as a member of the 
 mitogen - activated protein kinase ( mapk ) family , 
 the c - jun n - terminal kinases ( jnks ) regulate the serine / threonine 
 phosphorylation of several transcription factors when they are activated in response to various stimuli 
 such as oxidative stress , neurotoxins , cytokines , and fatty acids . jnk1 and jnk2 are ubiquitously expressed in most tissues , while 
 jnk3 is primarily expressed in the brain and , to a lesser extent , 
 in the heart and testes . recent studies have shown that jnk3 plays a central role in the 
 brain to mediate neurodegeneration , such as -amyloid processing 
 and neuronal apoptosis in alzheimer s disease , as well as the mediation of neurotoxicity in rodent models 
 of parkinson s disease . the selective expression of jnk3 in the brain , along with findings 
 that jnk3 knockout mice exhibit amelioration of neurodegeneration 
 in animal models of parkinson and alzheimer s disease , makes 
 inhibiting this isoform a particularly promising therapeutic target 
 for neurodegenerative diseases . identifying potent and selective inhibitors of jnk3 may contribute 
 toward neuroprotective therapies with reduced untoward side effect 
 profiles if jnk1 inhibition has such negative effects . to date , however , 
 the development of specific small molecule inhibitors with high isoform 
 selectivity for jnk3 , especially against jnk1 , is still a relatively 
 untapped area as the three jnk isoforms share more than 90% sequence 
 identity in the atp pocket . many published jnk3 inhibitors are also 
 potent for jnk1 , jnk2 , and some inhibit p38 as well , because 
 of their high degree of amino acid sequence similarity , which might 
 lead to potential side effect profiles on immune and inflammatory 
 systems . thus , developing isoform - specific jnk3 inhibitors 
 as therapeutics has gained considerable interest over the past few 
 years despite most reports being centered on pan - jnk inhibitors . our previous effort toward the development of isoform selective 
 jnk3 inhibitors led to the identification of a class of aminopyrazole 
 compounds . these aminopyrazole - based jnk3 inhibitors not only had 
 high selectivity against p38 but also showed noticeable isoform 
 selectivity against jnk1 ( compound sr-4326 , 18.5-fold , figure 1 ) . herein , we describe 
 extensive sar studies in a continuing effort to develop highly isoform 
 selective , efficacious , and pharmacologically viable jnk3 inhibitors 
 from this novel aminopyrazole scaffold . the focused medicinal chemistry 
 efforts led to the discovery of several highly potent and isoform 
 selective jnk3 inhibitors with an isoform selectivity of > 50-fold 
 over jnk1 . in addition , these jnk3 inhibitors generally had high selectivity 
 against the closely related protein kinase p38 , were potent 
 in protecting against ros generation and mitochondrial dysfunction , 
 and were optimized to have good dmpk properties for topical use and/or 
 in iv dosing . several short routes were 
 used to synthesize inhibitors 68 which possessed different substituents on 
 the urea moiety ( scheme 1 ) . ullman coupling 
 of 4-nitro-1h - pyrazole 1 with 3-bromobenzoic 
 acid 2 yielded acid 3 . amide formation to 3 gave intermediate 4 , followed by a pd / c mediated 
 hydrogenation to afford amine 5 . inhibitor 7 was formed via a one - step coupling reaction 
 from amine 5 under microwave reaction conditions . inhibitors 8a  l were synthesized by treating amine 5 with different isocyanates or with triphosgene and a secondary 
 amine , respectively . reagents and conditions : ( a ) 
 cui , trans - n , n - dimethylcyclohexane-1,2-diamine , 
 cs2co3 , dmf , 90 c ; ( b ) 6-methylpyridin-3-amine , 
 edc , hobt , diea , ch2cl2 , 25 c ; ( c ) pd / c , 
 meoh , h2 ; ( d ) k2co3 , thf or edc , 
 hobt , diea , ch2cl2 , 25 c ; ( e ) 2-chloro-1h - benzo[d]imidazole , etoh , hcl , microwave , 
 140 c ; ( f ) isocyanate , ch2cl2 , 25 c , 
 or secondary amine ; triphosgene , 50 c , or cdi , thf , microwave , 
 150 c . the preparation of inhibitor 12 is described in scheme 2 . treatment 
 of ethyl 1h - pyrazole-4-carboxylate 9 with 3-bromobenzoic acid 2 followed by amide 
 formation gave amide 10 . amide 10 was hydrolyzed 
 under basic conditions to yield acid 11 , which was then 
 subjected to an amide formation reaction using edc as the coupling 
 reagent to furnish the final product 12 . reagents and conditions : 
 ( a ) 
 cui , trans - n , n - dimethylcyclohexane-1,2-diamine , 
 cs2co3 , dmf , 90 c ; ( b ) 6-methylpyridin-3-amine , 
 edc , hobt , diea , ch2cl2 , 25 c ; ( c ) lioh , 
 thf / h2o ( 1:1 ) , 100 c ; ( d ) 2-chloroaniline , edc , hobt , 
 diea , ch2cl2 , 25 c . inhibitors 16a  d , all of which 
 have a fluoro substitution on the middle phenyl ring , were synthesized 
 following procedures shown in scheme 3 . the 
 commercially available 4-nitro-1h - pyrazole 1 was n - arylated with ethyl 3-bromobenzoate 13 using standard ullman procedures , followed by ester hydrolysis under 
 basic conditions to form acid 14 . amide formation followed 
 by pd / c mediated reduction of 14 was applied to yield 
 amine 15 . finally , intermediate 15 was subjected 
 to a urea formation to furnish product 16 . reagents and conditions : 
 ( a ) 
 cui , trans - n , n - dimethylcyclohexane-1,2-diamine , 
 cs2co3 , dmf , 90 c ; ( b ) lioh , thf / h2o ( 1:1 ) , 100 c ; ( c ) 6-methylpyridin-3-amine , edc , hobt , 
 diea , ch2cl2 , 25 c ; ( d ) pd / c , meoh , h2 ; ( e ) 1-chloro-2-isocyanatobenzene , ch2cl2 , 25 c . the preparation of inhibitors 2022 is outlined in scheme 4 . ullman coupling 
 of 4-nitro-1h - pyrazole 1 with 1-bromo-3-iodobenzene 
 ( for synthesis of compounds 20 ) or 3-bromobenzonitrile 
 ( for synthesis of compounds 21 ) or 3-bromobenzoic acid 
 ( for synthesis of compounds 22 ) , followed by a pd / c mediated 
 reduction , afforded the intermediate amine 18 . target compounds 20 were prepared through a suzuki coupling reaction between 
 intermediate 19a and a boronic acid pinacol ester catalyzed 
 by pd(pph3)4 . target compounds 21a and 21b were prepared through treatment of intermediate 19b with hydrazine monohydrate , followed by a cyclization 
 reaction with acetimidamide or acetic acid . target compounds 22 were prepared through amide formation in the presence of 
 hatu in dmf . reagents and conditions : ( a ) 
 cui , trans - n , n - dimethylcyclohexane-1,2-diamine , 
 cs2co3 , dmf , 100 c ; ( b ) pd / c , meoh , h2 ; ( c ) 1-chloro-2-isocyanatobenzene , ch2cl2 , 25 c ; ( d ) r1b(oh)2 , ph(pph3)4 , k2co3 , dioxane / h2o ( 3:1 ) , 95 c ; ( e ) n2h4xh2o , etoh , reflux ; ( f ) acetimidamide , etoh , 
 naome or acetic acid , propylphosphonic anhydride , tea , dmf , 110 c ; 
 ( g ) r3nh2 , edc , hobt , diea , ch2cl2 , 25 c . applications of n - substituted 
 pyrazole amines led to inhibitors 26 . as shown in scheme 5 , the n1 alkylation 
 of 3-nitropyrazole with a primary iodo- or bromoalkane using potassium 
 carbonate as the base was first used to furnish n - substituted pyrazoles , 
 followed by reduction of the nitro group to give pyrazole amines 24 . amide formation between 24 and acid 3 , followed by a pd / c mediated reduction furnished amine 25 . a one - pot urea formation of 25 with 1-chloro-4-isocyanobenzene 
 in dcm afforded the target inhibitor 26 which was purified 
 by reverse - phase preparative hplc . reagents and conditions : ( a ) 
 k2co3 , dmf , 40 c ; ( b ) pd / c , meoh , h2 ; ( c ) edc , hobt , diea , ch2cl2 , 25 c ; 
 ( d ) pd / c , meoh , h2 ; ( e ) 1-chloro-2-isocyanatobenzene , ch2cl2 , 25 c . in the crystal structures published in our previous work , the aniline  urea 
 moiety ( figure 1 ) binds to the hydrophobic 
 selectivity pocket ( hydrophobic pocket i ) and is responsible for the 
 isoform selectivity of jnk3 over jnk1 . urea 
 moiety in region a ( figure 1 ) with selected 
 bioisosters . as shown in figure 2 , very few 
 replacements to the urea moiety were tolerated at region a , as all 
 compounds had jnk3 ic50 values of > 10 m . for 
 example , 
 replacement of the aniline nh group with a carbon ( 6a ) or oxygen ( 6b ) or a direct heterocyclic ring connection 
 ( 6c , d ) all resulted in complete loss of 
 jnk3 inhibition . similarly , structural changes to the other nh group 
 of urea were not tolerated either . some other replacements of the 
 urea moiety were also investigated ( such as compound 7 ; for more data see supporting information ) , but no improvement was observed . the observed results for modifications 
 to the urea moiety suggested that the urea core was essential for 
 achieving a high jnk3 affinity . therefore , this urea moiety will be 
 retained in all further optimizations . a previously disclosed 
 isoform selective jnk3 inhibitor sr-4326 . we next investigated 
 a series of replacements and substitutions 
 on the phenyl group of the urea moiety . as shown in table 1 , replacing the aromatic group with an alkyl group 
 was not tolerated , as indicated by the significant loss of jnk inhibition 
 activity for 8a . compound 8b , which had 
 no substitutions on the urea phenyl ring , showed a jnk3 inhibition 
 activity similar to the lead compound sr-4326 but with almost no isoform 
 selectivity . in the case of chloro substitutions , both the meta- and 
 the para - cl substitutions ( 8d and 8e ) led 
 to a decrease in jnk affinity , which could be due to space limitation 
 inside the binding pocket . however , when a cl substituent was introduced 
 at the ortho - position of the phenyl ring ( 8c ) , where 
 there should be enough space to accommodate a cl group based on the 
 crystal structures , higher jnk3 potency 
 was obtained probably because of the extra hydrophobic interactions 
 from the cl group . more interestingly , some isoform selectivity ( jnk3 
 over jnk1 ) was achieved as well ( 4.4-fold ) . on the other hand , fluoro 
 substitutions on this phenyl ring exhibited a significantly different 
 sar profile . compared to the lead compound sr-4326 and compound 8c , a mono - f - substitution at all three positions resulted 
 in reduction of jnk3 inhibition activity , although these substitutions 
 could improve the isoform selectivity ( compounds 8f , 8 g , 8h ) . while the meta - f compound 8 g and the para - f compound 8h showed a slight decrease 
 of jnk3 potency , a substitution at the ortho - position resulted in 
 around 3-fold loss of potency as compared to the unsubstituted analog 8b ( table 1 ) . ic50 values are the mean 
 of two or more experiments ( with triplicate replicates for each experiment ) 
 with errors within 80% of the mean . no inhibition up to 10 m . the unusual sar observed for f - substituted compounds 
 in table 1 might be due to the f - bonding / interactions . different types of interactions between fluorine atoms with protein 
 functional groups have been reported ( not found in other halogens ) , 
 and in some protein environments , compounds with a fluorine substitution 
 behave very differently from other halogen - substituted compounds . such fluorine interactions include f  h 
 hydrogen bonds , -interactions with aromatic or guanidine groups , 
 multipolar interactions , and/or f - nitrogen interactions ( halogen bonding ) . for the aminopyrazole based jnk inhibitors , 
 these special f - interactions could result in a change of inhibitor 
 conformations in the pocket or a twist of the orientation of the phenyl 
 ring inside hydrophobic pocket i. these changes in conformation or 
 orientation could produce unfavorable binding of the inhibitor in 
 the atp pocket , and therefore cause a decrease in inhibition of jnk 
 activity for compounds 8f  h . significant loss of potency also occurred with replacement of the o - cl - phenyl moiety by an o - cl - benzyl group 
 ( 8i , table 1 ) . this result indicated 
 that the hydrophobic selectivity pocket might not have enough room 
 to tolerate a larger group or that the phenyl ring of the benzyl group 
 could not assume a conformation / orientation that could give maximal 
 hydrophobic interactions . unfortunately , any substitution to this 
 nh group greatly reduced the jnk3 potency ( table 1 ) . for example , a simple methyl substitution resulted in compound 8j which exhibited a jnk3 ic50 of 3063 nm . larger 
 substituents yielded even lower jnk3 inhibitory activity ( compounds 8k and 8l , ic50 > 10 m ) . 
 as 
 is demonstrated in the crystal structure ( see figure 4 ) , this urea nh is involved in h - bonding interactions with 
 protein residues , and its alkylation is predicted to reduce the jnk3 
 inhibitory activity . the feasibility of applying some minor 
 structural modifications 
 to the central phenyl group attached to the pyrazole n1 ( region b , 
 figure 1 ) was explored next . we envisioned that introduction of a fluoro 
 substitution on the benzene ring might introduce additional interactions 
 that could perturb the jnk3 activity and selectivity , as well as improve 
 dmpk properties . therefore , compounds with a simple fluoro substitution 
 at different positions of this benzene ring were investigated ( table 2 ) . although improvement in isoform selectivity was 
 achieved ( compared to sr-4326 and 8c ) for the 2-fluoro 
 substitution ( 16a ) and the 6-fluoro substitution ( 16d ) , loss of jnk3 inhibition potency was observed for all 
 fluoro - substituted compounds 16a  d , especially for the 3-fluoro - derivative 16b , which 
 showed an ic50 value against jnk3 in the micromolar range 
 ( 4588 nm ) . the reduction in jnk inhibitory potency could be due to 
 the f - bondings / interactions previously mentioned 
 and as observed in compounds 8f  h ( table 1 ) . in addition , the 2-f analog 16a exhibited potent inhibition of cytochrome p450 isoforms 
 cyp2c9 and cyp2d6 ( see table 5 ) . because of 
 these detrimental features , f - substitutions on this benzene ring will 
 not be applied in future optimizations . ic50 values are the mean 
 of two or more experiments ( with triplicate replicates for each experiment ) 
 with errors within 80% of the mean . no inhibition up to 10 m . the crystal structure published in our previous work 
 indicated 
 that these compounds were type i kinase inhibitors and showed that 
 the amide carbonyl and the aromatic ring ( in the amide moiety ) did 
 not have any significant interactions with the protein ( only an h - bond 
 interaction was observed between the amide nh and the protein backbone ) . thus , structural modifications in this part 
 will likely not affect the binding mode significantly . to test this 
 observation , a series of replacements for the amide moiety in region 
 c ( figure 1 ) with some amide bioisosters were 
 investigated . as shown in figure 3 , the pyridine 
 and pyrimidine based structures ( 20a  b ) , which have heteroatoms on the aromatic ring to form h - bonds , showed 
 no jnk inhibitory activity . the triazole ( 21a ) and the 
 oxodiazole ( 21b ) based amide isosters , which mimic the 
 binding of the anilinoamide in region c , also gave no inhibition for 
 jnk3 and jnk1 . moreover , alkylation of the amide nh ( 22a ) led to complete loss of jnk3 inhibitory activity . these results 
 indicated that the amide structure was critical for efficient binding 
 and the amide moiety conh was indispensable for this 
 scaffold to achieve a high jnk3 affinity . sar studies of the amide moiety are a very important 
 part of our 
 efforts to improve both jnk3 inhibitory potency and isoform selectivity 
 ( against jnk1 ) for this aminopyrazole - based scaffold . the effect of 
 substitution patterns on the pyridine ring for lead compound 8c was first explored . as shown in table 3 , moving the methyl group from 4-position to 2-position 
 reduced jnk3 inhibitory potency by 3.7-fold but it reduced the jnk1 
 potency as much as 15.8-fold providing a benefit for the overall jnk3/jnk1 
 isoform selectivity ( 22b vs 8c ) . the dimethylpyridine 
 compound 22c showed a jnk3 potency similar to that of 
 the monosubstituted analogue 22b but a reduced isoform 
 selectivity . nevertheless , lead compound 8c still had 
 the best jnk3 potency ( 38 nm ) . while the 2-pyridine analogs 22d and 22e exhibited a significant loss of jnk3 
 inhibition potency , the 4-pyridine compound , 22f , presented just a slightly lower jnk3 potency ( 98 nm vs 38 nm ) but 
 a better isoform selectivity ( 28-fold vs 4.4-fold , 22f vs 8c ) . the dimethyl-4-pyridine compound 22 g exhibited an even higher isoform selectivity ( 35-fold ) , 
 although with a sacrifice in jnk3 potency . interestingly , the pyrimidine 
 amide 22h showed a similar jnk3 potency but had isoform 
 selectivity slightly better than compound 8c . it is also 
 important to note that the benzyl type amide 22i gave 
 a weaker jnk3 inhibition but improved isoform selectivity as compared 
 to the corresponding aniline type amide 8c and sr-4326 . in summary , for pyridine and/or pyrimidine based amides , 3-n 
 derivatives gave better jnk3 potency but not satisfactory isoform 
 selectivity ( 8c and 22h ) . on the other hand , 
 4-n analogs exhibited good isoform selectivity but lower jnk3 
 inhibition activity ( 22f and 22 g ) . nevertheless , 
 no compounds from this class could present both good jnk3 inhibition 
 potency ( ic50 < 50 nm ) and excellent isoform selectivity 
 ( > 50-fold ) . ic50 values are the mean 
 of two or more experiments ( with triplicate replicates for each experiment ) 
 with errors within 80% of the mean . although the benzamide moiety ( region c , figure 1 ) extends away from hydrophobic pocket ii and is 
 exposed to 
 solvents , results in table 3 indicated that 
 structures in region c can still significantly affect both jnk3 inhibitory 
 activity and isoform selectivity . considering that pyridine / pyrimidine 
 amide compounds might also have cytotoxicity and poor microsomal stabilities 
 ( see table 5 and table 6 ) , we expanded sar studies for this part to other amide structures . thus , amides based on five - membered heterocyclic ring systems were 
 investigated . as shown in table 4 , the n - methylpyrazole compounds 26a  d showed a similar potency and isoform selectivity compared 
 to lead compound sr-4326 . application of an n - methyl - triazole 
 ( 26e ) resulted in a 5-fold reduction in jnk3 inhibitory 
 activity . for example , an n - isopropyl 
 substitution on the pyrazole ring yielded a jnk3 inhibitor ( 26f ) having better isoform selectivity compared to the n - methyl analog ( 26a ) . remarkably , those 
 analogs with a substitution containing a dimethylamino , 
 diethylamino , piperidine , or pyrrolidine group ( 26g  k ) exhibited high jnk3 potency ( ic50 of 2134 
 nm ) and good to excellent isoform selectivity ( 22- to 60-fold over 
 jnk1 ) . one compound , 26k , which had a substituent containing 
 a pyrrolidine moiety , stood out and showed the highest jnk3 potency 
 ( ic50 < 1 nm ) and isoform selectivity ( > 500-fold 
 over 
 jnk1 and > 210-fold over jnk2 ; see table 5 ) . careful inspection 
 of the crystal structure of 26k indicated the potential 
 for a hydrogen bond between the 
 carbonyl of n89 in jnk3 with the pyrrolidinyl nh of 26k with an h - bond distance of 2.9  ( figure 4 ) . this potential for 
 tight interaction with optimal hydrogen bond distance and optimal 
 h - bond angle was likely the reason for the potent inhibition of 26k ( the energy minimized state of a computational model showed 
 the n - pyrrolidinyl of 26k could rotate 
 90 to make a h - bond with n89 ) . in contrast , 26j , which contained the six - member pipyridinyl ring and was not optimized 
 for h - bond distance and angle , had weaker inhibition to jnk3 than 26k . this observation is likely true for all of the other 
 analogs presented in table 4 . excellent jnk3 
 isoform selective inhibitors were also obtained from amides containing 
 no aromatic groups ( alkylamides , 26l  q ) . as shown in table 4 , all the inhibitors 
 prepared gave excellent isoform selectivity and fair to excellent 
 jnk3 inhibitory activity . the much better jnk3 potency and the higher 
 isoform selectivity ( jnk3 over jnk1 ) for these compounds containing 
 a side chain amino group could be due to extra h - bond interactions 
 from the amino group , and a combination of these interactions and 
 the hydrophobic interaction inside hydrophobic pocket i might be more 
 favorable for jnk3 than for jnk1 . these interactions are indicated 
 in the crystal structure of 26k with jnk3 ( figure 4 ) . ic50 values are the mean 
 of two or more experiments ( with triplicate replicates for each experiment ) 
 with errors within 80% of the mean . no inhibition at 10 m . to better understand the observed sar for these jnk3 
 isoform selective 
 inhibitors , the crystal structure for 26k with jnk3 was 
 solved at 1.8  ( figure 4 ) . this crystal 
 structure showed that 26k ( the r - enantiomer ) 
 interacted with jnk3 through h - bonding with the hinge residue m149 
 with a bond distance of 2.73.2  . the hydrogen bond between 
 the benzamide nh of 26k and the main - chain atoms of m149 
 in jnk3 would explain why substitution on the amide nh ( 22a ) resulted in no jnk3 affinity ( shown as yellow dotted lines ) . an 
 extra h - bond also occurred between the urea nh ( on the aniline side ) 
 and the side chain of residue k93 bridged by a water molecule . the 
 existence of this interaction explains why n - alkylation of the urea 
 nh led to loss of jnk3 inhibitory activity ( compounds 8j , 8k , and 8l , table 1 ) . as mentioned , the n - pyrrolidinylpyrazole group 
 made optimal h - bond interactions based on length and angle with residue 
 n89 , and the rest of the ring structure of 26k made tight 
 hydrophobic interactions with residues in atp binding pocket . the 
 center phenyl ring made hydrophobic interactions with side chain of 
 i70 and backbone of a151 . the central pyrazole ring also made hydrophobic 
 interactions with the side chains of a91 and v196 . the most 
 significant hydrophobic interactions occurred at the terminal o - cl - phenyl ring ( attached to the urea moiety ) and were 
 tightly sandwiched between the side chains of k93 and m146 . nearby 
 hydrophobic residues v78 , m115 , l126 , l144 , and l206 construct a hydrophobic 
 pocket for the o - cl - phenyl ring . these structural 
 observations are consistent with the observed sar results indicating 
 that there was almost no jnk3 affinity if the urea moiety was replaced 
 or the urea nh was alkylated . this structure also explains why there 
 was a reduction of jnk3 inhibitory activity when the phenyl ring was 
 substituted with a cl on its meta- or para - position ( 8d and 8e ) , since there was not enough space inside this 
 hydrophobic pocket . the isoform selectivity of jnk3 over jnk1 for 
 aminopyrazole - based jnk3 inhibitors was also mainly due to interactions 
 in this pocket , which has been documented in our previous publications . x - ray crystal structure of 26k in jnk3 . to evaluate the selectivity of 
 these aminopyrazole based jnk3 inhibitors , several selected compounds were subjected to additional counter screening 
 against jnk2 and p38 . as shown in table 5 , almost all aminopyrazole compounds had no inhibition on the most closely 
 related kinase p38 at a concentration of 10 m with the exception 
 of the n - alkyl substituted pyrazole amide 26f ( 3623 nm for p38 ) . moreover , these compounds had inhibitory activity 
 for jnk2 with potency similar to that for jnk3 with only a few exceptions 
 ( 22f , 22 g , 22i , 26k , and 26l ) . to further evaluate the general kinase selectivity 
 of these aminopyrazole based jnk3 inhibitors , compound 26n was subjected to a profiling study at a concentration of 10 m 
 in the full panel kinomescan ( ambit , san diego , ca ) , a high throughput method for screening kinase inhibitors against 
 a panel of 464 kinases . the treespot maps in figure 5 revealed that 26n possessed an extremely high 
 selectivity with significant inhibitions ( > 80% at 10 m ) 
 observed 
 only for jnk1/2/3 , clk2 , haspin , mek3 , and ysk4 ( see supporting information for detailed profiling data ) . inhibition of four selected cytochrome p450 isoforms ( 1a2 , 2c9 , 
 2d6 , and 3a4 ) was also tested at 10 m , and results demonstrated 
 that most of these aminopyrazole - based jnk3 inhibitors had generally 
 low inhibition against all four enzymes . generally , the pyrazole - amide 
 compounds 26f , 26j , 26 g , 26k presented lower inhibition for p450 isoforms compared 
 to those pyridineamide based compounds ( 22e , 22f , 22i , 16a ) . compounds 26l and 26n exhibited almost no inhibition for all four 
 selected isoforms even when tested at 10 m . it is noteworthy 
 that the f - substituted compound 16a exhibited potent 
 inhibitions for isoforms cyp2c9 and cyp2d6 . ic50 values are the mean 
 of two or more experiments ( with triplicate replicates for each experiment ) 
 with errors within 80% of the mean . no inhibition up to 10 m . with fluoro - substitution on the 
 middle phenyl ring at region b. the microsomal stability of these compounds in both human and mouse 
 liver microsomes were evaluated . briefly , the microsomal stabilities 
 of most inhibitors based on the pyridine amide ( 16a , 22f , 22 g , 22i ) or the n - alkyl substituted pyrazoleamide ( 26f ) were low with 
 the exception of the f - substituted compound 16a ( half - life 
 was 50 min in human and 86 min in mouse ) . the presence of a secondary 
 amine pyrrolidine ring or piperidine ring at the amide part was clearly 
 a favorable factor for metabolic stability . for example , inhibitors 26 g and 26l , which had no secondary amine substitutions , 
 exhibited a stability of t1/2 = 3540 
 min in human microsomes and only 46 min in mouse microsomes , 
 while the corresponding inhibitors containing a secondary amine moiety 
 ( 26j , 26k , 26n ) had a much 
 greater microsomal stability ( t1/2 = 113408 
 min in human and t1/2 = 3372 min 
 in mouse ) . the aqueous solubility was also tested for these compounds 
 at both ph 3.5 and ph 7.4 . as shown in table 5 , while the solubility for compounds containing the pyridineamide 
 ( 22f , 22 g , 22i ) and n - alkyl substituted pyrazoleamide 26f were 
 low , inhibitors with substituents containing a secondary or tertiary 
 amine exhibited excellent solubility at both ph 3.5 and ph 7.4 . indeed , 26n had excellent solubility at both ph 3.5 and ph 7.4 ( table 5 ) . treespot interaction maps for 26n ( also coded 
 as sr-11935 ) 
 in the panel profiling of 464 kinases . the cytotoxicity in shsy5y cells was evaluated for some selected 
 lead inhibitors . as shown in table 6 , pyridineamide based inhibitors 22f and 22 g showed significant cytotoxicity at both 10 
 and 30 m , while the benzylamide based inhibitor 22i and the simple alkyl substituted pyrazoleamide 26f exhibited 
 significant toxicity only at 30 m . remarkably , other pyrazoleamide 
 and alkylamide based compounds were all nontoxic even at a high concentration 
 of 30 m in shsy5y cells , indicating that they were relatively 
 safe jnk3 inhibitors . this was especially true for 26n which had > 90% cell viability at both 10 and 30 m . some compounds were also evaluated in vitro for their ability to 
 inhibit the phosphorylation of c - jun in neuronal 
 shsy5y cells . in - cell western blot analysis is a direct measure of 
 jnk activity and was employed to investigate the ability of these 
 jnk3 isoform selective inhibitors to inhibit c - jun phosphorylation . as shown in table 6 , most of the compounds had an ic50 of around 1 m 
 or higher in this assay , indicating that they were reasonable jnk 
 inhibitors . it is noteworthy that there was a large right shift between 
 jnk3 biochemical potency and cell - based potency for these isoform 
 selective jnk3 inhibitors ( table 6 ) . the lack 
 of jnk1 inhibition and the cell types might be part of the reasons . indeed , because c - jun phosphorylation is dependent on all three 
 isoforms and shsy5y cells contain all three isoforms , the uninhibited 
 jnk1 activity in these cells was the likely reason for the high cell - based 
 ic50 values . in contrast , these same compounds showed good 
 cell potency in other types of cell assays ( table 6 ) which were not as dependent on jnk1 nuclear activity for 
 substrate phosphorylation but rather more centered on the role of 
 jnk3 in mitochondrial dysfunction . ic50 values are the mean 
 of two or more experiments ( with triplicate replicates for each experiment ) 
 with errors within 80% of the mean . these compounds showed protection from 6-ohda - induced 
 mitochondrial 
 membrane depolarization and cell death in shsy5y cells , both of which 
 are regulated and promoted by jnk activation . improved cell potency was obtained in these two assays as compared 
 to that of the in - cell western assay . interestingly , very good cell 
 potency was seen in the mitochondrial membrane potential assays ( ic50 < 50 nm ) for most compounds ( table 6 ) . the lower ic50 value in mitochondrial membrane potential 
 assays compared to that in in - cell western blot assays for these compounds 
 ( table 6 ) could be due to the fact that jnk2/3 
 has a greater contribution to mitochondrial dysfunction than jnk1 . this interpretation is consistent with the in vivo observation of 
 zhao and herdegen where minimal to no jnk1 activity is seen on the 
 mitochondria in neurodegenerative models . thus , a small dose of these jnk2/3 isoform selective inhibitors 
 is sufficient to inhibit jnk2/3 activity and to rescue mitochondrial 
 membrane potential . the high cell potency for isoform selective 
 jnk2/3 inhibitors was 
 further confirmed in our mitochondrial ros inhibition assays in shsy5y 
 cells . as shown in figure 6 , compound 26n significantly reduced the ros generated by 6-ohda ( 35 
 m ) at a concentration as low as 40 nm ( ic50 < 
 40 nm ) . similar cell potency in this specific assay was also observed 
 for compound 26j ( ic50 < 40 nm ) , showing 
 this phenomenon is shared among the compounds in this class . indeed , 
 in contrast to biochemical assays , ic50 determinations 
 of functional end points in cell - based assays often have steep slopes 
 indicative of the functional readout that is being measured as opposed 
 to the direct substrate competitive inhibition seen in a biochemical 
 assay , and this could be why the cell - based ic50 is at 
 the biochemical ic50 for 26k and lower than 
 that for 26n . we have seen similar behavior in our aminopyrimidine 
 class where the inhibition of ros generation 
 ( 1 nm ) was lower than that of the biochemical ic50 value ( 50 nm ) . thus , it may be that only a small amount of jnk3 
 and jnk2 inhibition goes a long way toward shutting down ros generation 
 and jnk mitochondrial function . study ( 9l , sr-3562 ) 
 had exquisite selectivity over a panel of kinases . thus , while we 
 can not rule out off - target kinase activity as a possible explanation 
 for the lower ic50 in the cell - based assays , it is unlikely 
 given the high selectivity of these compounds . finally , we can not 
 rule out alternative interpretations such as other signaling pathways 
 that do not have a kinase component contributing to ros or mmp changes 
 in the mitochondria , so this remains a possibility for the greater 
 potency in ros generation and mmp dissipation for these compounds . shsy5y cells were treated with 35 m 6-ohda 
 for 5 h , and mitochondrial ros was measured by normalized mitosox 
 fluorescence in the presence or absence of 40 nm , 370 nm , and 3 m 
 inhibitor . statistical significance ( p < 0.05 ) 
 between control , untreated group , and 6-ohda treated group is shown 
 by . significance ( p < 0.05 ) between 6-ohda - treated 
 groups and different concentrations of the inhibitor and 6-ohda - treated 
 groups is shown by . in order to evaluate the feasibility of using these jnk3 
 isoform 
 selective inhibitors for in vivo applications , in vivo pharmacokinetic 
 ( pk ) properties for selected compounds were studied in mice . as demonstrated 
 in table 7 , these select lead compounds showed 
 good pk properties in iv dosing . they had good cmax and auc ( area under curve ) values and fair half - lives ( 11.5 
 h ) . the clearance ( cl ) and the volume of distribution ( vd ) properties were also good for 22i , 26j , and 26k but were only fair for compound 26n . on the other hand , these compounds showed poor pk properties 
 in oral dosing . for example , all of them exhibited almost no oral 
 bioavailability ( f values are all 1% ) . therefore , 
 the focus of future optimizations will be to improve oral pk properties 
 if oral administration is preferred . however , these compounds , as 
 jnk2/3 inhibitors for topical applications and/or in iv dosing , may 
 be very useful to protect against retinal degeneration in glaucoma and could conceivably be used in this indication . finally , since jnk3 selective inhibitors have the potential to be 
 utilized in cns disorders , we measured the brain penetration properties 
 of 26n . after ip dosing ( 10 mg / kg in mice ) brain levels 
 at 2 h after dosing was approximately 55% of that found in the plasma . in addition , we also measured the brain concentration of 26n at 2 and 6 h after po and iv dosing . because of the poor oral bioavailability 
 of 26n ( see table 7 ) the brain 
 levels at 2 and 6 h were below the lower limit of quantitation ( 5 
 nm ) after oral dosing but were found to be 60 and 40 nm at 2 and 6 
 h , respectively , after iv dosing of 2 mg / kg ( table 8) . dosed at 
 0.5 mg / kg for iv and at 
 3 mg / kg for po . we have developed a series of aminopyrazoleamide based jnk3 isoform 
 selective inhibitors starting from the initial lead compound sr-4326 . sar investigation and optimization successfully yielded potent jnk3 
 inhibitors with high selectivity over p38 kinase , as well as high 
 isoform selectivity against jnk1 and in some cases over jnk2 . for 
 example , compound 26n had an isoform selectivity of > 50-fold 
 for jnk3 against jnk1 and were potent in all three types of cell - based 
 assays in shsy5y cells ( in - cell western target modulation , inhibition 
 of 6-ohda induced cell death , and inhibition of 6-ohda induced mitochondrial 
 membrane depolarization ) . another lead inhibitor 26k was 
 highly potent and selective with an ic50 value against 
 jnk3 in the subnanomolar range and possessed an isoform selectivity 
 of > 500-fold over jnk1 and > 20000-fold against p38. in 
 addition , 26n and 26k were noncytotoxic , 
 potently inhibited 
 mitochondrial dysfunction , and possessed good pk properties in iv 
 dosing . in addition , 26n showed a plasma / brain ratio 
 of 2:1 . the x - ray crystal structure of 26k in 
 jnk3 revealed a binding mode that can very well support the observed 
 sar , and this structure was also helpful for designing potent and 
 selective jnk3 inhibitors . future optimizations for these aminopyrazole - based 
 compounds will be mainly focused on improving the pharmacokinetic 
 properties in order to obtain better jnk3 isoform selective inhibitors 
 for oral dosing . these studies will be reported in due course . commercially 
 available reagents and anhydrous solvents were used 
 without further purification unless otherwise specified . thin layer 
 chromatography ( tlc ) analyses were performed with precoated silica 
 gel 60 f254 . the mass spectra were recorded by lc / ms with finnigan 
 lcq advantage max spectrometer of thermo electron . flash chromatography 
 was performed on prepacked columns of silica gel ( 230400 mesh , 
 4063 m ) by combiflash with etoac / hexane or meoh / dcm 
 as eluents . the preparative hplc was performed on sunfire c18 obd 10 m ( 30 mm  250 mm ) with ch3cn + 50% 
 meoh / h2o + 0.1% tfa as eluents to purify the targeted compounds . analytic hplc was performed on agilent technologies 1200 series with 
 ch3cn ( solvent b)/h2o + 0.9% ch3cn 
 + 0.1% tfa ( solvent a ) as eluents , and the targeted products were 
 detected by uv in the detection range of 215310 nm . nmr spectra were 
 recorded 
 with a bruker 400 mhz spectrometer at ambient temperature with the 
 residual solvent peaks as internal standards . the line positions of 
 multiplets were given in ppm (  ) , and the coupling constants 
 ( j ) were given in hertz . the high - resolution mass 
 spectra ( hrms , electrospray ionization ) experiments were performed 
 with thermo finnigan orbitrap mass analyzer . data were acquired in 
 the positive ion mode at resolving power of 100  000 at m / z 400 . calibration was performed with 
 an external calibration mixture immediately prior to analysis . a mixture of 4-nitro-1h - pyrazole 1 ( 10 mmol ) , 3-bromobenzoic acid 2 ( 20 mmol ) , 
 cui ( 2.0 mmol ) , trans - n , n - dimethylcyclohexane-1,2-diamine ( 4.0 
 mmol ) , and cs2co3 ( 30 mmol ) in dmf ( 20 ml ) was 
 purged with argon and stirred for 12 h at 100 c in a sealed 
 tube . the reaction mixture was cooled , and filtered through a pad 
 of silica gel , and rinsed with etoac . the resulting solution was concentrated 
 in vacuo to yield a crude residue which was purified by chromatography 
 on silica gel ( etoac / hexane ) to provide 3-(4-nitro-1h - pyrazol-1-yl)benzoic acid 3 . to a solution of 
 3-(4-nitro-1h - pyrazol-1-yl)benzoic acid 3 ( 5.0 mmol ) in ch2cl2 ( 20 ml ) were added edc 
 ( 10 mmol ) , hobt ( 10 mmol ) , and diisopropylethylamine ( 15 mmol ) , and 
 the mixture was stirred for 30 min . then the 6-methylpyridin-3-amine 
 ( 5.5 mmol ) was added , and the resulting mixture was stirred overnight . water ( 50 ml ) was added to the reaction mixture and extracted with 
 etoac ( 2  100 ml ) . the resulting solution was concentrated in 
 vacuo to yield a crude product ( 4 ) . this intermediate 
 was hydrogenated in anhydrous methanol ( 100 ml ) 
 in the presence of 10% pd / c ( 1.0 g ) under a balloon of hydrogen for 
 3 h. the mixture was filtered through a celite pad and evaporated . the residue was purified by chromatography on silica gel ( dichloromethane / methanol ) 
 to give the 3-(4-amino-1h - pyrazol-1-yl)-n-(6-methylpyridin-3-yl)benzamide 5 . 1-chloro-2-isocyanatobenzene 
 ( 0.12 mmol ) was added to 3-(4-amino-1h - pyrazol-1-yl)-n-(6-methylpyridin-3-yl)benzamide 
 ( 0.1 mmol ) in ch2cl2 ( 1.0 ml ) at room temperature 
 and stirred for 1 h. the solvent was evaporated and the residue was 
 purified by reverse - phase preparative hplc to give 3-(4-(3-(2-chlorophenyl)ureido)-1h - pyrazol-1-yl)-n-(6-methylpyridin-3-yl)benzamide 8c as a white powder . 66% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.93 ( s , 1h ) , 10.51 ( s , 1h ) , 9.08 ( d , j = 2.0 hz , 1h ) , 8.70 ( s , 1h ) , 8.42 ( dd , j = 8.4 , 2.0 hz , 1h ) , 8.38 ( s , 1h ) , 8.108.03 ( m , 1h ) , 7.89 
 ( d , j = 8.0 hz , 1h ) , 7.82 ( s , 1h ) , 7.68 ( t , j = 8.0 hz , 2h ) , 7.427.47 ( m , 2h ) , 7.377.28 
 ( m , 2h ) , 3.85 ( s , 2h ) , 2.61 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h20cln5o2 , 446.1 ; found , 446.4 58% 
 yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 
 mhz , dmso )  10.6010.54 ( m , 2h ) , 8.82 ( d , j = 2.4 hz , 1h ) , 8.52 ( s , 1h ) , 8.388.33 ( m , 1h ) , 8.11 ( dd , j = 8.4 , 2.6 hz , 1h ) , 8.077.98 ( m , 1h ) , 7.907.83 
 ( m , 1h ) , 7.79 ( s , 1h ) , 7.65 ( t , j = 80 hz , 1h ) , 7.60 
 ( d , j = 7.6 hz , 1h ) , 7.43 ( m , 2h ) , 7.387.28 
 ( m , 2h ) , 2.47 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h18cln5o3 , 448.1 ; found , 448.4 62% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso ) 11.00 ( s , 1h ) , 10.76 
 ( s , 1h ) , 10.18 ( s , 1h ) , 9.12 ( s , 1h ) , 8.88 ( s , 1h ) , 8.538.37 
 ( m , 2h ) , 8.23 ( t , j = 4.9 hz , 2h ) , 8.12 ( dd , j = 8.0 , 1.2 hz , 1h ) , 7.90 ( d , j = 7.8 
 hz , 2h ) , 7.71 ( dd , j = 16.4 , 8.4 hz , 2h ) , 7.537.40 
 ( m , 1h ) , 7.237.12 ( m , 2h ) , 2.63 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c25h20n6o2 , 437.2 ; found , 437.4 . 35% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  11.10 ( s , 1h ) , 10.89 
 ( s , 1h ) , 9.80 ( s , 1h ) , 9.69 ( s , 1h ) , 9.04 ( s , 1h ) , 8.83 ( s , 1h ) , 8.48 
 ( s , 1h ) , 8.37 ( s , 1h ) , 8.17 ( dd , j = 8.4 , 1.6 hz , 
 1h ) , 8.00 ( d , j = 9.8 hz , 2h ) , 7.847.71 ( m , 
 1h ) , 7.59 ( s , 1h ) , 7.37 ( m , 3h ) , 4.49 ( s , 2h ) , 3.563.52 ( m , 
 1h ) , 3.49 ( dd , j = 9.2 , 4.4 hz , 1h ) , 3.20 ( dd , j = 16.8 , 12.2 hz , 1h ) , 2.62 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c26h24n6o2 , 453.2 ; found , 453.3 . 48% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  12.98 ( s , 1h ) , 10.90 
 ( s , 1h ) , 10.80 ( s , 1h ) , 10.58 ( s , 1h ) , 9.01 ( s , 1h ) , 8.96 ( s , 1h ) , 
 8.50 ( s , 1h ) , 8.28 ( s , 1h ) , 8.13 ( d , j = 8.0 hz , 
 1h ) , 8.04 ( s , 1h ) , 8.00 ( d , j = 8.0 hz , 1h ) , 7.76 
 ( t , j = 8.0 hz , 1h ) , 7.36 ( ddd , j = 56.6 , 6.0 , 3.2 hz , 4h ) , 2.53 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h18brn7o , 517.4 ; found , 517.6 . 45% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.78 ( s , 1h ) , 8.96 ( s , 1h ) , 8.41 ( s , 1h ) , 
 8.28 ( s , 2h ) , 8.17 ( s , 1h ) , 7.95 ( dd , j = 8.1 , 1.3 
 hz , 1h ) , 7.79 ( d , j = 8.1 hz , 1h ) , 7.66 ( s , 1h ) , 
 7.637.49 ( m , 2h ) , 6.22 ( s , 1h ) , 3.87 ( dd , j = 13.4 , 6.7 hz , 1h ) , 2.51 ( s , 3h ) , 1.76 ( dt , j = 
 11.6 , 5.8 hz , 2h ) , 1.611.51 ( m , 2h ) , 1.431.48 ( m , 
 2h ) , 1.361.24 ( m , 2h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c22h24n6o2 , 405.2 ; found , 405.3 . 58% 
 yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 
 mhz , dmso )  10.82 ( s , 1h ) , 8.99 ( s , 1h ) , 8.81 ( s , 1h ) , 8.69 
 ( s , 1h ) , 8.52 ( s , 1h ) , 8.32 ( s , 2h ) , 8.077.96 ( m , 1h ) , 7.81 
 ( d , j = 8.0 hz , 1h ) , 7.78 ( d , j = 
 14.4 hz , 1h ) , 7.61 ( t , j = 8.0 hz , 2h ) , 7.41 ( d , j = 7.6 hz , 2h ) , 7.267.17 ( m , 2h ) , 6.90 ( t , j = 7.2 hz , 1h ) , 2.52 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h20n6o2 , 413.2 ; found , 413.3 . 63% yield in four steps ( tfa salt , white solid ) ; h nmr 
 ( dmso - d6 , 400 mhz )  10.88 ( s , 1h ) , 
 9.45 ( s , 1h ) , 9.04 ( s , 1h ) , 8.62 ( s , 1h ) , 8.40 ( m , 3h ) , 8.20 ( dd , j = 8.2 , 1.6 hz , 1h ) , 8.09 ( d , j = 8.2 
 hz , 1h ) , 7.947.81 ( m , 2h ) , 7.69 ( t , j = 8.0 
 hz , 1h ) , 7.63 ( s , 1h ) , 7.47 ( dd , j = 8.0 , 1.4 hz , 
 1h ) , 7.387.25 ( m , 1h ) , 7.106.96 ( m , 1h ) , 2.59 ( s , 
 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19fn6o2 , 447.1 ; found , 447.4 . 64% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.89 ( s , 1h ) , 9.21 ( s , 1h ) , 9.05 ( s , 1h ) , 
 8.99 ( s , 1h ) , 8.61 ( s , 1h ) , 8.39 ( d , j = 8.6 hz , 
 2h ) , 8.08 ( dd , j = 8.1 , 1.3 hz , 1h ) , 7.88 ( d , j = 8.2 hz , 1h ) , 7.84 ( s , 1h ) , 7.78 ( s , 1h ) , 7.68 ( t , j = 8.0 hz , 1h ) , 7.64 ( d , j = 7.9 hz , 1h ) , 
 7.347.27 ( m , 2h ) , 7.066.92 ( m , 1h ) , 2.58 ( d , j = 10.6 hz , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19fn6o2 , 447.1 ; found , 447.4 . h nmr ( 400 mhz , 
 dmso )  10.87 ( s , 1h ) , 9.04 ( s , 2h ) , 8.82 ( s , 1h ) , 8.59 ( s , 
 1h ) , 8.38 ( s , 2h ) , 8.07 ( d , j = 7.8 hz , 1h ) , 7.88 
 ( d , j = 7.8 hz , 1h ) , 7.83 ( s , 1h ) , 7.67 ( t , j = 8.0 hz , 2h ) , 7.52 ( d , j = 8.8 hz , 2h ) , 
 7.377.22 ( m , 2h ) , 2.58 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19fn6o2 , 447.1 ; found , 447.3 . 56% yield in four steps ( tfa salt , white solid ) ; h nmr 
 ( 400 mhz , dmso )  10.92 ( s , 1h ) , 9.21 ( s , 1h ) , 9.07 ( s , 1h ) , 
 8.93 ( s , 1h ) , 8.61 ( s , 1h ) , 8.40 ( s , 2h ) , 8.08 ( dd , j = 8.1 , 1.4 hz , 1h ) , 7.89 ( d , j = 8.1 hz , 1h ) , 7.84 
 ( s , 1h ) , 7.67 ( dd , j = 15.0 , 7.0 hz , 2h ) , 7.54 ( dt , j = 12.0 , 2.4 hz , 1h ) , 7.387.22 ( m , 1h ) , 7.15 ( dd , j = 8.2 , 1.1 hz , 1h ) , 6.78 ( td , j = 8.4 , 
 2.4 hz , 1h ) , 2.58 ( d , j = 13.2 hz , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19fn6o2 , 431.2 ; found , 
 431.3 . 55% yield in four steps ( tfa salt , white solid ) ; h nmr 
 ( 400 mhz , dmso )  10.98 ( s , 1h ) , 9.14 ( d , j = 7.2 hz , 2h ) , 8.74 ( d , j = 2.3 hz , 1h ) , 8.68 ( s , 
 1h ) , 8.46 ( dd , j = 5.2 , 3.6 hz , 2h ) , 8.22 ( td , j = 8.3 , 1.6 hz , 1h ) , 8.198.11 ( m , 1h ) , 7.997.90 
 ( m , 2h ) , 7.73 ( dd , j = 16.4 , 8.8 hz , 2h ) , 7.307.34 
 ( m , 1h ) , 7.277.14 ( m , 1h ) , 7.147.01 ( m , 1h ) , 2.66 
 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19fn6o2 , 431.2 ; found , 431.4 . 58% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.94 ( s , 1h ) , 9.08 ( s , 1h ) , 9.03 ( s , 1h ) , 
 8.87 ( s , 1h ) , 8.59 ( s , 1h ) , 8.468.36 ( m , 2h ) , 8.07 ( dd , j = 8.1 , 1.3 hz , 1h ) , 7.89 ( d , j = 8.2 
 hz , 1h ) , 7.83 ( s , 1h ) , 7.68 ( t , j = 8.0 hz , 2h ) , 
 7.567.45 ( m , 2h ) , 7.197.08 ( m , 2h ) , 2.61 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19fn6o2 , 431.2 ; found , 431.3 . 32% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.97 ( s , 1h ) , 9.45 ( s , 1h ) , 9.12 ( d , j = 2.0 hz , 1h ) , 8.74 ( s , 1h ) , 8.59 ( s , 1h ) , 8.46 ( dd , j = 8.6 , 2.0 hz , 1h ) , 8.42 ( s , 1h ) , 8.148.07 ( m , 
 1h ) , 7.93 ( d , j = 8.0 hz , 1h ) , 7.87 ( s , 1h ) , 7.72 
 ( t , j = 8.0 hz , 2h ) , 7.50 ( ddd , j = 8.0 , 4.8 , 2.0 hz , 2h ) , 7.427.32 ( m , 2h ) , 3.89 ( s , 2h ) , 
 2.65 ( s , 3h ) . lc / ms ( esi ) m / z : [ m 
 + h ] calcd for c24h21n6o2 , 461.1 ; found , 461.4 . 21% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  10.96 ( s , 1h ) , 9.10 
 ( s , 1h ) , 8.51 ( s , 1h ) , 8.488.37 ( m , 2h ) , 8.34 ( t , j = 1.8 hz , 1h ) , 8.057.97 ( m , 1h ) , 7.86 ( dd , j = 6.8 , 1.5 hz , 1h ) , 7.757.70 ( m , 2h ) , 7.63 ( m , 
 2h ) , 7.557.34 ( m , 3h ) , 3.18 ( s , 3h ) , 2.62 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h21n6o2 , 461.1 ; found , 461.2 . 23% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  10.59 ( s , 1h ) , 8.84 
 ( s , 1h ) , 8.52 ( s , 1h ) , 8.31 ( s , 1h ) , 8.308.23 ( m , 1h ) , 8.168.08 
 ( m , 1h ) , 8.027.94 ( m , 1h ) , 7.84 ( s , 1h ) , 7.73 ( s , 1h ) , 7.63 
 ( d , j = 4.0 hz , 2h ) , 7.54 ( s , 1h ) , 7.46 ( s , 2h ) , 
 7.377.29 ( m , 1h ) , 3.513.47 ( m , 2h ) , 3.403.44 
 ( m , 2h ) , 3.17 ( s , 1h ) , 2.48 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c25h23n6o3 , 491.2 ; found , 491.4 . 28% yield in four steps ( tfa salt , white solid ) ; h 
 nmr ( 400 mhz , dmso )  11.46 ( s , 1h ) , 8.98 ( s , 1h ) , 8.66 ( s , 
 1h ) , 8.40 ( s , 1h ) , 8.12 ( dd , j = 8.0 , 1.6 hz , 1h ) , 
 8.007.91 ( m , 3h ) , 7.917.82 ( m , 2h ) , 7.71 ( t , j = 8.0 hz , 1h ) , 7.21 ( d , j = 6.8 hz , 1h ) , 
 7.13 ( t , j = 7.6 hz , 1h ) , 6.91 ( dd , j = 11.6 , 4.2 hz , 1h ) , 6.57 ( s , 1h ) , 4.10 ( t , j = 
 8.8 hz , 2h ) , 3.21 ( t , j = 8.6 hz , 2h ) , 2.66 ( s , 3h ) . 
 lc / ms ( esi ) m / z : [ m + h ] calcd for c25h22n6o2 , 439.1 ; found , 439.4 . 71% 
 yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 
 mhz , dmso )  10.93 ( s , 1h ) , 9.12 ( s , 1h ) , 9.08 ( s , 1h ) , 8.83 
 ( t , j = 5.8 hz , 1h ) , 8.48 ( t , j = 
 1.8 hz , 1h ) , 8.42 ( d , j = 9.2 hz , 2h ) , 8.30 ( s , 1h ) , 
 8.15 ( dd , j = 8.0 , 1.6 hz , 1h ) , 7.98 ( d , j = 6.8 hz , 1h ) , 7.75 ( t , j = 8.0 hz , 1h ) , 
 7.70 ( d , j = 6.2 hz , 1h ) , 7.48 ( dd , j = 7.6 , 1.6 hz , 1h ) , 7.42 ( dd , j = 7.4 , 2.0 hz , 
 1h ) , 7.34 ( m , 2h ) , 4.55 ( d , j = 5.7 hz , 2h ) , 2.61 
 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h20cln5o2 , 446.1 ; found , 446.3 . 45% 
 yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 
 mhz , dmso )  10.88 ( s , 1h ) , 9.02 ( s , 1h ) , 8.54 ( s , 1h ) , 8.40 
 ( t , j = 1.8 hz , 1h ) , 8.34 ( d , j = 
 8.4 hz , 1h ) , 8.02 ( dd , j = 8.1 , 1.4 hz , 1h ) , 7.94 
 ( d , j = 8.0 hz , 1h ) , 7.867.77 ( m , 1h ) , 7.69 
 ( t , j = 8.0 hz , 1h ) , 7.64 ( dd , j = 7.6 , 1.6 hz , 1h ) , 7.507.56 ( m , 3h ) , 4.61 ( s , 2h ) , 4.404.45 
 ( m , 2h ) , 3.103.14 ( m , 2h ) , 2.56 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c26h22cln5o2 , 472.2 ; found , 
 472.5 . 47% yield in five steps ( tfa salt , white solid ) ; h nmr 
 ( 400 mhz , dmso )  10.92 ( s , 1h ) , 9.46 ( s , 1h ) , 9.05 ( s , 1h ) , 
 8.50 ( dd , j = 7.6 , 2.4 hz , 1h ) , 8.468.31 
 ( m , 3h ) , 8.18 ( dd , j = 8.4 , 1.4 hz , 1h ) , 8.02 ( ddd , j = 8.6 , 4.4 , 2.4 hz , 1h ) , 7.91 ( s , 1h ) , 7.747.69 
 ( m , 1h ) , 7.697.60 ( m , 1h ) , 7.47 ( dd , j = 
 8.0 , 1.4 hz , 1h ) , 7.357.26 ( m , 1h ) , 7.116.93 ( m , 1h ) , 
 2.60 ( s , 3h ) . lc / ms ( esi ) m / z : [ m 
 + h ] calcd for c23h18clfn6o2 , 465.1 ; found , 465.3 . 53% yield in five steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.58 ( s , 1h ) , 9.45 ( s , 1h ) , 8.80 ( d , j = 2.4 hz , 1h ) , 8.65 ( s , 1h ) , 8.39 ( s , 1h ) , 8.27 ( s , 1h ) , 
 8.21 ( dd , j = 8.4 , 1.2 hz , 1h ) , 8.07 ( dd , j = 8.4 , 2.4 hz , 1h ) , 7.94 ( dt , j = 10.0 , 
 2.0 hz , 1h ) , 7.90 ( s , 1h ) , 7.66 ( d , j = 9.0 hz , 1h ) , 
 7.45 ( dd , j = 8.0 , 1.2 hz , 1h ) , 7.367.20 
 ( m , 2h ) , 7.007.04 ( m , 1h ) , 2.45 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h18clfn6o2 , 465.1 ; found , 
 465.2 . 22% yield in five steps ( tfa salt , white solid ) ; h nmr 
 ( 400 mhz , dmso )  10.91 ( s , 1h ) , 9.44 ( s , 1h ) , 8.54 ( d , j = 6.4 hz , 1h ) , 8.48 ( s , 1h ) , 8.39 ( s , 1h ) , 8.248.14 
 ( m , 2h ) , 8.148.07 ( m , 1h ) , 8.04 ( s , 1h ) , 7.94 ( d , j = 6.4 hz , 1h ) , 7.85 ( s , 1h ) , 7.57 ( t , j = 9.2 hz , 1h ) , 7.47 ( dd , j = 8.0 , 1.6 hz , 1h ) , 
 7.367.23 ( m , 1h ) , 7.096.97 ( m , 1h ) , 2.67 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h18clfn6o2 , 465.1 ; found , 465.4 . 15% yield in five steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.79 ( s , 1h ) , 9.53 ( s , 1h ) , 8.93 ( s , 1h ) , 
 8.74 ( s , 1h ) , 8.48 ( s , 1h ) , 8.37 ( s , 1h ) , 8.27 ( s , 2h ) , 8.05 ( s , 1h ) , 
 7.947.99 ( m , 1h ) , 7.56 ( d , j = 8.0 hz , 1h ) , 
 7.39 ( d , j = 8.0 , 1h ) , 7.29 ( s , 1h ) , 7.147.18 
 ( m , 1h ) , 7.107.13 ( m , 1h ) , 7.007.06 ( m , 1h ) , 2.60 
 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h18clfn6o2 , 465.1 ; found , 465.4 . 38% yield 
 in four 
 steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , dmso )  
 9.43 ( s , 1h ) , 8.84 ( d , j = 4.8 hz , 2h ) , 8.69 ( s , 
 1h ) , 8.39 ( s , 1h ) , 8.28 ( s , 1h ) , 8.248.09 ( m , 3h ) , 8.02 ( d , j = 8.1 hz , 1h ) , 7.877.76 ( m , 2h ) , 7.68 ( t , j = 8.0 hz , 1h ) , 7.46 ( dd , j = 8.0 , 1.2 
 hz , 1h ) , 7.367.24 ( m , 1h ) , 7.03 ( td , j = 
 8.0 , 1.6 hz , 1h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c21h16cln5o , 390.1 ; found , 390.2 . 25% yield 
 in four steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , 
 dmso )  10.72 ( s , 1h ) , 9.41 ( s , 1h ) , 8.87 ( s , 1h ) , 8.44 ( s , 
 1h ) , 8.37 ( s , 2h ) , 8.18 ( d , j = 6.8 hz , 2h ) , 7.81 
 ( s , 1h ) , 7.58 ( d , j = 7.6 hz , 2h ) , 7.51 ( d , j = 8.0 hz , 1h ) , 7.46 ( d , j = 7.0 hz , 1h ) , 
 7.30 ( s , 1h ) , 7.03 ( s , 1h ) , 6.98 ( d , j = 6.6 hz , 
 1h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c21h16cln7o , 406.1 ; 
 found , 406.3 . 28% yield 
 in five steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , 
 dmso )  9.40 ( s , 1h ) , 8.52 ( s , 1h ) , 8.39 ( s , 2h ) , 8.20 ( d , j = 8.0 , 1h ) , 7.87 ( d , j = 8.0 , 1h ) , 7.83 
 ( m , 2h ) , 7.58 ( t , j = 7.8 hz , 1h ) , 7.47 ( d , j = 8.0 , 1h ) , 7.31 ( t , j = 7.2 hz , 1h ) , 
 7.16 ( s , 1h ) , 7.04 ( t , j = 7.6 hz , 1h ) , 2.42 ( s , 
 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c19h16cln7o , 394.1 ; 
 found , 394.3 . 37% yield 
 in five steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , 
 dmso )  9.50 ( s , 1h ) , 8.59 ( s , 1h ) , 8.42 ( s , 1h ) , 8.29 ( s , 1h ) , 
 8.20 ( d , j = 7.1 hz , 1h ) , 8.04 ( d , j = 8.0 hz , 1h ) , 7.83 ( s , 1h ) , 7.78 ( d , j = 7.4 hz , 
 1h ) , 7.63 ( t , j = 7.8 hz , 1h ) , 7.47 ( d , j = 8.0 hz , 1h ) , 7.31 ( t , j = 7.8 hz , 1h ) , 7.04 ( t , j = 7.8 hz , 1h ) , 2.45 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c19h15cln6o2 , 395.1 ; found , 395.2 . 48% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( dmso - d6 , 400 
 mhz )  9.88 ( s , 1h ) , 9.42 ( s , 1h ) , 8.59 ( s , 1h ) , 8.368.39 
 ( m , 1h ) , 8.32 ( s , 1h ) , 8.21 ( dd , j = 8.4 , 1.6 hz , 
 1h ) , 8.01 ( d , j = 9.6 hz , 1h ) , 7.90 ( s , 1h ) , 7.867.78 
 ( m , 2h ) , 7.62 ( t , j = 8.0 hz , 1h ) , 7.47 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.31 ( t , j = 7.2 
 hz , 1h ) , 7.03 ( dd , j = 10.8 , 4.4 hz , 1h ) , 3.77 ( s , 
 3h ) , 2.17 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h21cln6o2 , 461.2 ; found , 461.5 . 55% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  10.80 ( s , 1h ) , 9.63 ( s , 1h ) , 8.778.64 
 ( m , 2h ) , 8.52 ( dd , j = 4.8 , 3.0 hz , 2h ) , 8.44 ( d , j = 8.0 hz , 1h ) , 8.26 ( dd , j = 8.3 , 1.5 
 hz , 1h ) , 8.17 ( dd , j = 8.4 , 1.2 hz , 1h ) , 7.98 ( d , j = 8.0 hz , 1h ) , 7.93 ( s , 1h ) , 7.887.80 ( m , 1h ) , 
 7.75 ( t , j = 8.0 hz , 1h ) , 7.53 ( dd , j = 8.0 , 1.6 hz , 1h ) , 7.427.32 ( m , 1h ) , 7.147.06 ( m , 
 1h ) , 2.73 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19cln6o2 , 447.1 ; found , 447.4 . 48% yield in four steps ( tfa salt , white 
 solid ) ; h 
 nmr ( 400 mhz , dmso )  10.55 ( s , 1h ) , 9.45 ( s , 1h ) , 8.62 ( s , 
 1h ) , 8.41 ( d , j = 7.6 hz , 2h ) , 8.19 ( d , j = 8.3 hz , 2h ) , 8.09 ( d , j = 8.4 hz , 1h ) , 7.89 ( d , j = 7.6 hz , 1h ) , 7.85 ( s , 1h ) , 7.68 ( t , j = 8.0 hz , 1h ) , 7.57 ( s , 1h ) , 7.47 ( dd , j = 8.0 , 
 1.6 hz , 1h ) , 7.377.26 ( m , 1h ) , 7.087.00 ( m , 1h ) , 2.63 
 ( s , 3h ) , 2.57 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h21cln6o2 , 461.1 ; found , 461.4 . 32% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  11.10 ( s , 1h ) , 9.60 ( s , 1h ) , 8.72 ( s , 1h ) , 
 8.43 ( d , j = 14.2 hz , 2h ) , 8.248.18 ( m , 1h ) , 
 8.118.01 ( m , 2h ) , 7.90 ( d , j = 7.8 hz , 1h ) , 
 7.847.74 ( m , 2h ) , 7.62 ( t , j = 8.0 hz , 1h ) , 
 7.46 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.30 ( d , j = 7.2 hz , 1h ) , 7.147.05 ( m , 1h ) , 7.04 ( d , j = 7.8 hz , 1h ) , 2.53 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19cln6o2 , 447.1 ; found , 447.4 . 43% yield in five steps ( tfa salt , white solid ) ; h nmr 
 ( 400 mhz , dmso ) 10.74 ( s , 1h ) , 9.44 ( s , 
 1h ) , 9.08 ( s , 2h ) , 8.61 ( s , 1h ) , 8.40 ( d , j = 5.2 
 hz , 2h ) , 8.21 ( d , j = 8.2 hz , 1h ) , 8.08 ( d , j = 7.6 hz , 1h ) , 7.88 ( d , j = 12.4 hz , 
 2h ) , 7.68 ( t , j = 8.0 hz , 1h ) , 7.47 ( d , j = 7.8 hz , 1h ) , 7.31 ( t , j = 7.6 hz , 1h ) , 7.04 ( t , j = 7.2 hz , 1h ) , 2.62 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19cln6o2 , 447.1 ; found , 447.3 . 22% yield in four steps ( tfa salt , white solid ) ; h nmr 
 ( 400 mhz , dmso )  11.42 ( s , 1h ) , 9.45 ( s , 1h ) , 8.63 ( s , 2h ) , 
 8.40 ( s , 2h ) , 8.20 ( d , j = 6.8 hz , 1h ) , 8.12 ( s , 
 2h ) , 8.067.99 ( m , 1h ) , 7.87 ( s , 2h ) , 7.73 ( d , j = 8.0 hz , 1h ) , 7.48 ( d , j = 9.4 hz , 1h ) , 7.31 ( s , 
 1h ) , 7.06 ( d , j = 6.0 hz , 1h ) , 2.66 ( s , 3h ) . lc / ms 
 ( esi ) m / z : [ m + h ] calcd 
 for c23h19cln6o2 , 447.1 ; 
 found , 447.3 . 20% yield in four steps ( tfa salt , white 
 solid ) ; h 
 nmr ( 400 mhz , dmso )  11.10 ( s , 1h ) ,  9.58 ( s , 1h ) , 8.72 
 ( s , 1h ) , 8.478.37 ( m , 2h ) , 8.258.18 ( m , 1h ) , 8.128.02 
 ( m , 2h ) , 7.967.89 ( m , 1h ) , 7.847.74 ( m , 2h ) , 7.63 
 ( t , j = 7.6 hz , 1h ) , 7.517.42 ( m , 1h ) , 7.31 
 ( t , j = 7.8 hz , 1h ) , 7.10 ( d , j = 
 8.0 hz , 1h ) , 7.05 ( t , j = 7.6 hz , 1h ) , 2.532.52 
 ( m , 6h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h21cln6o2 , 461.1 ; found , 461.4 . 46% yield in four steps ( tfa salt , white 
 solid ) ; h 
 nmr ( 400 mhz , dmso )  10.74 ( s , 
 1h ) , 9.44 ( s , 1h ) , 9.08 ( s , 2h ) , 8.61 ( s , 1h ) , 8.40 ( d , j = 5.2 hz , 2h ) , 8.21 ( d , j = 8.2 hz , 1h ) , 8.08 ( d , j = 7.6 hz , 1h ) , 7.88 ( d , j = 12.4 hz , 
 2h ) , 7.68 ( t , j = 8.0 hz , 1h ) , 7.47 ( d , j = 7.8 hz , 1h ) , 7.31 ( t , j = 7.6 hz , 1h ) , 7.04 ( t , j = 7.2 hz , 1h ) , 2.62 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h19cln6o2 , 448.1 ; found , 448.4 . 66% yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , dmso )  9.46 ( s , 1h ) , 9.42 ( t , j = 5.6 hz , 1h ) , 8.76 ( s , 1h ) , 8.58 ( s , 1h ) , 8.41 ( s , 1h ) , 
 8.34 ( d , j = 8.2 hz , 1h ) , 8.30 ( s , 1h ) , 8.19 ( dd , j = 8.4 , 1.4 hz , 1h ) , 8.01 ( dd , j = 8.1 , 
 1.3 hz , 1h ) , 7.857.77 ( m , 3h ) , 7.61 ( t , j = 8.0 hz , 1h ) , 7.47 ( dd , j = 8.0 , 1.4 hz , 1h ) , 
 7.357.27 ( m , 1h ) , 7.04 ( td , j = 8.0 , 1.6 
 hz , 1h ) , 4.63 ( d , j = 5.6 hz , 2h ) , 2.67 ( s , 3h ) . 
 lc / ms ( esi ) m / z : [ m + h ] calcd for c24h21cln6o2 , 461.1 ; found , 461.2 . 72% yield in 
 four steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , dmso ) 
  10.61 ( s , 1h ) , 9.43 ( s , 1h ) , 
 8.59 ( s , 1h ) , 8.39 ( s , 1h ) , 8.34 ( s , 1h ) , 8.20 ( dd , j = 8.3 , 1.4 hz , 1h ) , 8.06 ( s , 1h ) , 8.067.98 ( m , 1h ) , 7.84 
 ( t , j = 3.6 hz , 2h ) , 7.697.58 ( m , 2h ) , 7.46 
 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.367.24 ( m , 1h ) , 
 7.04 ( td , j = 8.0 , 1.6 hz , 1h ) , 3.84 ( s , 3h ) . lc / ms 
 ( esi ) m / z : [ m + h ] calcd 
 for c21h18cln7o2 , 436.1 ; 
 found , 436.2 . 68% yield in 
 four steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , dmso ) 
  11.07 ( s , 1h ) , 9.42 ( s , 1h ) , 
 8.66 ( s , 1h ) , 8.38 ( s , 2h ) , 8.23 ( dd , j = 8.4 , 1.6 
 hz , 1h ) , 8.02 ( dd , j = 8.1 , 1.3 hz , 1h ) , 7.88 ( d , j = 8.0 hz , 1h ) , 7.82 ( s , 1h ) , 7.677.57 ( m , 2h ) , 
 7.47 ( dd , j = 8.0 , 1.5 hz , 1h ) , 7.387.26 
 ( m , 1h ) , 7.086.97 ( m , 1h ) , 6.63 ( d , j = 2.4 
 hz , 1h ) , 3.81 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c21h18cln7o2 , 436.1 ; found , 
 436.4 . 65% yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , dmso )  10.58 ( s , 1h ) , 9.50 ( s , 1h ) , 
 8.68 ( s , 1h ) , 8.45 ( s , 2h ) , 8.26 ( dd , j = 8.4 , 1.6 
 hz , 1h ) , 8.14 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.997.88 
 ( m , 2h ) , 7.72 ( t , j = 8.0 hz , 1h ) , 7.567.45 
 ( m , 2h ) , 7.417.32 ( m , 1h ) , 7.147.05 ( m , 1h ) , 6.33 
 ( d , j = 1.9 hz , 1h ) , 3.79 ( s , 3h ) . lc / ms ( esi ) m / z : [ m 
 + h ] calcd for c21h18cln7o2 , 436.1 ; found , 436.2 . 75% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , meod )  8.36 ( s , 1h ) , 8.19 
 ( s , 1h ) , 8.068.00 ( m , 1h ) , 7.86 ( d , j = 8.0 
 hz , 1h ) , 7.797.72 ( m , 2h ) , 7.65 ( s , 1h ) , 7.53 ( t , j = 8.0 hz , 1h ) , 7.31 ( dd , j = 8.0 , 1.2 
 hz , 1h ) , 7.217.17 ( m , 1h ) , 6.996.92 ( m , 1h ) , 3.74 
 ( s , 3h ) , 2.16 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c22h20cln7o2 , 450.1 ; found , 
 450.4 . 66% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  10.91 ( s , 1h ) , 9.43 
 ( s , 1h ) , 8.63 ( s , 1h ) , 8.39 ( t , j = 13.4 hz , 3h ) , 
 8.21 ( dd , j = 8.4 , 1.6 hz , 1h ) , 8.05 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.927.81 ( m , 2h ) , 7.63 ( t , j = 7.9 hz , 1h ) , 7.47 ( dd , j = 8.0 , 1.4 
 hz , 1h ) , 7.367.26 ( m , 1h ) , 7.086.99 ( m , 1h ) , 3.87 
 ( s , 3h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c20h17cln8o2 , 437.1 ; found , 437.3 . 44% yield in five steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  10.60 ( d , j = 7.2 hz , 1h ) , 9.44 ( s , 1h ) , 8.59 ( s , 1h ) , 8.448.32 
 ( m , 2h ) , 8.21 ( dd , j = 8.3 , 1.5 hz , 1h ) , 8.09 ( d , j = 3.6 hz , 1h ) , 8.067.97 ( m , 1h ) , 7.84 ( d , j = 6.8 hz , 2h ) , 7.707.58 ( m , 2h ) , 7.47 ( dd , j = 8.0 , 1.5 hz , 1h ) , 7.367.26 ( m , 1h ) , 7.097.00 
 ( m , 1h ) , 4.554.47 ( m , 1h ) , 1.42 ( dd , j = 
 6.8 , 2.0 hz , 7h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h22cln8o2 , 464.2 ; found , 464,4 . 54% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , meod )  8.36 ( s , 1h ) , 8.20 
 ( t , j = 1.8 hz , 1h ) , 8.13 ( s , 1h ) , 8.02 ( dd , j = 8.4 , 1.6 hz , 1h ) , 7.877.82 ( m , 1h ) , 7.74 ( d , j = 8.4 hz , 1h ) , 7.68 ( s , 1h ) , 7.64 ( s , 1h ) , 7.52 ( t , j = 8.0 hz , 1h ) , 7.31 ( dd , j = 8.0 , 1.4 
 hz , 1h ) , 7.18 ( d , j = 1.6 hz , 1h ) , 6.95 ( dd , j = 7.8 , 1.6 hz , 1h ) , 4.574.32 ( m , 2h ) , 3.59 ( t , j = 5.7 hz , 2h ) , 2.87 ( s , 6h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h25cln8o2 , 493.2 ; found , 493,5 . 54% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , meod )  8.48 ( s , 1h ) , 8.32 
 ( t , j = 2.0 hz , 1h ) , 8.28 ( s , 1h ) , 8.14 ( dd , j = 8.4 , 1.6 hz , 1h ) , 8.017.93 ( m , 1h ) , 7.86 ( dd , j = 5.4 , 4.0 hz , 1h ) , 7.78 ( d , j = 14.8 
 hz , 2h ) , 7.65 ( t , j = 8.0 hz , 1h ) , 7.43 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.337.24 ( m , 1h ) , 7.106.98 
 ( m , 1h ) , 4.61 ( t , j = 5.9 hz , 2h ) , 3.72 ( t , j = 6.0 hz , 2h ) , 3.323.28 ( m , 4h ) , 1.34 ( t , j = 7.2 hz , 6h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c26h29cln8o2 , 521.2 ; found , 521.3 . 55% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  10.74 ( s , 1h ) , 9.49 
 ( s , 1h ) , 8.59 ( s , 1h ) , 8.42 ( s , 1h ) , 8.36 ( s , 1h ) , 8.28 ( s , 1h ) , 8.20 
 ( dd , j = 8.4 , 1.6 hz , 1h ) , 8.03 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.86 ( d , j = 7.0 hz , 2h ) , 7.74 
 ( s , 1h ) , 7.65 ( t , j = 7.9 hz , 1h ) , 7.47 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.367.27 ( m , 1h ) , 7.097.00 
 ( m , 1h ) , 4.52 ( t , j = 6.0 hz , 2h ) , 3.64 ( s , 2h ) , 
 3.49 ( s , 2h ) , 3.01 ( s , 2h ) , 1.901.94 ( m , 4h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h22cln8o2 , 519.2 ; found , 
 519.5 . 62% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , meod )  8.50 ( s , 1h ) , 8.33 
 ( s , 1h ) , 8.25 ( s , 1h ) , 8.15 ( dd , j = 8.4 , 1.6 hz , 
 1h ) , 7.99 ( d , j = 6.0 hz , 1h ) , 7.87 ( d , j = 8.4 hz , 1h ) , 7.76 ( s , 1h ) , 7.73 ( s , 1h ) , 7.66 ( t , j = 7.9 hz , 1h ) , 7.467.39 ( m , 1h ) , 7.30 ( d , j = 6.9 hz , 1h ) , 7.117.01 ( m , 1h ) , 3.59 ( d , j = 13.6 hz , 2h ) , 3.513.46 ( m , 1h ) , 3.293.19 ( m , 2h ) , 
 2.402.27 ( m , 4h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c25h25cln8o2 , 505.2 ; found , 505.4 . 53% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  10.72 ( s , 1h ) , 9.48 
 ( s , 1h ) , 9.10 ( s , 1h ) , 8.59 ( d , j = 7.7 hz , 1h ) , 
 8.42 ( s , 1h ) , 8.36 ( s , 1h ) , 8.28 ( s , 1h ) , 8.20 ( dd , j = 8.4 , 1.6 hz , 1h ) , 8.02 ( dt , j = 11.4 , 5.6 hz , 
 1h ) , 7.86 ( d , j = 5.2 hz , 1h ) , 7.75 ( s , 1h ) , 7.62 
 ( dt , j = 19.8 , 8.0 hz , 1h ) , 7.47 ( dt , j = 8.0 , 2.0 hz , 1h ) , 7.367.22 ( m , 1h ) , 7.04 ( ddd , j = 11.0 , 4.6 , 2.3 hz , 1h ) , 5.335.12 ( m , 1h ) , 3.773.27 
 ( m , 5h ) , 2.42 ( m , 1h ) , 2.26 ( m , 1h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h23cln8o2 , 496.2 ; found , 496.4 . 73% yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 mhz , ch3od )  8.44 ( s , 1h ) , 8.23 
 ( d , j = 1.7 hz , 1h ) , 8.12 ( dd , j = 8.2 , 1.4 hz , 1h ) , 7.977.91 ( m , 1h ) , 7.817.72 ( m , 
 2h ) , 7.60 ( t , j = 7.9 hz , 1h ) , 7.41 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.337.25 ( m , 1h ) , 7.097.02 
 ( m , 1h ) , 3.53 ( t , j = 6.4 hz , 2h ) , 3.273.20 
 ( m , 4h ) , 2.05 ( dt , j = 19.2 , 5.6 hz , 2h ) , 1.33 ( t , j = 7.2 hz , 6h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h27cln6o2 , 455.2 ; found , 455.4 . 69% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , meod )  8.45 ( s , 1h ) , 8.23 
 ( t , j = 1.8 hz , 1h ) , 8.13 ( dd , j = 8.4 , 1.5 
 hz , 1h ) , 8.007.89 ( m , 1h ) , 7.847.71 ( m , 2h ) , 7.60 
 ( t , j = 8.0 hz , 1h ) , 7.43 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.30 ( ddd , j = 8.0 , 7.6 , 1.5 
 hz , 1h ) , 7.126.92 ( m , 1h ) , 3.783.62 ( m , 2h ) , 3.55 
 ( t , j = 6.5 hz , 2h ) , 3.29 ( d , j = 
 8.0 hz , 2h ) , 3.11 ( dd , j = 10.8 , 8.0 hz , 2h ) , 2.232.13 
 ( m , 2h ) , 2.131.90 ( m , 4h ) . lc / ms 
 ( esi ) m / z : [ m + h ] calcd 
 for c24h27cln6o2 , 467.2 ; 
 found , 467.5 . 67% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  9.45 ( s , 1h ) , 8.79 ( t , j = 
 6.0 hz , 1h ) , 8.56 ( s , 1h ) , 8.40 ( s , 1h ) , 8.24 ( t , j = 1.7 hz , 1h ) , 8.19 ( dd , j = 8.3 , 1.5 hz , 1h ) , 
 7.98 ( dd , j = 8.0 , 1.2 hz , 1h ) , 7.82 ( s , 1h ) , 7.75 
 ( d , j = 8.0 hz , 1h ) , 7.58 ( t , j = 
 8.0 hz , 1h ) , 7.47 ( dd , j = 8.0 , 1.6 hz , 1h ) , 7.347.27 
 ( m , 1h ) , 7.077.00 ( m , 1h ) , 3.43 ( d , j = 11.3 
 hz , 2h ) , 3.22 ( t , j = 6.0 hz , 2h ) , 2.90 ( q , j = 10.4 hz , 2h ) , 2.75 ( s , 1h ) , 1.89 ( d , j = 14.2 hz , 2h ) , 1.801.84 ( m , 1h ) , 1.38 ( dd , j = 24.8 , 10.8 hz , 2h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c23h25cln6o2 , 453.2 ; found , 453.9 . 72% yield in four steps ( tfa salt , white 
 solid ) ; h nmr ( 400 mhz , dmso )  9.47 ( s , 1h ) , 8.80 
 ( t , j = 6.0 hz , 1h ) , 8.56 ( d , j = 
 6.4 hz , 2h ) , 
 8.41 ( s , 1h ) , 8.24 ( d , j = 1.6 hz , 2h ) , 8.19 ( dd , j = 8.3 , 1.5 hz , 1h ) , 7.97 ( dd , j = 8.0 , 
 1.2 hz , 1h ) , 7.81 ( s , 1h ) , 7.75 ( d , j = 8.0 hz , 1h ) , 
 7.58 ( t , j = 8.0 hz , 1h ) , 7.46 ( dd , j = 8.0 , 1.6 hz , 1h ) , 7.337.27 ( m , 1h ) , 7.116.98 ( m , 
 1h ) , 3.35 ( s , 3h ) , 3.28 ( d , j = 12.6 hz , 2h ) , 3.23 
 ( t , j = 6.1 hz , 2h ) , 2.942.77 ( m , 2h ) , 1.83 
 ( d , j = 12.4 hz , 2h ) , 1.35 ( d , j = 12.4 hz , 2h ) , 1.291.23 ( m , 1h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c24h27cln6o2 , 467.2 ; found , 467.6 . 58% 
 yield in four steps ( tfa salt , white solid ) ; h nmr ( 400 
 mhz , dmso )  9.51 ( s , 1h ) , 9.32 ( d , j = 6.4 
 hz , 1h ) , 8.56 ( s , 1h ) , 8.44 ( s , 1h ) , 8.28 ( t , j = 
 2.0 hz , 1h ) , 8.238.15 ( m , 1h ) , 8.057.98 ( m , 1h ) , 7.84 
 ( s , 1h ) , 7.77 ( d , j = 8.0 hz , 1h ) , 7.667.59 
 ( m , 1h ) , 7.47 ( dd , j = 8.0 , 1.4 hz , 1h ) , 7.367.27 
 ( m , 1h ) , 7.086.99 ( m , 1h ) , 4.85 ( m , 1h ) , 4.18 ( m , 4h ) , 3.26 
 ( m , 1h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c20h19cln6o2 , 411.1 ; found , 411.4 . 65% yield in four steps ( tfa salt , white 
 solid ) ; h nmr 
 ( 400 mhz , dmso )  9.45 ( s , 1h ) , 8.80 ( s , 1h ) , 8.56 ( s , 1h ) , 
 8.41 ( s , 1h ) , 8.25 ( s , 1h ) , 8.19 ( d , j = 8.4 hz , 
 1h ) , 8.00 ( d , j = 8.1 hz , 1h ) , 7.84 ( s , 1h ) , 7.76 
 ( d , j = 7.6 hz , 1h ) , 7.61 ( t , j = 
 8.0 hz , 1h ) , 7.527.40 ( m , 1h ) , 7.30 ( dd , j = 11.6 , 4.4 hz , 1h ) , 7.106.95 ( m , 1h ) , 4.54 ( d , j = 6.1 hz , 1h ) , 3.47 ( d , j = 6.3 hz , 3h ) , 
 3.30 ( s , 1h ) , 3.253.16 ( m , 1h ) , 2.23 ( dd , j = 13.2 , 7.6 hz , 1h ) , 2.122.00 ( m , 1h ) . lc / ms ( esi ) m / z : [ m + h ] calcd for c21h21cln6o2 , 425.1 ; found , 
 425.4 . enzyme inhibition studies were performed 
 in 384-well polystyrene homogeneous time - resolved fluorescence plates 
 ( grainier ) for 1 h at ambient temperature ( 22 c ) with 
 0.5 m biotinylated fl - atf-2 , 1.25 m atp , 0.75 nm activated 
 jnk31 or jnk2 or jnk1 ( with a control in the absence of kinase 
 for determining the basal signal ) in 10 l volumes containing 
 the final concentrations of the following : 50 mm hepes , ph 7.0 , 2.5 
 mm mgcl2 , 0.1 mg / ml bovine serum albumin , 1 mm dl - dithiothreitol , 0.01% triton x-100 ( all from sigma - aldrich ) , and 
 5% dmso ( with or without compound ) . a 10-point titration of all compounds 
 was carried out in 3-fold dilutions from 10 m to 0.5 nm . after 
 22 min , the kinase reaction was terminated by addition of 10 l 
 of quenching solution ( 1 lance buffer , detection reagents , streptavidin - xlapc 
 ( 200 nm ) , and europium cryptate - labeled rabbit polyclonal anti - phospho - atf-2 
 ( 1 nm ) were from cis - bio ) . the homogeneous time - resolved fluorescence 
 signal was detected using an envision plate reader 1 h after quenching . the data from four different experiments were averaged and presented 
 as the mean  sd . ic50 values were determined by fitting 
 the data to the equation for a four - parameter logistic . shsy5y cells ( atcc ) were grown at 37 c 
 and 5% co2 in dmem / f:12 ( invitrogen ) supplemented with 
 10% fetal bovine serum ( fbs ) and penicillin / streptomycin . to ensure 
 that the cells were actively growing , only cells at 80% confluency 
 and between passages five and 15 were used in the experiments . the 
 cells were serum starved for 24 h in dmem / f:12 medium containing 2% 
 fbs before any treatment . mitochondrial 
 membrane depolarization was monitored by mitotracker orange cmtros 
 ( invitrogen ) fluorescence . shsy5y cells ( 60  000 cells / well ) 
 were seeded in black walled , clear bottomed 96-well plates . cells 
 were incubated with compounds for 30 min before the addition of 35 
 m 6-hydroxydopamine ( 6-ohda ) for 4 h. after the incubation , 
 cells were stained with 500 nm mitotracker orange cmtros for 30 min 
 under growth conditions . cells were washed twice in hank s 
 buffer salt solution ( hbss ) , and placed in prewarmed hbss for fluorescent 
 recordings . fluorescence was detected at 576 nm ( exciting at 554 nm ) 
 on a spectramax e5 plate reader ( molecular devices ) . mitochondrial 
 membrane depolarization was normalized to cell abundance by staining 
 the cells with hoechst 33342 ( excitation , 350 nm ; emission , 450 nm ) . . cells ( 60  000 cells / well ) were 
 seeded in a 96-well plate ( clear bottom ) and treated as described 
 in the text . at the culmination of each treatment shsy5y 
 cells ( 60  000 cells / well ) were seeded in a 96-well plate ( clear 
 bottom ) and incubated with different concentrations ( 030 m ) 
 of compounds for 48 h. at the culmination of the treatment the cells 
 were treated with the mtt reagent . shsy5y cells ( 60  000 cells / well ) 
 were plated in a clear - bottomed packard view black 96-well plate in 
 100 l of 10% fbs dmem : f12 medium and were allowed to attach 
 overnight . next day , the cells were treated with the compounds for 
 1 h prior to induction of the jnk pathway activation . the cells were 
 treated with 35 m 6-ohda for 45 h. cells were then 
 fixed in 4% paraformaldehyde in pbs for 20 min at room temperature 
 with no shaking . they were then washed once with 0.1 m glycine to 
 neutralize paraformaldehyde for 5 min . cells were permeabilized with 
 0.2% triton x-100 in pbs for 20 min at room temperature on orbital 
 shaker after which they were washed once with pbs for 5 min . they 
 were then incubated with licor blocking buffer in pbs ( 1:1 dilution 
 in pbs ) for 11.5 h rocking at room temperature . cells were 
 incubated with primary antibody p - c - jun s63 ab ( cell signaling no . 9261 ) 1:100 dilution in licor blocking buffer overnight at 4 c . 
 next day , they were washed twice with pbs0.1% tween 20 ( pbst ) 
 washing solution for 5 min each at room temperature on the orbital 
 shaker , followed by one wash with licor blocking buffer containing 
 0.05% tween-20 for 5 min on the shaker at room temperature . the cells 
 were then incubated with secondary antibody goat anti - rabbit ir800 
 ( 1:500 dilution ) for 1 h at room temperature in the dark ( covered 
 the plate with foil ) in licor blocking buffer - containing tween-20 . 
 following this , cells were washed twice with pbst for 5 min each at 
 room temperature and then once with licor blocking buffer - containing 
 0.05% tween-20 . the wells were then incubated with topro 3 stain ( nucleic 
 acid staining ) , diluted 1:4000 in licor blocking buffer or licor blocking 
 buffer with 0.05% tween-20 for 30 min at room temperature in the dark . finally the plates were washed twice with pbs and analyzed using the 
 odyssey licor infrared scanner . purification of 
 jnk3 39 - 402 and its 
 crystallization with amppnp was done following previously published 
 procedure . compound 26k was soaked into the crystal by adding 
 2 mm compound into the crystallization drop and incubating for 24 
 h. the crystal was then transferred to a mounting loop , and excess 
 soaking solution was removed and flash frozen by plunging into liquid 
 nitrogen . a 
 diffraction data set 
 was collected at ls - cat beamline 21-id - g ( aps ) using marmosiac 300 
 ccd detector ( mar research ) . the data set was processed with autoproc 
 with xds as the data reduction engine . quick molecular replacement using phaser 
 in phenix suite properly positoned jnk3 , 26k was identified 
 as positive densities in atp binding pocket . restraints and coordintates 
 for the compound was generated using elbow in phenix suite and incorporated 
 into the jnk3 coordinate using the graphics program coot . the model 
 was then refined using autobuster with tls ( translation , libration , 
 and screw - motion ) , water update , and unknown ligand search options 
 turned on . the coordinate and the structure factor are 
 deposited to wwpdb with the pdb code 4wh2 .
the 
 c - jun n - terminal kinase 3 ( jnk3 ) is expressed primarily in 
 the brain . 
 numerous reports have shown that inhibition of jnk3 is 
 a promising strategy for treatment of neurodegeneration . 
 the optimization 
 of aminopyrazole - based jnk3 inhibitors with improved potency , isoform 
 selectivity , and pharmacological properties by structure  activity 
 relationship ( sar ) studies utilizing biochemical and cell - based assays , 
 and structure - based drug design is reported . 
 these inhibitors had 
 high selectivity over jnk1 and p38 , minimal cytotoxicity , potent 
 inhibition of 6-ohda - induced mitochondrial membrane potential dissipation 
 and ros generation , and good drug metabolism and pharmacokinetic ( dmpk ) 
 properties for iv dosing . 
 26n was profiled against 464 
 kinases and was found to be highly selective hitting only seven kinases 
 with > 80% inhibition at 10 m . 
 moreover , 26n showed 
 good solubility , good brain penetration , and good dmpk properties . 
 finally , the crystal structure of 26k in complex with 
 jnk3 was solved at 1.8  to explore the binding mode of aminopyrazole 
 based jnk3 inhibitors .