) , 3.16 ( m , 1h ) , 2.06 
 ( br d , j = 11.5 hz , 2h ) , 1.97 ( br d , j = 12.0 hz , 2h ) , 1.34 ( m , 4h ) .
c nmr ( cd3od )  ( ppm ) 169.57 , 164.14 ( d , j = 159.5 
 hz ) , 134.23 , 132.98 , 130.64 ( d , j = 10.9 hz ) , 125.75 , 
 123.77 , 123.20 ( d , j = 18.2 hz ) , 121.96 , 116.48 
 ( d , j = 21.7 hz ) , 79.69 , 56.14 , 48.68 , 37.61 , 31.41 , 
 31.27 .
hrms ( es ) : calcd for c19h24fn4o3 [ m + h ] 375.1827 , found 375.1817 .
h nmr ( dmso - d6 )  ( ppm ) 13.13 ( s , 1h ) , 9.76 ( s , 1h ) , 8.15 ( s , 1h ) , 
 8.00 ( d , j = 8.3 hz , 1h ) , 7.317.23 ( m , 2h ) , 
 7.02 ( d , j = 8.0 hz , 1h ) , 6.93 ( t , j = 7.4 hz , 1h ) , 3.82 ( s , 3h ) , 3.793.70 ( m , 1h ) , 3.24 ( s , 
 3h ) , 3.07 ( m , 1h ) , 2.01 ( d , j = 11.4 hz , 2h ) , 1.79 
 ( d , j = 11.4 hz , 2h ) , 1.45 ( m , 2h ) , 1.21 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 
 167.30 , 162.43 , 157.02 , 132.38 , 130.90 , 128.59 , 123.21 , 122.30 , 120.44 , 
 119.70 , 110.88 , 77.70 , 55.41 , 55.04 , 46.70 , 38.17 , 30.26 , 29.73 .
lrms(es ) : m / z 387 [ m + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , found 387.2028 .
h nmr ( dmso - d6 )  ( ppm ) 13.18 ( br s , 1h ) , 9.86 ( s , 1h ) , 8.14 ( s , 1h ) , 
 8.05 ( d , j = 7.9 hz , 1h ) , 7.51 ( d , j = 8.2 hz , 2h ) , 7.36 ( t , j = 8.2 hz , 1h ) , 4.10 
 ( s , 2h ) , 3.76 ( m , 1h ) , 3.24 ( s , 3h ) , 3.08 ( s , 1h ) , 2.01 ( m , 2h ) , 1.81 
 ( m , 2h ) , 1.45 ( m , 2h ) , 1.19 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 170.16 , 162.31 , 135.49 , 131.62 , 
 129.68 , 128.30 , 128.10 , 122.23 , 122.09 , 77.68 , 55.04 , 46.77 , 38.23 , 
 30.25 , 29.74 .
hrms ( es ) : calcd for c19h23cl2n4o3 [ m 
 + h ] 425.1142 , found 425.1147 .
h nmr ( cdcl3 )  ( ppm ) 
 10.89 ( d , j = 12.2 hz , 1h ) , 8.45 ( s , 1h ) , 8.07 ( m , 
 1h ) , 7.44 ( m , 1h ) , 7.22 ( m , 1h ) , 7.13 ( m , 1h ) , 6.73 ( d , j = 8.3 hz , 1h ) , 3.96 ( m , 1h ) , 3.29 ( s , 3h ) , 3.11 ( m , 1h ) , 2.05 ( m , 
 4h ) , 1.31 ( m , 4h ) .
c nmr ( cdcl3 )  ( ppm ) 
 162.80 , 160.84 ( d , j = 246.9 hz ) , 160.53 , 133.81 
 ( d , j = 10.9 hz ) , 131.77 , 124.79 , 123.19 , 121.42 , 
 120.65 ( d , j = 10.7 hz ) , 116.46 ( d , j = 21.8 hz ) , 78.19 , 55.88 , 47.27 , 30.75 , 30.14 . lrms ( es ) :
m / z 361 [ m + h ] . 
 hrms ( es ) : calcd for c18h22fn4o3 [ m + h ] 361.1670 , found 361.1645 .
h nmr ( dmso - d6 )  ( ppm ) 13.40 ( s , 1h ) , 10.22 ( s , 1h ) , 8.32 ( s , 1h ) , 
 8.29 ( d , j = 8.6 hz , 1h ) , 7.907.88 ( m , 1h ) , 
 7.847.81 ( m , 1h ) , 7.787.75 ( m , 2h ) , 3.72 ( m , 1h ) , 
 3.23 ( s , 3h ) , 3.06 ( m , 1h ) , 2.00 ( br d , j = 12.5 
 hz , 2h ) , 1.76 ( br d , j = 12.5 hz , 2h ) , 1.45 ( m , 
 2h ) , 1.14 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 163.34 , 162.73 , 135.06 , 133.01 , 132.78 , 130.73 , 
 128.39 , 126.63 ( d , j = 5.2 hz ) 126.04 ( d , j = 29.1 hz ) , 123.57 ( d , j = 276.3 hz ) , 
 122.06 , 120.30 , 77.66 , 55.06 , 46.97 , 30.28 , 29.67 .
lrms ( es ) : m / z 411 [ m + h ] . 

hrms ( es ) : calcd for c19h22f3n4o3 [ m + h ] 411.1639 , found 
 411.1621 .
h nmr ( cdcl3 )  ( ppm ) 
 10.04 ( s , 1h ) , 8.39 ( s , 1h ) , 7.53 ( d , j = 7.6 hz , 
 1h ) , 7.39 ( t , j = 7.6 hz , 1h ) , 7.30 ( d , 7.7 hz , 
 1h ) , 7.25 ( t , j = 7.5 hz , 1h ) , 6.87 ( d , j = 8.3 hz , 1h ) , 3.92 ( m , 1h ) , 3.36 ( s , 3h ) , 3.16 ( m , 1h ) , 
 2.90 ( q , j = 7.6 hz , 2h ) , 2.08 ( br d , j = 10.0 hz , 4h ) , 1.35 ( m , 4h ) , 1.26 ( t , j = 7.6 
 hz , 3h ) .
c nmr ( cdcl3 )  ( ppm ) 167.60 , 
 163.29 , 142.86 , 134.89 , 133.19 , 130.65 , 129.69 , 127.15 , 126.11 , 123.25 , 
 120.94 , 78.18 , 55.86 , 74.45 , 30.58 , 30.12 , 26.44 , 15.84 .
hrms ( es ) : calcd for c20h27n4o3 [ m + h ] 371.2078 , found 371.2078 .
h nmr : ( dmso - d6 )  ( ppm ) 13.25 ( s , 1h ) , 11.77 ( s , 1h ) , 8.44 ( s , 1h ) , 
 8.15 ( s , 1h ) , 8.04 ( s , 1h ) , 7.63 ( s , 1h ) , 7.29 ( s , 1h ) , 7.18 ( s , 1h ) , 
 4.15 ( s , 3h ) , 3.88 ( m , 1h ) , 3.30 ( s , 3h ) , 3.16 ( m , 1h ) , 2.08 ( m , 2h ) , 
 1.91 ( m , 2h ) , 1.55 ( m , 2h ) , 1.29 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.43 , 160.94 , 157.47 , 133.49 , 
 131.33 , 122.29 , 120.83 , 120.59 , 120.28 , 112.32 , 77.73 , 56.05 , 55.05 , 
 46.72 , 30.28 , 29.83 . lrms ( es ) :
m / z 373 [ m + h ] . hrms ( es ) : calcd 
 for c19h25n4o4 [ m + h ] 373.1870 , found 373.1873 .
h nmr ( dmso - d6 )  ( ppm ) 13.32 ( br s , 1h ) , 10.93 ( s , 1h ) , 9.53 ( s , 
 1h ) , 8.31 ( s , 1h ) , 8.28 ( d , j = 8.5 hz , 1h ) , 7.68 
 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.43 ( m , 1h ) , 7.337.28 
 ( m , 3h ) , 7.167.14 ( m , 2h ) , 7.016.96 ( m , 2h ) , 3.82 
 ( m , 1h ) , 3.35 ( s , 1h ) , 3.25 ( s , 3h ) , 3.09 ( m , 1h ) , 2.03 ( br d , j = 12.0 hz , 2h ) , 1.81 ( br d , j = 12.0 
 hz , 2h ) , 1.48 ( m , 2h ) , 1.20 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 164.84 , 162.97 , 144.57 , 141.62 , 
 132.81 , 129.33 , 128.04 , 122.32 , 122.00 , 120.01 , 119.63 , 119.26 , 118.30 , 
 116.49 , 77.74 , 55.06 , 46.95 , 30.33 , 29.73 . lrms ( es ) : m / z 434 [ m + h ] .
hrms ( es ) : calcd for c24h28n5o3 [ m + h ] 434.2187 , found 434.2165 .
h nmr ( dmso - d6 )  ( ppm ) 13.35 ( br 
 s , 1h ) , 10.89 ( s , 1h ) , 9.53 ( s , 1h ) , 8.33 ( s , 1h ) , 7.63 ( dd , j = 8.1 , 1.4 hz , 1h ) , 7.34 ( m , 1h ) , 7.117.09 ( m , 
 2h ) , 6.996.97 ( m , 1h ) , 6.87 ( m , 1h ) , 6.82 ( dd , j = 8.4 , 1.0 hz , 1h ) , 3.84 ( m , 1h ) , 3.35 ( s , 1h ) , 3.25 ( s , 3h ) , 3.09 
 ( m , 1h ) , 2.29 ( s , 3h ) , 2.14 ( s , 3h ) , 2.03 ( br d , j = 12.0 hz , 2h ) , 1.82 ( br d , j = 12.0 hz , 2h ) , 
 1.49 ( m , 2h ) , 1.21 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 165.36 , 163.08 , 147.05 , 138.87 , 137.74 , 
 132.95 , 132.72 , 130.25 , 127.52 , 125.96 , 125.74 , 122.41 , 120.79 , 119.98 , 
 117.44 , 115.36 , 114.63 , 77.74 , 55.06 , 46.95 , 30.33 , 29.75 , 20.25 , 
 13.64 . lrms ( es ) : m / z 462 [ m + h ] .
hrms ( es ) : calcd for c26h32n5o3 [ m + h ] 462.2500 , found 462.2503 .
h nmr ( dmso - d6 )  ( ppm ) 13.28 ( s , 1h ) , 9.75 ( s , 1h ) , 8.29 ( s , 1h ) , 
 8.20 ( d , j = 8.4 hz , 1h ) , 7.39 ( t , j = 8.5 hz , 1h ) , 6.75 ( d , j = 8.5 hz , 2h ) , 3.76 
 ( s , 6h ) , 3.70 ( m , 1h ) , 3.22 ( s , 3h ) , 3.06 ( m , 1h ) , 2.00 ( m , 2h ) , 1.76 
 ( m , 2h ) , 1.45 ( m , 2h ) , 1.13 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.79 , 161.21 , 156.88 , 132.21 , 
 131.08 , 122.52 , 119.81 , 115.03 , 104.28 , 77.66 , 55.81 , 55.08 , 46.93 , 
 30.29 , 29.66 . lrms ( es ) :
. hrms ( es ) : calcd for c20h27n4o5 [ m + h ] 403.1976 , found 403.1960 .
h nmr ( cd3od )  ( ppm ) 
 8.36 ( s , 1h ) , 6.17 ( s , 2h ) , 3.84 ( s , 3h ) , 3.82 ( s , 6h ) , 3.35 ( s , 3h ) , 
 3.22 ( m , 1h ) , 2.08 ( m , 4h ) , 1.36 ( m , 4h ) .
c nmr ( cd3od )  ( ppm ) 164.90 , 164.72 , 164.35 , 160.74 , 134.07 , 
 124.36 , 122.58 , 108.39 , 92.09 , 79.74 , 57.05 , 56.89 , 56.62 , 48.71 , 
 31.61 , 31.40 . lrms ( es ) :
m / z 433 [ m + h ] . hrms ( es ) : calcd for c21h29n4o6 [ m + h ] 433.2082 , found 433.2065 .
h nmr ( dmso - d6 )  ( ppm ) 13.24 ( s , 1h ) , 10.89 ( d , j = 10.2 hz , 1h ) , 8.33 ( s , 1h ) , 8.09 ( q , j = 8.7 
 hz , 1h ) , 8.05 ( d , j = 8.4 hz , 1h ) , 7.39 ( m , 1h ) , 
 7.24 ( m , 1h ) , 3.82 ( m , 1h ) , 3.25 ( s , 3h ) , 3.10 ( m , 1h ) , 2.02 ( d , j = 10.3 hz , 2h ) , 1.84 ( d , j = 11.6 hz , 
 2h ) , 1.47 ( q , j = 12.0 hz , 2h ) , 1.22 ( q , j = 12.5 hz , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 164.3 ( q , j = 250.6 , 
 10.9 hz ) , 162.71 , 160.3 ( q , j = 250.8 , 14.5 hz ) , 
 158.03 , 133.26 , 133.18 , 132.82 , 122.12 , 120.50 , 117.39 , 117.31 , 112.59 , 
 112.42 , 104.96 , 104.75 , 104.52 , 79.12 , 78.84 , 78.58 , 77.71 , 55.07 , 
 46.89 , 30.09 , 29.76;. lrms ( es ) : m / z 379 [ m + h ] .
hrms ( es ) : calcd 
 for c18h21f2n4o3 [ m + h ] 379.1576 , found 379.1567 .
h nmr ( dmso - d6 )  ( ppm ) 13.39 ( s , 1h ) , 10.77 ( s , 1h ) , 8.34 ( d , j = 8.5 hz , 1h ) , 8.30 ( s , 1h ) , 7.59 ( m , 1h ) , 7.52 ( m , 2h ) , 
 3.84 ( m , 1h ) , 3.24 ( s , 3h ) , 3.09 ( m , 1h ) , 2.02 ( d , j = 10.7 hz , 2h ) , 1.81 ( d , j = 10.7 hz , 2h ) , 1.49 
 ( q , j = 11.8 hz , 2h ) , 1.20 ( q , j = 11.8 hz , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.8 , 162.5(q , j = 250.7 , 
 14.4 hz ) , 160.3 , 137.16 127.75 , 126.59 , 124.12 , 122.13 , 118.94 , 110.2 
 ( q , j = 22.0 , 14.5 hz ) , 107.5 ( t , j = 27.0 hz ) , 77.71 , 55.06 , 46.99 , 30.29 , 29.76 . lrms ( es ) :
m / z 379 [ m + h ] . 
 hrms ( es ) : calcd for c18h21f2n4o3 [ m + h ] 379.1576 , found 
 379.1568 .
h nmr ( cdcl3 )  
 ( ppm ) 10.34 ( s , 1h ) , 8.23 ( s , 1h ) , 7.60 ( d , j = 
 1.9 hz , 2h ) , 7.31 ( t , j = 1.8 hz , 1h ) , 7.05 ( s , 
 1h ) , 6.58 ( d , 1h ) , 3.77 ( m , 1h ) , 3.15 ( s , 3h ) , 2.98 ( m , 1h ) , 1.91 
 ( m , 4h ) , 1.18 ( m , 4h ) .
c nmr ( dmso - d6 )  ( ppm ) 163.12 , 161.74 , 136.46 , 135.73 , 131.89 , 
 128.34 , 126.03 , 125.79 , 121.09 , 78.10 , 55.91 , 47.48 , 30.69 , 30.08 . 
 lrms ( es ) : m / z 411 [ m 
 + h ] .
hrms ( es ) : calcd for c18h21cl2n4o3 [ m + h ] 411.0985 , found 411.0966 .
h nmr ( cdcl3 )  ( ppm ) 
 10.65 ( s , 1h ) , 8.48 ( s , 1h ) , 8.02 ( d , j = 8.6 hz , 
 2h ) , 7.24 ( d , j = 8.6 hz , 2h ) , 6.84 ( d , j = 8.3 hz , 1h ) , 6.62 ( t , j = 73.2 hz , 
 1h ) , 4.00 ( m , 1h ) , 3.39 ( s , 3h ) , 3.22 ( m , 1h ) , 2.15 ( m , 4h ) , 1.42 
 ( m , 4h ) .
c nmr ( cdcl3 )  ( ppm ) 163.28 , 
 163.20 , 154.0 ( t , j = 2.9 hz ) , 133.56 , 130.28 , 129.24 , 
 123.64 , 120.74 , 119.22 , 118.94 , 115.47 , 112.01 , 78.11 , 55.94 , 47.53 , 
 30.67 , 30.12 .
hrms ( es ) : calcd for c19h23f2n4o4 [ m 
 + h ] 409.1682 , found 409.1649 .
h nmr ( cdcl3 )  ( ppm ) 
 10.44 ( s , 1h ) , 8.49 ( s , 1h ) , 7.89 ( d , j = 8.8 hz , 
 2h ) , 6.85 ( d , j = 8.3 hz , 1h ) , 6.62 ( d , j = 8.8 hz , 2h ) , 4.01 ( m , 1h ) , 3.39 ( m , 7h ) , 3.20 ( m , 1h ) , 
 2.15 ( m , 4h ) , 2.06 ( m , 4h ) , 1.41 ( m , 4h ) .
c nmr ( cdcl3 )  ( ppm ) 164.74 , 163.48 , 150.26 , 133.26 , 128.99 , 124.26 , 
 120.52 , 119.42 , 111.20 , 78.20 , 55.92 , 47.66 , 47.39 , 30.73 , 30.18 , 
 25.46 .
hrms ( es ) : calcd for c22h30n5o3 [ m + h ] 412.2343 , found 412.2338 .
h nmr ( cdcl3 )  ( ppm ) 
 10.48 ( s , 1h ) , 8.47 ( s , 1h ) , 7.90 ( d , j = 8.8 hz , 
 2h ) , 6.94 ( d , j = 8.8 hz , 2h ) , 6.83 ( m , 1h ) , 4.00 
 ( m , 1h ) , 3.39 ( s , 3h ) , 3.37 ( t , j = 5 hz , 4h ) , 3.20 
 ( m , 1h ) , 2.60 ( t , j = 5 hz , 4h ) , 2.38 ( s , 3h ) , 2.14 
 ( m , 4h ) , 1.41 ( m , 4h ) .
c nmr ( cdcl3 )  
 ( ppm ) 164.17 , 163.44 , 153.54 , 133.33 , 128.81 , 124.05 , 122.92 , 120.61 , 
 114.27 , 78.17 , 55.92 , 54.79 , 47.62 , 47.41 , 46.11 , 30.72 , 30.17 .
lrms 
 ( es ) : m / z 441 [ m + h ] .
hrms ( es ) : calcd for c23h33n6o3 [ m + h ] 441.2609 , found 441.2597 .
h nmr ( cdcl3 )  
 ( ppm ) 10.76 ( s , 1h ) , 8.53 ( s , 1h ) , 7.70 ( d , j = 
 7.5 hz , 1h ) , 7.66 ( d , j = 8.4 hz , 1h ) , 7.59 ( d , j = 0.9 hz , 1h ) , 7.46 ( t , j = 7.8 hz , 
 1h ) , 7.33 ( t , j = 7.5 hz , 1h ) , 6.85 ( d , j = 8.4 hz , 1h ) , 4.07 ( m , 1h ) , 3.40 ( s , 3h ) , 3.22 ( m , 1h ) , 
 2.16 ( m , 4h ) , 1.43 ( m , 4h ) .
c nmr ( cdcl3 ) 
  ( ppm ) 163.03 , 156.21 , 155.22 , 148.18 , 133.76 , 129.23 , 127.56 , 
 127.24 , 123.77 , 122.59 , 121.20 , 112.41 , 111.23 , 78.15 , 55.91 , 47.41 , 
 30.74 , 30.14 .
hrms ( es ) : calcd for c20h23n4o4 [ m + h ] 383.1714 , found 383.1703 .
h nmr ( dmso - d6 )  ( ppm ) 13.30 ( s , 
 1h ) , 11.01 ( s , 1h ) , 8.83 ( d , j = 7.0 hz , 1h ) , 8.37 
 ( s , 1h ) , 8.15 ( d , j = 8.4 hz , 1h ) , 7.82 ( d , j = 8.9 hz , 1h ) , 7.34 ( t , j = 7.7 hz , 
 1h ) , 7.09 ( t , j = 6.9 hz , 1h ) , 3.85 ( m , 1h ) , 3.26 
 ( s , 3h ) , 3.11 ( m , 1h ) , 2.04 ( d , j = 10.3 hz , 2h ) , 
 1.84 ( d , j = 10.7 hz , 2h ) , 1.49 ( m , 2h ) , 1.23 ( m , 
 2h ) .
c nmr ( dmso - d6 )  
 ( ppm ) 162.70 , 158.15 , 146.85 , 141.15 , 132.80 , 128.99 , 124.60 , 122.00 , 
 120.08 , 119.14 , 114.53 , 97.79 , 77.74 , 55.06 , 46.95 , 30.33 , 29.79 . 
 lrms ( es ) : m / z 383 [ m 
 + h ] .
hrms ( es ) : calcd for c19h23n6o3 [ m + h ] 383.1826 , 
 found 383.1812 .
h 
 nmr ( meod - d4 )  ( ppm ) 9.57 ( d , j = 6.9 hz , 1h ) , 8.31 ( d , j = 8.3 hz , 
 2h ) , 7.74 ( d , j = 9.0 hz , 1h ) , 7.57 ( td , j = 6.9 , 1.2 hz , 1h ) , 7.19 ( t , j = 6.9 , 
 1.2 hz , 1h ) , 3.94 ( m , 1h ) , 3.39 ( s , 3h ) , 3.27 ( m , 1h ) , 2.12 ( m , 4h ) , 
 1.50 ( m , 2h ) , 1.39 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.89 , 156.52 , 147.37 , 136.35 , 127.61 , 
 127.41 , 122.12 , 119.89 , 117.57 , 117.46 , 114.43 , 77.71 , 55.07 , 46.90 , 
 30.30 , 29.76 . lrms ( es ) :
m / z 383 [ m + h ] . hrms ( es ) : calcd for c19h23n6o3 [ m + h ] 383.1826 , found 383.1811 .
h nmr ( cdcl3 )  ( ppm ) 10.88 ( s , 1h ) , 10.24 ( s , 1h ) , 
 9.58 ( d , j = 6.9 hz , 1h ) , 8.51 ( s , 1h ) , 7.67 ( d , j = 9.0 hz , 1h ) , 7.41 ( t , j = 7.9 hz , 
 1h ) , 6.99 ( t , j = 6.9 hz , 1h ) , 6.80 ( d , j = 7.9 hz , 1h ) , 4.01 ( m , 1h ) , 3.39 ( s , 3h ) , 3.21 ( m , 1h ) , 
 3.02 ( s , 3h ) , 2.14 ( m , 4h ) , 1.41 ( m , 4h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.73 , 157.27 , 146.18 , 145.49 , 
 127.61 , 127.42 , 122.35 , 120.04 , 116.31 , 114.46 , 113.62 , 77.74 , 55.06 , 
 46.77 , 30.24 , 29.81 , 16.52 . lrms ( es ) :
m / z 397 [ m + h ] . hrms ( es ) : calcd for c20h25n6o3 [ m + h ] 397.1983 , found 397.1972 .
h nmr ( cdcl3 ) :  
 ( ppm ) 10.61 ( s , 1h ) , 8.49 ( s , 1h ) , 7.84 ( m , 2h ) , 7.48 ( t , j = 7.5 hz , 2h ) , 7.38 ( t , j = 7.4 hz , 
 1h ) , 7.31 ( t , j = 3.6 hz , 1h ) , 6.85 ( d , j = 8.5 hz , 1h ) , 6.80 ( d , j = 3.6 hz , 
 1h ) , 4.07 ( m , 1h ) , 3.40 ( s , 3h ) , 3.22 ( m , 1h ) , 2.16 ( m , 4h ) , 1.42 
 ( m , 4h ) .
c nmr ( cdcl3 )  ( ppm ) 163.03 , 
 156.39 , 155.72 , 146.33 , 133.74 , 129.49 , 128.94 , 128.80 , 124.72 , 122.85 , 
 120.91 , 117.13 , 107.30 , 78.18 , 55.88 , 47.25 , 30.78 , 30.14 . lrms ( es ) : m / z 409 [ m + h ] .
hrms ( es ) : calcd for c22h25n4o4 [ m + h ] 409.1870 , found 409.1832 .
h nmr ( dmso - d6 )  ( ppm ) 13.30 ( s , 1h ) , 11.20 ( s , 1h ) , 
 8.68 ( s , 1h ) , 8.38 ( s , 1h ) , 8.30 ( m , 2h ) , 8.14 ( d , j = 8.4 hz , 1h ) , 7.587.51 ( m , 3h ) , 3.83 ( m , 1h ) , 3.26 ( s , 
 3h ) , 3.11 ( m , 1h ) , 2.04 ( d , j = 10.5 hz , 2h ) , 1.84 
 ( d , j = 11.0 hz , 2h ) , 1.49 ( q , j = 12.5 hz , 2h ) , 1.23 ( q , j = 11.5 hz , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 
 162.64 , 157.34 , 151.99 , 150.43 , 132.90 , 130.23 , 128.50 , 128.07 , 127.92 , 
 126.62 , 121.89 , 120.02 , 77.74 , 55.06 , 47.00 , 30.33 , 29.76 . lrms ( es ) :
m / z 410 [ m + h ] . hrms ( es ) : calcd for c21h24n5o4 [ m + h ] 410.1823 , found 410.1807 .
a 100 ml three - necked round - bottomed flask equipped 
 with a magnetic stirring bar and fitted with a dropping funnel was 
 charged with 4-nitro-1h - pyrazole-3-carboxylic acid 
 ( 4.0 g , 25.5 mmol ) and methanol ( 40 ml ) .
the flask was cooled to 0 
 c , and thionyl chloride ( 2.1 ml , 28.9 mmol ) was added to the 
 vigorously stirred solution over a period of 10 min .
the mixture was 
 stirred for an additional 12 h at room temperature , after which time 
 tlc indicated complete consumption of the starting acid .
the reaction 
 mixture was concentrated under reduced pressure at 40 c and 
 the residue treated with toluene and reconcentrated ( 3  20 ml ) 
 under reduced pressure at 40 c to give methyl ester 5 as an off - white solid .
h nmr ( dmso - d6 )  ( ppm ) 14.39 ( br s , 1h ) , 9.98 ( s , 
 1h ) , 3.90 ( s , 3h ) .
c nmr ( dmso - d6 )  ( ppm ) 161.15 , 138.13 , 133.20 , 130.90 , 52.84 .
lrms 
 ( es ) : m / z 172 [ m + h ] . a 100 ml round - bottomed flask equipped with 
 digital 
 thermometer and stirrer
was charged with 10% palladium on carbon ( 0.621 
 g ) under argon . in a separate vessel ,
a slurry of methyl ester 5 ( 4.42 g , 25.8 mmol ) in ethanol ( 45 ml ) was warmed to 35 
 c to effect dissolution and the solution added to the catalyst 
 under argon . following a nitrogen  hydrogen purge sequence , 
 an atmosphere of hydrogen was introduced and the reaction mixture 
 maintained at 30 c until the reaction completion ( 6 h ) was noted 
 by h nmr analysis . following a purge cycle ,
the reaction 
 mixture under argon was filtered and the liquors concentrated under 
 reduced pressure to give amine 6 as a solid .
h nmr ( dmso - d6 )  
 ( ppm ) 12.83 ( br s , 1h ) , 7.10 ( s , 1h ) , 4.83 ( br s , 2h ) , 3.78 ( s , 3h ) .
c nmr ( dmso - d6 )  ( ppm ) 
 160.39 , 136.94 , 128.43 , 115.59 , 50.88 . lrms ( es ) :
a solution of amine 6 ( 3.57 
 g , 25.3 mmol ) in 1,4-dioxane ( 50 ml ) under argon was treated with 
 triethylamine ( 4.3 ml , 31 mmol ) followed by 2,6-dimethoxybenzoyl chloride 
 ( 6.13 g , 30.6 mmol ) such that the internal temperature was maintained 
 in the range 2025 c .
the reaction mixture was stirred 
 at 25 c until the reaction was complete ( 12 h ) by tlc analysis . 

the reaction mixture was filtered , the filter - cake washed with 1,4-dioxane , 
 and the combined filtrates progressed to next stage without further 
 isolation . to obtain analytical data for compound 7 and also 
 to determine the yield of this reaction ,
a 2 g sample was taken out 
 of the homogeneous filtrate solution ( total weight of this solution 
 is 91 g ) .
the crude product ( 192 mg ) was purified by 
 column chromatography ( dcm / meoh ) .
evaporation of the appropriate fractions 
 yielded finally the desired compound 7 as an amorphous solid ( 161 
 mg ) .
a 5 mg sample was used for to obtain analytical data ; the rest 
 was redissolved for use in the next reaction .
h nmr ( dmso - d6 ) 13.68 ( br s , 1h ) , 
 9.16 ( s , 1h ) , 8.31 ( s , 1h ) , 7.41 ( t , j = 8.4 hz , 
 1h ) , 6.76 ( d , j = 8.4 hz , 2h ) , 3.83 ( s , 3h ) , 3.77 
 ( s , 6h ) .
c nmr ( dmso - d6 ) 
  ( ppm ) 163.86 , 161.55 , 157.07 , 131.27 , 129.97 , 123.61 , 120.41 , 
 114.66 , 104.35 , 55.84 , 51.63 .
hrms ( es ) : calcd for c14h16n3o5 [ m + h ] 306.1084 , found 306.1081 . to a solution of sodium hydroxide ( 3.32 
 g , 83 mmol ) in water ( 20 ml ) was charged a solution of ester 7 in one portion ( 7.33 g , 24.0 mmol ; the solution of crude 7 from the previous reaction , plus 156 mg of redissolved pure 7 ) .
the reaction mixture was stirred at 25 c until completion 
 as determined by tlc analysis .
the reaction mixture was concentrated 
 under reduced pressure at 45 c , the oily residue diluted with 
 water and acidified to ph 1 with concentrated hydrochloric acid , such 
 that the temperature was maintained below 30 c .
the resulting 
 precipitate was collected by filtration , washed with water , pulled 
 dry on the filter , and subsequently washed with heptanes .
the filter 
 cake was charged to a 200 ml rotary evaporator flask and drying completed 
 azeotropically with toluene .
h nmr 
 ( dmso - d6 ) 13.44 ( br s , 2h ) , 9.17 ( br s , 
 1h ) , 8.29 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.76 
 ( d , j = 8.4 hz , 2h ) , 3.77 ( s , 6h ) .
hrms ( es ) : calcd for c13h14n3o5 [ m + h ] 292.0928 , found 292.0920 .
a mixture of carboxylic 
 acid ( 50 mg , 0.17 mmol , 1.2 equiv ) , amine ( 14 mg , 0.14 mmol , 1.0 equiv ) , 
 hydroxybenzotriazole ( 19 mg , 0.14 mmol , 1.0 equiv ) , polymer supported - carbodiimide 
 ( 105 mg , 0.14 mmol , 1.0 equiv ) , and acetonitrile was heated by microwave 
 irradiation for 10 min at 100 c .
the final product ( 9c ) was isolated from the reaction mixture by filtering through a short 
 column of si - carbonate under gravity , which scavenged the excess carboxylic 
 acid and hydroxybenzotriazole .
removal of the solvent under reduced pressure yielded the required 
 compounds as amorphous solids .
h nmr ( cd3od )  ( ppm ) 8.33 ( s , 1h ) , 7.42 ( t , j = 8.5 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 
 2h ) , 3.86 ( s , 6h ) , 3.82 ( m , 1h ) , 1.88 ( m , 4h ) , 1.68 ( d , j = 12.8 hz , 1h ) , 1.40 ( m , 4h ) , 1.27 ( m , 1h ) .
c nmr 
 ( cd3od )  ( ppm ) 165.02 , 164.71 , 159.24 , 134.17 , 132.97 , 
 123.89 , 121.82 , 115.64 , 105.24 , 56.50 , 49.36 , 33.77 , 26.58 , 26.19 . 
 lrms ( es ) : m / z 373 [ m 
 + h ] .
hrms ( es ) : calcd for c19h25n4o4 [ m + h ] 373.1870 , 
 found 373.1850 .
h nmr ( cd3od )  ( ppm ) 
 8.32 ( s , 1h ) , 7.42 ( t , j = 8.4 hz , 1h ) , 6.76 ( d , j = 8.4 hz , 2h ) , 3.86 ( s , 6h ) , 2.79 ( m , 1h ) , 0.80 ( m , 2h ) , 
 0.65 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 161.32 , 161.20 , 131.10 , 122.44 , 122.30 , 115.01 , 104.28 , 
 55.82 , 22.13 , and 22.02 ( d , rotamers ) , 5.55 .
hrms ( es ) : calcd for c16h19n4o4 [ m + h ] 331.1401 , found 331.1385 .
h nmr ( dmso - d6 )  ( ppm ) 13.28 ( s , 1h ) , 9.71 ( s , 1h ) , 8.60 ( d , j = 8.1 hz , 1h ) , 8.30 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.74 ( d , j = 8.4 hz , 2h ) , 4.37 
 ( sex , j = 8.3 , 1h ) , 3.76 ( s , 6h ) , 2.13 ( m , 4h ) , 
 1.63 ( m , 2h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.53 , 161.23 , 156.93 , 132.14 , 131.07 , 122.61 , 119.84 , 
 115.07 , 104.32 , 55.82 , 54.86 , 43.39 , 29.81 , 14.62 . lrms ( es ) :
m / z 345 [ m + h ] . 
 hrms ( es ) : calcd for c17h21n4o4 [ m + h ] 345.1557 , found 345.1548 .
h nmr ( cdcl3 )  ( ppm ) 
 12.19 ( br s , 1h ) , 9.94 ( s , 1h ) , 8.41 ( s , 1h ) , 7.29 ( t , j = 8.5 hz , 1h ) , 6.98 ( d , j = 8.1 hz , 1h ) , 6.57 
 ( d , j = 8.5 hz , 2h ) , 4.05 ( m , 1h ) , 3.80 ( s , 6h ) , 
 2.001.94 ( m , 2h ) , 1.661.45 ( m , 10h ) .
c nmr ( cdcl3 )  ( ppm ) 161.75 , 160.98 , 155.95 , 131.18 , 
 129.57 , 121.16 , 119.75 , 112.84 , 102.24 , 54.19 , 48.30 , 33.28 , 26.12 , 
 22.39 . lrms ( es ) :
m / z 387 [ m + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , found 387.2043 .
h nmr ( dmso - d6 )  ( ppm ) 11.80 ( br s , 1h ) , 9.72 ( s , 1h ) , 8.28 ( s , 1h ) , 
 8.00 ( br d , j = 6.9 hz , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 3.76 
 ( s , 6h ) , 3.66 ( m , 1h ) , 2.21 ( br s , 1h ) , 2.15 ( br s , 1h ) , 1.631.37 
 ( m , 5h ) , 1.160.99 ( m , 3h ) .
c nmr ( cd3od )  ( ppm ) 165.11 , 164.82 , 159.28 , 133.97 , 133.00 , 124.01 , 
 122.33 , 115.79 , 105.34 , 56.59 , 53.91 , 43.80 , 40.38 , 37.06 , 36.28 , 
 29.28 , 27.54 . lrms ( es ) : m / z 385 [ m + h ] .
hrms ( es ) : calcd for c20h25n4o4 [ m + h ] 385.1870 , found 385.1857 .
h nmr ( dmso - d6 )  ( ppm ) 13.30 ( s , 1h ) , 9.66 ( s , 1h ) , 9.55 ( s , 1h ) , 
 8.31 ( s , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 6.75 ( d , j = 8.6 hz , 2h ) , 3.76 ( s , 6h ) , 3.62 ( m , 4h ) , 2.84 ( m , 4h ) .
c nmr ( dmso - d6 )  ( ppm ) 
 161.26 , 161.18 , 156.93 , 131.41 , 131.10 , 122.90 , 119.80 , 115.01 , 104.32 , 
 65.98 , 55.82 , 54.31 . lrms ( es ) :
m / z 376 [ m + h ] . hrms ( es ) : calcd 
 for c17h22n5o5 [ m + h ] 376.1615 , found 376.1620 .
h nmr ( dmso - d6 )  ( ppm ) 13.15 ( brs , 1h ) , 9.70 ( s , 1h ) , 8.99 ( t , j = 6.3 hz , 1h ) , 8.32 ( s , 1h ) , 7.38 ( t , j = 8.4 hz , 1h ) , 7.317.29 ( m , 4h ) , 7.22 ( m , 1h ) , 6.74 ( d , j = 8.4 hz , 2h ) , 4.41 ( d , j = 6.4 hz , 
 2h ) , 3.75 ( s , 6h ) .
c nmr ( dmso - d6 )  ( ppm ) 163.46 , 161.29 , 156.93 , 139.45 , 131.95 , 131.10 , 
 128.21 , 127.26 , 126.71 , 122.58 , 120.18 , 115.01 , 104.32 , 55.82 , 41.66 . 
 lrms ( es ) : m / z 381 [ m 
 + h ] .
hrms ( es ) : calcd for c20h21n4o4 [ m + h ] 381.1557 , 
 found 381.1543 .
h nmr ( dmso - d6 )  ( ppm ) 13.33 ( br s , 1h ) , 9.66 ( s , 1h ) , 8.96 ( t , j = 5.9 , 1h ) , 8.50 ( m , 1h ) , 8.34 ( s , 1h ) , 7.74 ( td , j = 7.7 , 1.9 hz , 1h ) , 7.39 ( t , j = 8.4 
 hz , 1h ) , 7.30 ( d , j = 7.8 hz , 1h ) , 7.25 ( m , 1h ) , 
 6.73 ( d , j = 8.4 hz , 2h ) , 4.53 ( d , j = 5.6 hz , 2h ) , 3.75 ( s , 6h ) .
c nmr ( dmso - d6 )  ( ppm ) 163.65 , 161.32 , 158.14 , 156.93 , 148.75 , 
 136.68 , 131.89 , 131.10 , 122.61 , 122.06 , 120.84 , 120.18 , 114.98 , 104.32 , 
 55.82 , 43.54 . lrms ( es ) :
h nmr ( dmso - d6 )  ( ppm ) 13.30 ( br s , 1h ) , 9.68 ( s , 1h ) , 8.72 ( t , j = 5.0 hz , 1h ) , 8.39 ( d , j = 5.0 hz , 
 1h ) , 8.34 ( s , 1h ) , 7.60 ( d , j = 7.6 hz , 1h ) , 7.39 
 ( t , j = 8.4 hz , 1h ) , 7.23 ( dd , j = 7.6 , 5.0 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 
 4.54 ( d , j = 5.0 hz , 2h ) , 3.76 ( s , 6h ) , 2.29 ( s , 
 3h ) .
c nmr ( dmso - d6 )  
 ( ppm ) 163.28 , 161.29 , 156.96 , 153.98 , 145.81 , 137.64 , 132.10 , 131.13 , 
 130.64 , 122.47 , 122.24 , 120.04 , 114.98 , 104.32 , 55.84 , 40.96 , 17 , 
 13 . lrms ( es ) : m / z 396 
 [ m + h ] .
hrms ( es ) : calcd for c20h22n5o4 [ m + h ] 396.1666 , 
 found 396.1656 .
h nmr ( cd3od )  ( ppm ) 
 8.35 ( s. 1h ) , 7.37 ( t , j = 8.4 hz , 1h ) , 6.71 ( d , j = 8.4 hz , 2h ) , 3.82 ( s , 6h ) , 3.17 ( d , j = 7.0 hz , 2h ) , 1.771.64 ( m , 5h ) , 1.55 ( m , 1h ) , 1.281.13 
 ( m , 3h ) , 1.000.89 ( m , 2h ) .
c nmr ( cd3od )  ( ppm ) 165.64 , 165.00 , 159.22 , 134.15 , 132.99 , 123.89 , 
 122.10 , 115.69 , 105.31 , 56.58 , 46.09 , 39.42 , 32.00 , 27.57 , 27.03 . 
 lrms ( es ) : m / z 386 [ m 
 + h ] . hrms ( es ) : calcd for c20h27n4o4 [ m + h ] 387.2027 , 
 found 387.2008 . yield : 
 48 mg ( solid ) , 76% .
h nmr ( dmso - d6 )  ( ppm ) 13.51 ( s , 1h ) , 10.31 ( s , 1h ) , 9.65 ( s , 1h ) , 
 8.40 ( s , 1h ) , 7.78 ( d , j = 7.7 hz , 2h ) , 7.41 ( t , j = 8.5 hz , 1h ) , 7.31 ( t , j = 7.8 hz , 
 2h ) , 7.09 ( t , j = 7.3 hz , 1h ) , 6.76 ( d , j = 8.5 hz , 2h ) , 3.77 ( s , 6h ) .
c nmr ( dmso - d6 )  ( ppm ) 162.29 , 161.40 , 157.02 , 138.10 , 
 132.32 , 131.19 , 128.49 , 123.81 , 123.02 , 120.69 , 120.37 , 114.98 , 104.40 , 
 55.88 . lrms ( es ) :
m / z 367 [ m + h ] . hrms ( es ) : calcd for c19h19n4o4 [ m + h ] 367.1401 , found 367.1402 .
h nmr ( dmso - d6 )  ( ppm ) 13.63 ( s , 1h ) , 10.73 ( s , 1h ) , 9.55 ( s , 1h ) , 
 8.43 ( d , j = 6.4 hz , 3h ) , 7.83 ( d , j = 5.5 hz , 2h ) , 7.42 ( t , j = 8.3 hz , 1h ) , 6.77 
 ( d , j = 8.4 hz , 2h ) , 3.78 ( s , 6h ) .
hrms ( es ) : calcd for c18h18n5o4 [ m + h ] 368.1353 , found 368.1347 .
h nmr ( dmso - d6 )  ( ppm ) 13.61 ( s , 1h ) , 9.63 ( s , 1h ) , 9.49 ( s , 1h ) , 
 8.42 ( s , 1h ) , 8.38 ( br d , j = 4.6 hz , 1h ) , 8.09 
 ( d , j = 8.4 hz , 1h ) , 7.83 ( m , 1h ) , 7.42 ( t , j = 8.4 hz , 1h ) , 7.18 ( m , 1h ) , 6.77 ( d , j = 8.5 hz , 2h ) , 3.78 ( s , 6h ) .
c nmr ( dmso - d6 )  ( ppm ) 161.87 , 161.55 , 157.01 , 150.45 , 148.29 , 
 138.41 , 131.62 , 131.30 , 123.04 , 120.90 , 120.15 , 114.66 , 113.86 , 104.35 , 
 55.84 . lrms ( es ) : m / z 368 [ m + h ] .
hrms ( es ) : calcd for c18h18n5o4 [ m + h ] 368.1353 , found 368.1335 .
h nmr ( dmso - d6 )  ( ppm ) 13.21 ( br 
 s , 1h ) , 9.75 ( s , 1h ) , 8.28 ( s , 1h ) , 8.09 ( d , j = 
 8.4 hz , 1h ) , 7.39 ( t , j = 8.4 hz , 1h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 4.53 ( br d , j = 3.8 
 hz , 1h ) , 3.76 ( s , 6h ) , 3.66 ( m , 1h ) , 3.403.33 ( m , 1h ) , 1.82 
 ( m , 2h ) , 1.73 ( m , 2h ) , 1.44 ( m , 2h ) , 1.19 ( m , 2h ) .
c 
 nmr ( dmso - d6 ) 162.67 , 161.25 , 156.91 , 
 131.07 , 122.56 , 115.10 , 104.32 , 68.11 , 55.82 , 47.00 , 34.19 , 30.00 . 

lrms ( es ) : m / z 389 [ m 
 + h ] .
hrms ( es ) : calcd for c19h25n4o5 [ m + h ] 389.1819 , 
 found 389.1800 .
h nmr ( cd3od )  ( ppm ) 8.36 ( s , 0.5h ) , 8.33 ( s , 0.5h ) , 
 7.39 ( t , j = 8.5 hz , 1h ) , 6.73 ( d , j = 8.5 hz , 2h ) , 4.05 ( m , 0.5h ) , 3.84 ( s , 6h ) , 3.76 ( m , 0.5h ) , 1.961.32 
 ( m , 8h ) , 1.06 ( m , 1h ) , 0.98 ( d , j = 6.5 hz , 1.5 
 h ) , 0.92 ( d , j = 6.6 hz , 1.5h ) .
c nmr 
 ( cd3od )  ( ppm ) 165.06 , 165.02 , 164.76 , 164.64 , 159.25 , 
 134.07 , 134.01 , 132.97 , 123.99 , 123.91 , 122.30 , 122.12 , 115.70 , 115.66 , 
 105.39 , 105.29 , 56.54 , 49.52 , 46.86 , 35.15 , 33.67 , 33.12 , 31.53 , 31.01 , 
 30.09 , 22.63 , 21.25 . lrms ( es ) :
m / z 387 [ m + h ] . hrms ( es ) : calcd 
 for c20h27n4o4 [ m + h ] 387.2027 , found 387.2007 .
h nmr 
 ( cd3od )  ( ppm ) 8.35 ( s , 1h ) , 7.37 ( t , j = 8.4 hz , 1h ) , 6.71 ( d , j = 8.4 hz , 2h ) , 4.083.99 
 ( m , 3h ) , 3.82 ( s , 6h ) , 2.90 ( m , 2h ) , 1.89 ( m , 2h ) , 1.51 ( m , 2h ) , 1.46 
 ( s , 9h ) .
c nmr ( cd3od )  ( ppm ) 165.00 , 
 164.89 , 159.22 , 156.39 , 134.00 , 132.99 , 124.03 , 122.16 , 115.72 , 105.35 , 
 81.17 , 56.61 , 47.67 , 44.39 , and 43.61 ( br d , rotamers ) , 32.69 , 28.79 . 

lrms ( es ) : m / z 474 [ m 
 + h ] .
hrms ( es ) : calcd for c23h32n5o6 [ m + h ] 474.2347 , 
 found 474.2324 .
h nmr ( cd3od )  ( ppm ) 
 8.32 ( s , 1h ) , 7.36 ( t , j = 8.4 hz , 1h ) , 7.327.22 
 ( m , 5h ) , 6.68 ( d , j = 8.4 hz , 2h ) , 4.52 ( m , 1h ) , 
 3.78 ( s , 6h ) , 3.58 ( d , j = 2.5 hz , 2h ) , 2.76 ( m , 
 2h ) , 2.58 ( dd , j = 10.0 , 4.3 , 1h ) , 2.43 ( q , j = 8.1 hz , 1h ) , 2.27 ( m , 1h ) , 1.75 ( m , 1h ) .
c nmr ( cd3od )  ( ppm ) 165.06 , 165.00 , 159.22 , 139.26 , 
 133.92 , 132.99 , 130.33 , 129.50 , 128.49 , 124.01 , 122.16 , 115.67 , 105.32 , 
 61.26 , 61.17 , 56.58 , 53.83 , 49.19 , 32.54 . lrms ( es ) :
m / z 450 [ m + h ] . hrms ( es ) : calcd for c24h28n5o4 [ m + h ] 450.2136 , found 450.2113 .
h nmr ( cd3od )  ( ppm ) 
 8.35 ( s. 1h ) , 7.38 ( t , j = 8.4 hz , 1h ) , 6.72 ( d , 
 8.4 j = 8.4 hz , 2h ) , 3.83 ( s , 6h ) , 3.53 ( s , 4h ) , 
 3.37 ( s , 3h ) .
c nmr ( cd3od )  ( ppm ) 
 165.76 , 165.05 , 159.26 , 134.10 , 132.99 , 115.71 , 105.32 , 72.07 , 59.03 , 
 56.56 , 39.52 . lrms ( es ) :
m / z 349 [ m + h ] . hrms ( es ) : calcd for c16h21n4o5 [ m + h ] 349.1506 , found 349.1504 .
h nmr ( dmso - d6 )  ( ppm ) 13.23 ( br s , 1h ) , 9.74 ( s , 1h ) , 8.38 ( br s , 
 1h ) , 8.29 ( s , 1h ) , 7.39 ( t , j = 8.5 hz , 1h ) , 6.75 
 ( d , j = 8.5 hz , 2h ) , 3.76 ( s , 6h ) , 3.19 ( m , 2h ) , 
 1.49 ( quint , j = 7.2 hz , 2h ) , 1.311.22 ( m , 
 4h ) , 0.85 ( t , j = 7.0 hz , 3h ) .
c nmr 
 ( dmso - d6 )  ( ppm ) 163.39 , 161.20 , 
 156.93 , 132.25 , 131.07 , 122.35 , 119.79 , 115.05 , 104.32 , 55.82 , 37.95 , 
 28.77 , 28.57 , 21.78 , 13.87 . lrms ( es ) :
m / z 361 [ m + h ] . hrms ( es ) : calcd for c18h25n4o4 [ m + h ] 361.1870 , found 361.1868 .
h nmr ( cd3od )  ( ppm ) 
 8.35 ( s , 1h ) , 7.38 ( t , j = 8.5 hz , 1h ) , 6.71 ( d , j = 8.5 hz , 2h ) , 3.83 ( s , 6h ) , 3.58 ( sept , j = 6.1 hz , 1h ) , 3.53 ( t , j = 6.1 hz , 2h ) , 3.45 
 ( t , j = 6.6 hz , 2h ) , 1.83 ( quin , j = 6.4 hz , 2h ) , 1.15 ( d , j = 6.1 hz , 6h ) .
c nmr ( cd3od )  ( ppm ) 165.58 , 165.00 , 159.22 , 
 134.17 , 132.99 , 123.82 , 121.99 , 115.69 , 105.32 , 73.08 , 67.50 , 56.61 , 
 38.06 , 30.72 , 22.49 . lrms ( es ) : m / z 391 [ m + h ] .
hrms ( es ) : calcd 
 for c19h27n4o5 [ m + h ] 391.1976 , found 391.1961 .
h nmr ( cd3od )  ( ppm ) 
 8.23 ( s , 1h ) , 7.26 ( t , j = 8.4 hz , 1h ) , 6.59 ( d , j = 8.4 hz , 2h ) , 3.71 ( s , 6h ) , 3.26 ( t , j = 7.5 hz , 2h ) , 2.26 ( t , j = 7.5 hz , 2h ) , 2.11 
 ( s , 6h ) , 1.65 ( q , j = 7.5 hz , 2h ) .
c nmr ( cd3od )  ( ppm ) 165.70 , 164.94 , 159.22 , 134.15 , 
 132.99 , 123.92 , 122.05 , 115.75 , 105.35 , 58.25 , 56.61 , 45.52 , 38.23 , 
 28.32 . lrms ( es ) :
m / z 376 [ m + h ] . hrms ( es ) : calcd for c18h26n5o4 [ m + h ] 376.1979 , found 376.1965 .
h nmr ( cd3od )  ( ppm ) 
 8.34 ( s , 1h ) , 7.38 ( t , j = 8.4 hz , 1h ) , 6.71 ( d , j = 8.4 hz , 2h ) , 3.83 ( s , 6h ) , 3.51 ( t , j = 6.9 hz , 2h ) , 2.69 ( t , j = 6.9 hz , 2h ) , 2.59 
 ( m , 4h ) , 1.80 ( m , 4h ) .
c nmr ( cd3od )  
 ( ppm ) 165.69 , 164.97 , 159.22 , 134.09 , 132.99 , 123.89 , 122.01 , 115.72 , 
 105.32 , 56.58 , 56.32 , 55.08 , 38.64 , 24.31 .
hrms ( es ) : calcd for c19h26n5o4 [ m + h ] 388.1979 , found 388.1965 .
h nmr ( cd3od )  ( ppm ) 
 8.34 ( s , 1h ) , 7.40 ( t , j = 8.4 hz , 1h ) , 6.73 ( d , j = 8.4 hz , 2h ) , 3.84 ( s , 6h ) , 3.70 ( t , j = 4.5 hz , 4h ) , 3.5 ( t , j = 6.6 , 2h ) , 2.57 ( t , j = 6.6 hz , 2h ) , 2.52 ( br t , j = 4.5 
 hz , 4 hz ) .
c nmr ( cd3od )  ( ppm ) 165.67 , 
 165.00 , 159.23 , 134.06 , 132.96 , 123.86 , 122.02 , 115.72 , 105.32 , 67.80 , 
 58.60 , 56.55 , 54.67 , 36.50 . lrms ( es ) :
m / z 404 [ m + h ] . hrms ( es ) : calcd for c19h26n5o5 [ m + h ] 404.1928 , found 404.1929 .
 glycogen 
 synthase kinase 3 ( gsk3 ) is a genetically validated drug 
 target for human african trypanosomiasis ( hat ) , also called african 
 sleeping sickness . 
 we report the synthesis and biological evaluation 
 of aminopyrazole derivatives as trypanosoma brucei gsk3 short inhibitors . 
 low nanomolar inhibitors , which had high 
 selectivity over the off - target human cdk2 and good selectivity over 
 human gsk3 enzyme , have been prepared . 
 these potent kinase 
 inhibitors demonstrated low micromolar levels of inhibition of the trypanosoma brucei brucei parasite grown in culture . 
Introduction
Results
Discussion
Conclusion
Experimental Section