deoxycytidine 
 kinase ( dck ) is a deoxyribonucleoside kinase capable 
 of phosphorylating deoxycytidine , deoxyadenosine , and deoxyguanosine 
 to their monophosphate forms using either atp or utp as phosphoryl 
 donors .
phosphorylation by dck is responsible 
 for converting salvaged deoxycytidine into deoxycytidine monophosphate 
 ( dcmp ) , a precursor for both dctp and dttp pools .
apart from the physiological 
 role of generating dntps , dck plays a crucial role in activating multiple 
 nucleoside analog prodrugs that are widely used in anticancer and 
 antiviral therapy .
recently , we and others identified a requirement 
 for dck in hematopoiesis in lymphoid and erythroid progenitors .
the 
 kinase has also been implicated in regulating the g2/m transition 
 in response to dna damage in cancer cells .
more recently , we have shown that partial inhibition of dck activity , 
 combined with perturbations of nucleotide de novo synthesis pathways , 
 was synthetically lethal to acute lymphoblastic leukemia cells but 
 not to normal hematopoietic cells .
these 
 aspects of dck s biology , and its potential role as a new therapeutic 
 target in cancer , prompted us to develop small molecule inhibitors 
 of its enzymatic activity . in earlier publications we reported the discovery 
 of hit compounds from a high throughput screen and subsequent optimization 
 of the molecules to lead compounds 1 and 2 ( numbered 36 and 37 , respectively , in 
 ref ( 8) ) .
lead compounds 1 and 2 can be divided into four distinct structural 
 parts ( figure 1a ) .
part a is the pyrimidine 
 ring , which is connected by a linker ( part b ) to a 5-subsituted - thiazole 
 ring ( part c ) , which in turn is connected to a phenyl ring ( part d ) . 

conceptually , each of these parts can be modified to attain desired 
  druglike  properties . in previous work , we focused 
 on the thiazole portion of the inhibitor .
the crystal structure of 
 dck with one of the early compounds suggested that the ring 5-position 
 could accommodate hydrophobic substituents , which led to the discovery 
 that a propyl group at the 5-position is strongly favored over a methyl 
 group .
part a indicates the pyrimidine ring , 
 and part b is the linker connecting to a 5-substituted - thiazole ring 
 ( part c ) , which is followed by a phenyl ring ( part d ) .
compounds 1 and 2 differ at the substituent present at 
 the phenyl meta position ( rm ) .
( b ) in vitro ( ic50 and ki ) 
 and cell ( ic50 ) properties for 1 and 2 . to guide and rationalize the medicinal 
 chemistry efforts in other 
 parts of the molecule
, we solved the crystal structures of human dck 
 with several of the inhibitors we developed .
the crystal structures 
 illuminate the relationship between the enzyme structure , the small 
 molecule structure , and its inhibition potency . in the first part 
 of this manuscript we report the in vitro binding affinities ( ic50 and ki ) , cellular ic50 values , and crystal structures of dck 
 in complex with compounds that differ in the pyrimidine and phenyl 
 rings . unfortunately , despite nanomolar affinity for dck , when tested 
 in a liver microsomal assay , these compounds exhibited low metabolic 
 stability ( data not shown ) .
this shortcoming was recapitulated by 
 pharmacokinetic studies in mice . to identify inhibitors 
 with improved in vivo properties
, we set 
 out to explore additional chemical modifications , specifically , those 
 that maintain the low nanomolar binding affinity of the lead compounds . 

in the second part of the manuscript , we report novel chiral derivatives 
 of our inhibitors . crystal structures of these chiral compounds bound 
 to dck played a key role in elucidating the chirality of the active 
 form of the inhibitor . by combining organic chemistry intuition with 
 detailed structural information on the target  inhibitor complex , 
 we have identified a lead compound that retains the nanomolar affinity 
 for dck but has gained significant in vivo metabolic stability .
this 
 compound could play a vital role in any therapeutic strategy based 
 on induction of dna replication stress overload by perturbing a cancer 
 cell s dntp pools .
the pyrimidine ring 
 ( part a of the molecules , figure 1a ) was predicted 
 to be the part of the molecule most difficult to improve .
this is 
 because , as observed in the crystal structures of dck in complex with 
 lead compounds 1 and 2 ( pdb codes 4l5b and 4kcg , respectively ) , 
 the inhibitor s pyrimidine ring binds to dck at a position 
 nearly identical to that adopted by the pyrimidine ring of the physiological 
 substrate dc , making several hydrogen bonds , hydrophobic , and  
 stacking interactions ( supporting information 
 figure s1 ) .
this binding mode suggested an already quite optimized 
 enzyme  pyrimidine ring interaction . for compounds 1 and 2 ,
both pyrimidine ring exocyclic amino groups 
 formed hydrogen - bonding interactions with side chains of glu53 , gln97 , 
 and asp133 .
hence , not surprisingly , simultaneous removal of both 
 amino groups resulted in complete loss of dck inhibition .
in contrast , removal of a single amino group 
 to generate compound 3 ( figure 2a ) , which is identical to 1 except for having a single 
 exocyclic amino group in the pyrimidine ring ( figure 1a ) , resulted in similarly tight binding affinity as measured 
 for 2 ( figures 1b and 2b ) . to explain how the affinity of 3 for dck is maintained with only a single exocyclic amino group
, 
 we sought the crystal structure of the complex , but unfortunately , 
 we were unable to obtain diffraction quality crystals .
we speculate 
 that the sole exocyclic amino group present in compound 3 is oriented in the dck active site such that it maintains its interaction 
 with asp133 , since only in that orientation can the neighboring pyrimidine 
 ring n atom maintain its interaction with the side chain of gln97 
 ( supporting information figure s1 ) .
the 
 conclusion here is that the interaction with glu53 made by an exocyclic 
 amino group , when present , provides only moderate additional binding 
 energy . while a single exocyclic pyrimidine ring amino group is sufficient 
 for a tight interaction with dck , in our cem cell - based assay compound 3 exhibited a much - increased ic50 value ( 21.8 nm , 
 figure 2b ) relative to compound 2 ( 4.9 nm , figure 1b ) .
this result showcases 
 the importance of evaluating the interaction between an inhibitor 
 and its target in using both an enzymatic in vitro assay and a cell - based 
 assay . because of the reduced inhibition of dck activity of 3 in the cell - based assay , all future compounds contained 
 the two exocyclic amino groups .
( a ) schematic representation 
 of compound 3 that has a single exocyclic amino group 
 and of compound 4 that has a ring nitrogen atom between 
 the two exocyclic amino groups .
( b ) in vitro ( ic50 and ki ) and cell 
 ( ic50 ) properties for 3 and 4 .
( c ) overlay of the dck4 ( orange , pdb code 4q18 ) and dck-1 ( green , pdb code 4l5b ) structures with a focus on the pyrimidine ring .
note the 0.4 
  shifted position of 4 relative to 1 that is due to the presence of a water molecule ( orange sphere ) . 
 binding of this water molecule
next , we assessed the importance of the position of the pyrimidine 
 ring n atoms by synthesizing compound 4 ( figure 2a ) .
this compound was measured to bind with 50-fold 
 higher ic50 relative to the very similar 
 lead compound 1 ( figure 1a ) , which 
 only differs in the position of one pyrimidine ring nitrogen atom . 

we solved the 2.0  resolution crystal structure of the dck4 complex to understand how this subtle change so drastically 
 impacted the interaction with the enzyme ( see table 1 for the data collection and refinement statistics ) .
all of the examined compounds bind to the open state 
 of the enzyme , 
 which is also the catalytically incompetent state ( for a discussion 
 about the open and closed states of dck , see refs ( 10 ) and ( 11 ) ) .
inhibitors bind within 
 a deep cavity , with the pyrimidine ring of the inhibitors positioned 
 deepest and occupying the same position occupied by the pyrimidine 
 ring of the nucleoside substrate .
while preventing 
 the binding of the nucleoside substrate , our inhibitors do not interfere 
 with binding of nucleotide to the phosphoryl donor - binding site .
in 
 fact , all crystal structures of dck in complex with inhibitors also 
 contained udp at the donor site . despite significantly different 
 ic50 values 
 between compound 1 ( 14.5 nm ) and compound 4 ( 754 nm ) ,
the pyrimidine ring of these related molecules interacts 
 with the enzyme via very similar hydrophobic and polar interactions . 

however , the entire molecule 4 is displaced about 0.4  away from the floor of the 
 binding cavity relative to compound 1 .
the crystal structure suggests that the factor 
 responsible for this shift is the recruitment of a water molecule 
 ( orange sphere , figure 2c ) by the pyrimidine 
 ring n present in compound 4 .
in contrast , for compound 1 the ch group in this position eliminates the potential for 
 a hydrogen bond .
this water molecule is also held in place through 
 interactions with arg104 and asp133 . hence , despite formation of this 
 additional water - mediated interaction with the enzyme , the displacement 
 away from the enzyme caused by allowing the water molecule to bind 
 at that position ultimately reduces the binding affinity of 4 . on the basis of these results , we decided to maintain 
 the original 
 structure of the pyrimidine ring and to focus on the other parts of 
 the molecule as potential modification sites .
we next examined the 
 effect of various substituents at different phenyl group positions 
 ( part d of the molecule , figure 1a ) .
previously , we reported that a 
 compound with no phenyl ring substituents , but otherwise identical 
 to compound 1 , showed very modest potency in our cem 
 cell based assay ( ic50 = 37 nm  ) . adding a hydroxyl group at the meta position decreased 
 the ic50 in that assay by about half ( compound 5 , previously compound 31 , figure 3 ) .
the effect of adding the longer 
 hydroxyethoxy group at that position ( compound 6 , previously 
 compound 32(3 ) ) was more impressive , 
 yielding an ic50 of 1 nm ( figure 3 ) .
we are aware that primary hydroxyls as in 6 are prone to oxidation or glucuronidation , but these studies do inform us as to the importance of the type 
 of substituent at the phenyl meta position .
( a ) schematic representation 
 of compounds 5 and 6 that differ by the 
 nature of the meta position substituent . ( b ) in vitro ( ic50 and ki ) 
 and cell ( ic50 ) properties for 5 and 6 .
( c ) overlay of the dck5 ( magenta , 
 pdb code 4q19 ) and dck6 ( pale green , pdb code 4q1a ) structures with 
 a focus on the phenyl ring meta position .
the tighter binding of 6 relative to 5 can be rationalized by the interaction 
 of the longer meta substituent ( position highlighted with a gray background ) 
 with s144/s146 of dck .
to understand the difference in affinities to dck between 
 compounds 5 and 6 , we determined the structures 
 of dck 
 in complex with these molecules , solved at 2.09 and 1.9  resolution , 
 respectively ( table 1 ) .
the structure of dck 
 in complex with compound 5 reveals that the hydroxyl 
 group at the phenyl group meta position does not make any inhibitor 
in contrast , the structure of dck in complex with compound 6 shows that the hydroxyethoxy at this position is able to 
 interact with the side chains of ser144 and ser146 ( figure 3c and supporting information 
 figure s3 ) .
we attribute this added interaction to the superior 
 binding of compound 6 versus compound 5 . in terms of the importance of substituents at the phenyl meta position , 
 it is clear that having none or a short one such as a hydroxyl ( compound 5 ) diminishes the interaction with dck . on the other hand , 
 the binding affinity measured by both the in vitro kinetic assay and 
 by the cell - based cem assay of larger substituents ( as present in 
 compounds 1 , 2 , and 6 ) are 
 comparable .
previous crystal structures of dck in complex with compound 1 ( pdb code 4l5b ) and 2 ( pdb code 4kcg ) also show an interaction between the 
 substituent at the phenyl meta position and the enzyme , this time 
 to ser144 .
additional side chains such as 2-fluoroethoxy poly(ethylene 
 glycol ) ( n = 2 ) ( peg)2 ( s16 , s17 , s19 ) , 2-hydroxyethyl ( peg)2 ( s11 ) , 2-methoxyethyl ( peg)2 ( s20 , s22 , s23 , s25s29 ) , and 2-(4,6-diaminopyrimidine-2-thio)ethyl ( peg)2 ( s10 ) substituents were well tolerated at the 
 meta position ( data not shown and supporting information 
 table s1 ) .
we conclude that the precise nature of the 
 substituent at the phenyl 
 meta position is not critical as long as it contains a polar group 
 that can extend to the proximity of ser144/ser146 .
to determine the importance of substituent 
 at the phenyl group para position , we prepared compound 7 ( previously compound 28(3 ) ) , which only differs from compound 2 by lacking a para 
 position substituent ( figure 4a ) .
the in vitro 
 measured binding affinity values ( ic50 ; ki ) of compound 7 are nearly identical 
 to that of 2 ( figure 4b ) , indicating 
 that substituents at the para position are not required for tight 
 binding .
this is explained by the crystal structures of dck in complex 
 with compounds 7 and 8 ( previously compound 30(3 ) ) , which show a nearly identical 
 binding mode , very similar to that observed for compound 2 ( figure 4c and supporting 
 information figure s4 ) .
this conclusion is supported by 
 the properties of compound 8 , which in contrast to the 
 methoxy group in compounds 1 and 2 has the 
 longer hydroxyethoxy group but similar binding affinity .
hence , the 
 in vitro binding affinities are largely unchanged between having no 
 substituent at the phenyl group para position , having a methoxy , or 
 the longer hydroxyethoxy .
however , we did notice a 10-fold 
 difference between compounds 7 and 8 in 
 the cem cell - based assay , with compound 7 being less 
 potent .
furthermore , substituents at the phenyl ring s para 
 position such as 2-fluoroethoxy ( s4 , s14 , s18 ) , fluoro ( s5 , s6 ) , methoxymethyl 
 terminated ( peg)2 ( s21 , s24 ) , 
 and n - substituted methanesulfonamide ( s29 , s30 ) were relatively well tolerated ( data not shown and supporting information table s1 ) .
groups attached 
 to the thiazole like 4-pyridinyl ( s7 ) , meta monosubstituted 
 phenyl ( s17 ) , and 3,5-disubstituted phenyl ring ( s31 ) substituents were also tolerated ( data not shown and supporting information table s1 ) .
therefore , 
 while not directly important for the binding affinity , having even 
 a small substituent at the phenyl group para position improves the 
 relevant cell - based measurements . as a result , most subsequent compounds 
 contained the methoxy group at that position .
( a ) schematic representation 
 of compounds 7 and 8 that differ by the 
 nature of the para position substituent .
( b ) in vitro ( ic50 and ki ) 
 and cell ( ic50 ) properties for 7 and 8 .
( c ) overlay of the dck7 ( teal , pdb 
 code 4q1b ) and 
 dck8 ( beige , pdb code 4q1c ) structures with a focus on the phenyl 
 ring para position .
the inhibitors bind very similarly ; the meta position 
 substituents make a direct interaction with the enzyme , but the para 
 substituent does not .
the very similar ic50 and ki values of 7 and 8 are explained by the lack of direct interactions 
 to the enzyme via the para position .
in contrast , the presence of 
 a para position substituent lowers the cell - based determined ic50 value .
in previous work we demonstrated that the nature 
 of the substituent at the thiazole ring 5-position ( part c of the 
 molecule , figure 1a ) plays a crucial role in 
 binding affinity . in short , we compared 
 having no substituent at that position to having a methyl , ethyl , 
 or propyl .
we found that propyl dramatically improved the binding 
 affinity , and as a result , compounds with a propyl at the 5-position 
 became our lead compounds ( i.e. , compounds 1 and 2 , figure 1 ) .
interestingly , compounds 
 with a small / no substituent at the thiazole 5-position were observed 
 to bind two inhibitor molecules per dck active site , to binding sites 
 that we refer to as position 1 and position 2 . in contrast , the tighter 
 binding propyl - containing molecules were observed to bind with a single 
 inhibitor molecule , at position 1 , per dck active site .
this revealed that binding of two molecules is 
 not required for high affinity . in our previous report , we analyzed 
 the implication of single versus double binding of inhibitor molecules 
 to dck and concluded that inhibition of dck is primarily caused by 
 the binding of the inhibitor at position 1 , whereas the molecule bound 
 at position 2 does not appreciably enhance the inhibition .
however , 
 when tested for metabolic stability , we discovered that the propyl - group - containing 
 compounds 1 and 2 are less stable relative 
 to those having the shorter methyl group , e.g. , compound 15a as reported by murphy et al .
we 
 also explored the activity of cyclopropyl and phenyl groups at the 
 thiazolyl 5-position ( supporting information table 
 s1 and data not shown ) .
the cyclopropyl analog ( s27 ) had a good ic50 value , but it failed in the pet l - fac 
 assay .
hence we were forced to revert to the 
 methylthiazole ring substituent despite a weaker interaction with 
 dck . to compensate for the loss of affinity provided by the thiazole 
 propyl group , we searched for a compensating modification that would 
 restore the in vitro binding affinity while maintaining acceptable 
 metabolic stability .
for that purpose , we decided to explore modifications 
 on the linker moiety ( part b of the compounds , figure 1a ) .
test concentration 
 of compounds 
 was 1 m . the 
 sch2 group acts to link the pyrimidine 
 and thiazole rings of our compounds .
we tested a variety of alternatives 
 to this linker , such as its deuterated analog ( scd2 ) , for the purpose of a kinetic isotope study .
we reasoned 
 that if the linker was implicated in hydrolytic metabolism , then , 
 because of the kinetic isotope effect , a deuterated ( scd2 ) analog would show an improvement in metabolic stability . 

the deuterium analogs ( s1 , s8 , s9 , s13 ) had affinity similar to their isotopologues , 
 as expected ( supporting information table s1 and data not shown ) . however , the deuterated compounds failed to 
 show an improvement in the pet l - fac liver assay , indicating that 
 a hydrolytic mechanism is probably not involved in the metabolism 
 of the sch2 linker .
we also tested the 
 replacement of the sulfur atom of the sch2 
 group with a methylene group ( ch2ch2 ) . replacing the sulfur atom of the linker with a carbon 
 atom resulted in a considerable decrease in dck affinity and metabolic 
 stability ( supporting information table s1 and data not shown ) .
we next tested a linker in which the methylene 
 was substituted to contain a methyl group ( sch(ch3) ) .
these racemic methyl - linker compounds showed very promising 
 biological results and increased metabolic stability ( see supporting information schemes 1 and 2 for the 
 synthesis of compounds 9 and 10 ) .
therefore , 
 we carefully examined the synthetic route in an attempt to reduce 
 the synthetic steps and improve the total yield .
we succeeded in developing 
 a six - step synthetic route toward 11 in an overall yield 
 of 43% ( scheme 1 ) .
commercially available 3-hydroxy-4-methoxybenzonitrile a was subjected to an aqueous ammonium sulfide solution under 
 basic conditions to provide thioamide b. cyclization 
 to form the thiazole core of c was achieved via condensation 
 of thioamide b with 4-bromopentane-2,3-dione in refluxing ethanol .
introduction of a peg 
 chain into the phenyl ring of compound d with 13-chloro-2,5,8,11-tetraoxatridecane under basic conditions was achieved in 89% yield . 

reduction of the resulting ketone - containing compound with diisobutylaluminum 
 hydride ( dibal - h ) afforded racemic secondary alcohol e in high yield .
the acyl chloride was 
 reacted in crude form with 4,6-diamino-2-mercaptopyrimidine to generate 
 product 11r / s .
reagents and conditions : ( a ) 
 ( nh4)2s ( 20% in h2o ) , pyridine , et3n , 60 c , 85% ; ( b ) 4-bromopentane-2,3-dione , etoh , reflux , 
 95% ; ( c ) 13-chloro-2,5,8,11-tetraoxatridecane , cs2co3 , dmf , 50 c , 89% ; ( d ) dibal - h , dcm , 78 c , 
 92% ; ( e ) socl2 , dcm , 0 c to rt ; ( f ) 4,6-diamino-2-mercaptopyrimidine , 
 k2co3 , dmf , 75 c , 65% in last two steps .
ch(ch3) linker was introduced to a compound that contained 
 the propyl group at the thiazole ring 5-position ( compound 9 ) and to a compound that , instead of the propyl group , contained 
 a methyl ( compound 10 ) ( figure 5a ) . as mentioned above , the rationale for compound 10 was the predicted improvement in metabolic stability .
interestingly , 
 whereas compounds with a propylthiazole ring previously showed tighter 
 binding to dck compared to the analogous methylthiazole compounds , 
 we now measured better binding with the methyl - containing compound 10 to the propyl - containing compound 9 ( figure 5b ) . hence , the proximity of the thiazole - ring substituent 
 ( propyl or methyl ) to the methyl - linker substituent resulted in the 
 larger propyl group being not as accommodating in the dck active site . 
 despite the improved in vitro binding parameters for 10 over 9 , the cell - based assay yielded similar ic50 values , yet consistent with 10 being superior 
 ( figure 5b ) .
both 
 compounds were synthesized as 
 the racemic mixture ( r / s ) ; the addition 
 of a methyl group ( arrow ) to the methylene linker group makes these 
 compounds chiral . whereas 9 has a propyl group at the 
 thiazole ring 5-position ( rt ) , 10 has a methyl 
 group .
( b ) in vitro ( ic50 and ki ) and cell ( ic50 ) properties 
 for 9 and 10 .
( c ) the propyl group at the 
 thiazole ring makes 9 bind as a single molecule to binding 
 site position 1 of dck ( see text for details ) .
notably , despite forming 
 the enzyme  inhibitor with racemic 9 , in the crystal 
 structure we observe only the r - isomer ( compound 9 in yellow , pdb code 4q1d , fo 
a 
 theoretical model of the s - isomer ( gray ) demonstrates 
 that only the r - isomer fits the electron density . 

( d ) the methyl group at the thiazole ring permits two molecules of 10 to bind to dck : one to position 1 and one to position 2 . 
 in position 1
we observe only the r - isomer ( 10r - p1 , 
 cyan , pdb code 4q1e ; fo  fc omit map contoured at 2 in green ) . a theoretical model 
 of the s - isomer at position 1 ( gray )
clearly demonstrates 
 that only the r - isomer fits the electron density 
 ( red arrow ) .
( e ) in position 2 we observe only the s - isomer ( 10s-p2 , plum , pdb code 4q1e ; fo  fc omit map contoured at 1.5 in green ) . 

a theoretical model of the r - isomer at position 2 
 ( gray ) clearly demonstrates that only the s - isomer 
 fits the electron density ( red arrow ) .
both compounds 9 and 10 were prepared 
 as racemic mixtures ; the introduced linker - methyl group makes that 
 position a new chiral center ( arrow , figure 5a ) . to elucidate which of the two enantiomers is the active dck inhibitor
, 
 we determined the crystal structure of dck in complex with compounds 9 and 10 ( solved at 2.0 and1.85  resolution , 
 respectively , table 1 ) . as expected , compound 9 binds as a single molecule to dck , specifically at position 
 1 , because of the presence of the propyl group in the thiazole ring . 

interestingly , despite the fact that a racemic mixture of 9 was used to form the complex to dck , the crystal structure provides 
 unambiguous evidence for the r - isomer binding at 
 position 1 ( figure 5c and supporting information figure s5 ) .
likewise , inspection of 
 the structure of the complex between racemic 10 and dck 
 shows that the r - isomer occupies the most relevant 
 position 1 binding site ( figure 5d and supporting information figure s5 ) . since compound 10 contains the methyl substituent in the thiazole ring , which 
 allows for a molecule to also occupy position 2 , we observe compound 10 at that position as well . however , whereas it is the r - isomer of 10 that binds to position 1 , it 
 is the s - isomer that binds to position 2 ( figure 5e and supporting information 
 figure s5 ) .
we previously concluded that position 1 is 
 the critical binding 
 site for this family of inhibitors .
this would suggest that the measured 
 in vitro inhibition values of racemic 10 are reflecting 
 the preferential binding of the r - isomer . to test 
 this , we synthesized compound 11 , which is a slight modification 
 of 10 ( the nature of the phenyl group substituents ) but 
 notably had the racemic mixture separated to yield the pure isomers 11r and 11s ( figure 6a ) . 

we determined the in vitro binding affinities of the enantiomerically 
 pure compounds and observed that 11s has 400-fold 
 weaker binding affinity relative to 11r ( figure 6b ) .
this result provides clear evidence that the r - form is responsible for the tight interaction with dck . 

this result also validates our structure - based interpretation that 
 position 1 is the one most relevant inhibitor binding site for dck 
 inhibition and that position 2 is occupied because of the high concentration 
 of the inhibitor used in the crystallization setups .
( a ) schematic representation of compounds 11s , 11r , and 12r ( r or s designate the chirality of the linker methylene carbon ; 
 arrows point at the added methyl group ) . ( b ) in vitro ( ic50 and ki ) 
 and cell ( ic50 ) properties for 11s , 11r , and 12r .
the r - isomer of 
 both 11 and 12 is responsible for the observed 
 inhibition of the enzyme .
( c ) dck was crystallized in the presence 
 of enantiomerically pure 12r , and the enzyme  inhibitor 
 complex structure was solved ( pdb code 4q1f ) .
fc omit map ( 1.6 ) for the position 1 binding 
 site clearly shows the presence of 12r ( brown ) . despite 
 the thiazole methyl group in 12r ( which is compatible 
 with molecules also binding to position 2 )
this is consistent with the results 
 with compound 10 ( figure 5 ) that 
 showed that only the s - isomer binds to positon 2 .
having 
 discovered that the r - isomers of compounds 9 , 10 , and 11 are responsible for 
 the dck inhibition , we set out to develop an asymmetric synthesis 
 ( scheme 2 ) .
the chiral synthesis developed 
 by our group for compound 12r , which is a close analog 
 of 10 , features a chiral corey 
shibata 
 ( cbs ) reaction of ketone d. chiral alcohol e was synthesized according to this 
 method with an enantiomeric excess of 96% , as determined via chiral 
 hplc . employing mesic or tosic anhydride to give the sulfonates under 
 different basic conditions such as et3n , pyridine , or
dmap 
 resulted in elimination to the alkene , presumably due to the stability 
 of the secondary benzylic - like carbocation .
the use of trifluoroacetic 
 anhydride ( tfaa ) at 0 c converted alcohol e into 
 the corresponding trifluoroacetate ( tfa ) f without a 
 significant decrease in the % ee of the ester .
finally , compound f was reacted with 4,6-diamino-2-mercaptopyrimidine to generate 12r in 61% yield over two steps with an enantiomeric excess 
 of 40% .
presumably , a portion of the reaction occurs via a direct 
 sn2 pathway , while another part occurs via an sn1 pathway , and thereby racemized material was obtained .
chiral resolution 
 via recrystallization generated 12r with an enantiomeric 
 excess of over 90% . likewise , ( s)-()-2-methyl - cbs - oxazaborolidine 
 was used in the cbs reduction to synthesize 12s .
reagents and conditions : ( a ) 
 ( nh4)2s ( 20% in h2o ) , pyridine , et3n , 60 c , 85% ; ( b ) 4-bromopentane-2,3-dione , etoh , reflux , 
 96% ; ( c ) n-(2-bromoethyl)methanesulfonamide , 
 cs2co3 , dmf , 50 c , 82% ; ( d ) ( r)-(+)-2-methyl - cbs - oxazaborolidine , bh3thf complex , 
 thf , 78 c , 77% , ( 96% ee ) ; ( e ) tfaa , dcm , 0 c , 
 ( f ) 4,6-diamino-2-mercaptopyrimidine , dmf , 80 c , 61% in last 
 two steps . compound 12r ( figure 6a ) was 
 measured to have very similar in vitro binding affinities to 11r ( figure 6b ) . significantly , just 
 as the affinity of 11s was much reduced relative to 11r , the affinity to dck of 12s was much reduced 
 relative to 12r .
this reiterated the preference of dck 
 for compounds that contain the r - isomer of the linker .
we expected 12r to bind only at position 1 based on the 
 previous structure with compound 10 ( observing 10r bound at position 1 ) and the kinetic results using enantiomerically 
 pure 11s , 11r , 12s , and 12r ( observing higher affinities for the r - isomers ) and since the crystals were formed with the enantiomerically 
 pure 12r .
additionally , lacking the s - isomer , we expected a vacant position 2 binding site .
indeed , the 
 crystal structure of the dck12r complex revealed 
 a single inhibitor molecule at position 1 ( figure 6c ) .
this result suggests that the r - isomer 
 has very low affinity to the binding site at position 2 .
notably , 
 while the interaction between the r - isomer and dck 
 is limited to the position 1 binding site , this does not diminish 
 the binding affinity for the enzyme . what could be behind 
 the dramatic selectivity of the dck position 1 binding site for the r - isomers of the inhibitors ?
likewise , what prevents the r - isomer from binding at position 2 , while this binding 
 site is compatible with the binding of the s - isomer ? 

the simple explanation would involve steric considerations relating 
 the inhibitor and enzyme , where the chiral methyl group of the linker 
 clashes with enzyme residues in the case of one isomer but not the 
 other .
however , inspection of the crystal structures solved with compounds 10(r / s ) and 12r does 
 not support this interpretation ; we could model the s - isomer bound to position 1 ( figure 5d ) and 
 the r - isomer bound at position 2 ( figure 5e ) with no apparent clashes .
comparison of 
 the binding mode between 10r and 10s reveals 
 that the relative orientation of the pyrimidine ring to the thiazolephenyl 
 part is strikingly different between the r and s isomers ( figure 7a and figure 7b ) .
that is , by a change of the angles of the linker 
 that connects the pyrimidine ring to the thiazole ring , each isomer 
 has adjusted its conformation to best fit its binding site ( i.e. , 
 induced fit ) .
this demonstrates that the enzyme dictates the relative 
 orientations between the pyrimidine ring , linker , and the thiazolephenyl 
 rings .
it also shows that the relative orientation between thiazole 
 and phenyl rings ( being coplanar ) is largely unchanged , not surprising 
 because of the resonance between the rings .
chiral selectivity is 
 due to conformational selection by the enzyme s 
 binding site .
( a ) observed orientation of 10r ( cyan ) 
 at position 1 ( 10r - p1 , pdb code 4q1e ) and 10s ( plum ) at position 
 2 ( 10s - p2 ) upon dck binding .
note the different relative 
 orientations of the thiazole and pyrimidine rings between 10r and 10s .
( c ) the conformation of 10r ( 10r - p1 ) 
 is dictated by the position 1 binding site . in this conformation
the 
 distance between the chiral linker methyl group and the thiazole ring 
 methyl group is 4.2  .
( d ) the theoretical model of 10s binding with the same conformation as 10r in position 
 1 ( 10s - p1 ) shows that the homologous distance is reduced to 2.5  . 

( e ) the conformation of 10s ( 10s - p2 ) is dictated by the 
 position 2 binding site . in this conformation
the distance between 
 the chiral linker methyl group and the thiazole ring methyl group 
 is 4.4  .
( f ) the theoretical model of 10r binding 
 with the same conformation as 10s in position 2 ( 10r - p2 ) 
 shows that the homologous distance is reduced to 2.6  .
( g ) for 
 10r - p1 , the observed torsion angle between the thiazole ring and the 
 linker is 59. scanning possible torsion angles shows 
 that this value represents a low energy conformation of 10r .
( h ) for 10s - p1 , the observed torsion angle is 189. this value 
 corresponds to a high - energy conformation .
( i ) for 10s - p2 , the observed 
 torsion angle is 326. scanning possible torsion angles 
 shows that this value is at a low energy conformation of 10s .
( j ) for 10r - p2 , the observed torsion angle is 147. this value 
 corresponds to a high - energy conformation . to further probe the observed chiral selectivity , we constructed 
 a theoretical model of 10s binding at position 1 with 
 the same orientation as 10r . whereas the observed distance 
 between the chiral methyl of the linker and the thiazole ring methyl 
 group for 10r in position 1 is 4.2  ( figure 7c ) , for the modeled 10s bound to position 1 , that distance would be an unfavorable 2.5  
 ( figure 7d ) . likewise , whereas the observed 
 distance between the chiral methyl and the thiazole methyl for 10s in position 2 is 4.4  ( figure 7e ) , for the modeled r - isomer adopting the same conformation 
 as 10s , that distance would be an unfavorable 2.6  
 ( figure 7f ) . hence
, the strict chiral selection 
 to either position 1 or position 2 is due to the enzyme dictating 
 a particular inhibitor orientation that is vastly different between 
 the binding sites . in the case of position 1 ,
that orientation is 
 not compatible with the s - isomer , and for position 
 2 , that orientation is not compatible with the r - isomer .
using computer simulations , we obtained a qualitative estimate 
 of the conformational penalty incurred by 10r and 10s upon binding with the protein .
the conformational penalty 
 is the energy difference between the preferred solution - phase geometry 
 of a substrate and the geometry that it assumes upon binding : e = esolution  ebound .
each enantiomer was docked with the solvated 
 protein at position 1 and allowed to equilibrate ( see details in experimental section and supporting 
 information figure s6 ) .
the equilibrated , docked inhibitor 
 structures were removed from the protein , and their energies were 
 assessed with the semiempirical pddg / pm3 method .
unbound structures of 10r and 10s were 
 optimized in implicit solvent to determine their low - energy solution - phase 
 conformations . as with the bound structures ,
the resulting energies were 
 used to obtain qualitative conformational penalties for each enantiomer . 

the conformational penalty for 10s was almost twice the 
 conformational penalty for 10r ( 45
kcal / mol larger penalty 
 for 10s ) , further demonstrating that 10r needs to undergo a much less unfavorable structural rearrangement 
 in order to bind with the protein at position 1 .
another way 
 of considering this issue is to examine the energy 
 of the inhibitor as a function of rotation around the bond that connects 
 the thiazole ring to the chiral linker atom ( bond marked with  
 in figure 7c  f ) . for 10r
bound to dck at position 1 , the observed dihedral angle that specifies 
 this rotation is 59 and fits a low energy conformation 
 ( figure 7 g ) .
in contrast , the modeled s - isomer at this binding site would have a torsion angle 
 of 189 , which is clearly a high - energy conformation ( figure 7h ) .
the same pattern is observed for position 2 , 
 with the s - isomer binding to dck with a torsion angle 
 of 326 , which is a low energy conformation , while the 
 modeled r - isomer at that position is a high - energy 
 conformation ( figure 7i and figure 7j ) .
hence , the chiral selectivity does not come 
 directly from the enzyme sterically favoring one isomer over the other . 

rather , the enzyme dictates a particular conformation , and the selectivity 
 comes from one isomer being able to adopt that particular conformation , 
 whereas the energy penalty for the other isomer precludes its binding .
in addition to explaining the chiral selectivity for the compounds 
 discussed here , this understanding can be used for the design of chiral 
 molecules that bind to either binding site .
specifically , the prediction 
 would be that replacing the thiazole methyl group with a hydrogen 
 atom would eliminate any steric clash to the chiral methyl group , 
 and hence either isomer could bind to either inhibitor binding site .
we 
 first determined the metabolic stability of 12r in a 
 standard microsomal liver clearance assay .
the nadph - dependent t1/2 of 12r was 37-fold 
 longer than that of our previous lead compound 2 ( table 2 ) .
we then tested compound 12 in mice , 
 using our previously described positron emission tomography ( pet ) 
 assay . whereas our earlier lead compound 2 retained only 25% inhibition of dck activity 4 h 
 after dosing by intraperitoneal injection , compound 12 ( given as the racemic mixture ) exhibited 
 > 50% inhibition of dck activity at this time point ( figure 8a ) .
furthermore , 8 h after treatment with compound 12 , dck inhibition was still above 30% .
we then determined 
 the pharmacokinetic properties of compound 12 to compare 
 with our previous lead compounds 1 and 2 .
as shown in figure 8b , the 
 pharmacokinetic properties of compound 12 were significantly 
 improved relative to the previously published values for compounds 1 and 2 .
collectively , these 
 findings demonstrate that introduction of the chiral linker plus replacement 
 of the thiazole ring propyl substituent by a methyl group yields a 
 dck inhibitor with improved metabolic stability . in vivo evaluation of 
 compound 12 .
( a ) quantification 
 of pet probe , f - l - fac , uptake in the liver of c57bl/6 
 female mice treated with compounds 12 ( 25 mg / kg ) via 
 intraperitoneal injection . dose formulation : 50% peg / tris , ph 7.4 . 

data are mean values  sem for at least n = 
 5 mice / time point .
c57bl/6 female mice were dosed via intraperitoneal injection 
 with 50 mg / kg compound 12 formulated in 50% peg / tris , 
 ph 7.4 .
data are mean values  sem for n = 4 
 mice / time point .
structural 
 and inhibition studies of the compounds discussed here , 
 performed using both the purified recombinant enzyme and a cell - based 
 assay , revealed and rationalized the essential determinants for binding 
 to dck and also guided the type and placement of substituents .
these compounds contain a propyl group at the 
 5-position of the thiazole ring , since , as shown earlier , the propyl 
 substituent provides improved affinity for dck compared to compounds 
 with a methyl group at that position .
unfortunately , this affinity - strengthening 
 propyl group compromised the metabolic stability relative to compounds 
 containing a methyl group at that position .
this forced us to revert 
 to the weaker - binding , but more metabolically stable , scaffold of 
 a methyl group at the thiazole ring .
with the goal of improving metabolic 
 stability , we tested a chiral methylene methyl sulfur linker between 
 the thiazole and pyrimidine moieties .
this linker was found to confer 
 two positive effects : ( 1 ) in terms of affinity for dck , the modified 
 linker compensated for the lack of the thiazole propyl group , and 
 ( 2 ) the compounds exhibited improved metabolic stability .
the interaction 
 of dck with compounds containing this linker is specific to the r - isomer .
this was proven by the dck - inhibitor crystal structure 
 and by comparing the binding affinities of the r versus s enantiomers .
the new lead compound 12r is 
 a promising dck inhibitor , which by perturbing the dntp pools and 
 inducing dna replication stress overload could be used in combination 
 with other drugs to specifically trigger synthetic lethality in cancer 
 cells .
general 
 laboratory reagents were purchased 
 from fisher ( pittsburgh , pa , usa ) and sigma - aldrich ( st . louis , mo , 
 usa ) .
( 800 north five points road , west 
 chester , pa 19380 , usa ) performed the separation of r and s enantiomers .
unless otherwise noted , 
 reactions were carried out in oven - dried glassware under an atmosphere 
 of nitrogen using commercially available anhydrous solvents .
4,6-diamino-2-mercaptopyrimidine 
 was obtained from drying the hydrate over dynamic vacuum at 110 c 
 for 20 h. all other reagents obtained from commercial suppliers were 
 reagent grade and used without further purification unless specified . 

reactions and chromatography fractions were analyzed by thin - layer 
 chromatography ( tlc ) using merck precoated silica gel 60 f254 glass plates ( 250 m ) .
visualization was carried out with 
 ultraviolet light , vanillin stain , permanganate stain , or p - anisaldehyde stain .
flash column chromatography was performed 
 using e. merck silica gel 60 ( 230400 mesh ) with compressed 
 air .
h and c nmr spectra were recorded on 
 a arx500 ( 500 mhz ) , avance 500 ( 500 mhz ) , or avance 300 ( 300 mhz ) 
 spectrometers .
chemical shifts are reported in parts per million ( ppm , 
  ) using the residual solvent peak as the reference .
the coupling 
 constants , j , are reported in hertz ( hz ) , and the 
 resonance patterns are reported with notations as the following : br 
 ( broad ) , s ( singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , and m 
 ( multiplet ) .
electrospray mass spectrometry data were collected with 
 a waters lct premier xe time - of - flight instrument controlled by masslynx 
 4.1 software .
samples were dissolved in methanol and infused using 
 direct loop injection from a waters acquity uplc into the multimode 
 ionization source .
analytical hplc analysis was performed on a knauer 
 smartline 
 hplc system with a phenomenex reverse - phase luna column ( 5 m , 
 4.6 mm  250 mm ) with inline knauer uv ( 254 nm ) detector .
mobile 
 phase : a , 0.1% tfa in h2o ; b , 0.1% tfa in mecn .
percent enantiomeric 
 excess ( % ee ) values were determined via chiral hplc with a chiralpak 
 ia-3/ia polysaccharide - based immobilized type column ( 3 m , 
 4.6 mm  150 mm ) with inline knauer uv ( 310 nm ) detector .
mobile 
 phase : a , 0.1% tfa in hexanes ; b , 0.1% tfa in propanol .
eluent gradient : 
 50% phase a and 50% phase b. chromatograms were collected by a ginastar 
 ( raytest usa , inc . ; wilmington , nc , usa ) analog to digital converter 
 and ginastar software ( raytest usa , inc . ) . to a mixture of 3-ethoxy-4-hydroxybenzonitrile a ( 2.50 g , 15.3 mmol ) in pyridine ( 35 ml ) and triethylamine 
 ( 2.5 ml ) was added ammonium sulfide solution ( 20 wt % in h2o , 15.65 ml , 46.0 mmol ) .
the organic layer was dried over anhydrous 
 na2so4 , concentrated in vacuo , and purified 
 by flash column chromatography over silica gel ( 3:1 ethyl acetate / hexanes ) 
 to yield b ( 2.56 g , 13.0 mmol , 85% ) as a yellow solid .
h nmr ( 300 mhz , cdcl3 )  7.68 ( d , j = 2.1 hz , 1h ) , 7.48 ( br s , 1h ) , 7.28 ( dd , j = 8.5 , 2.1 hz , 1h ) , 7.11 ( br s , 1h ) , 6.89 ( d , j = 8.5 hz , 1h ) , 6.03 ( s , 1h ) , 4.21 ( q , j = 6.9 hz , 
 2h ) , 1.47 ( t , j = 6.9 hz , 3h ) ; c nmr 
 ( 125 mhz , acetone - d6 )  200.5 , 150.3 , 
 145.8 , 131.0 , 121.0 , 114.0 , 112.6 , 64.3 , 14.1 . a mixture of thioamide b ( 1.50 
 g , 7.6 mmol ) and 4-bromopentane-2,3-dione ( 2.04 g , 11.4 mmol ) in ethanol 
 ( 40 ml )
was stirred under refluxing conditions for 4 h. the resulting 
 mixture was cooled and concentrated in vacuo to remove residual solvent . 

the crude residue was purified by flash column chromatography over 
 silica gel ( 10:3 hexanes / ethyl acetate ) to yield the desired thiazole 
 intermediate c ( 2.00 g , 7.2 mmol , 95% ) as a white solid .
h nmr ( 300 mhz , cdcl3 )  7.47 ( d , j = 1.8 hz , 1h ) , 7.35 ( dd , j = 8.2 , 1.8 
 hz , 1h ) , 6.96 ( d , j = 8.1 hz , 1h ) , 5.93 ( s , 1h ) , 
 4.23 ( q , j = 7.2 hz , 2h ) , 2.77 ( s , 3h ) , 2.71 ( s , 
 3h ) , 1.50 ( t , j = 6.9 hz , 3h ) ; c nmr 
 ( 75 mhz , cdcl3 )  196.0 , 162.8 , 148.9 , 148.0 , 146.3 , 
 142.9 , 125.9 , 120.5 , 114.8 , 109.4 , 64.9 , 29.5 , 14.9 , 13.6 . to a solution of thiazole intermediate c ( 1.66 g , 6.0 mmol ) in dmf ( 35 ml ) were added cs2co3 ( 3.13 g , 9.6 mmol ) and 13-chloro-2,5,8,11-tetraoxatridecane 
 ( 2.19 g , 12.0 mmol ) .
after concentration to remove residual solvent , the resulting residue 
 was washed with brine and extracted with ethyl acetate .
the organic 
 layer was washed with water three times , dried over anhydrous na2so4 , and concentrated in vacuo , and the crude residue 
 was purified by flash column chromatography over silica gel ( 1:1 ethyl 
 acetate / hexanes ) to yield desired ketone d ( 2.26 g , 5.3 
 mmol , 89% ) as a white solid .
h nmr ( 500 mhz , cdcl3 )  7.48 ( d , j = 2.0 hz , 1h ) , 7.38 
 ( dd , j = 8.5 , 2.0 hz , 1h ) , 6.94 ( d , j = 8.5 hz , 1h ) , 4.244.20 ( m , 2h ) , 4.17 ( q , j = 7.0 hz , 2h ) , 3.933.89 ( m , 2h ) , 3.793.75 ( m , 2h ) , 
 3.703.63 ( m , 4h ) , 3.573.53 ( m , 2h ) , 3.37 ( s , 3h ) , 
 2.77 ( s , 3h ) , 2.71 ( s , 3h ) , 1.47 ( t , j = 7.0 hz , 
 3h ) ; c nmr ( 125 mhz , cdcl3 )  196.0 , 
 162.5 , 150.8 , 149.4 , 149.0 , 143.1 , 126.9 , 119.8 , 114.0 , 111.4 , 72.1 , 
 71.1 , 70.8 , 70.7 , 69.7 , 69.0 , 64.9 , 59.2 , 29.5 , 15.0 , 13.6 . to a stirred solution of ketone d
( 1.06 g , 2.5 mmol ) in ch2cl2 ( 35 ml ) cooled 
 to 78 c was added slowly diisobutylaluminum hydride 
 ( 1.0 m in thf , 10 mmol , 10 ml ) .
the mixture was allowed to warm to 
 23 c and stirred for 1 h. the mixture was cooled to 0 c 
 and slowly quenched with a saturated aqueous solution of rochelle s 
 salt .
the cloudy solution was stirred for 1 h at 23 c until 
 the solution became clear again .
the resulting solution was extracted 
 with ethyl acetate , washed with brine , dried over anhydrous na2so4 , and concentrated in vacuo to give the desired 
 alcohol e ( 978 mg , 2.3 mmol , 92% ) as a pale yellow solid .
h nmr ( 500 mhz , cdcl3 )  7.44 ( d , j = 2.0 hz , 1h ) , 7.33 ( dd , j = 8.5 , 2.0 
 hz , 1h ) , 6.89 ( d , j = 8.5 hz , 1h ) , 4.91 ( q , j = 6.5 hz , 1h ) , 4.224.17 ( m , 2h ) , 4.13 ( q , j = 7.0 hz , 2h ) , 3.913.86 ( m , 2h ) , 3.763.72 
 ( m , 2h ) , 3.693.61 ( m , 4h ) , 3.553.51 ( m , 2h ) , 3.35 
 ( s , 3h ) , 2.37 ( s , 3h ) , 1.52 ( d , j = 6.0 hz , 3h ) , 
 1.44 ( t , j = 7.0 hz , 3h ) ; c nmr ( 125 
 mhz , cdcl3 )  164.3 , 155.1 , 150.0 , 149.0 , 127.2 , 
 125.8 , 119.3 , 113.8 , 111.0 , 71.8 , 70.8 , 70.6 , 70.4 , 69.5 , 68.7 , 64.6 , 
 64.4 , 58.9 , 24.0 , 14.7 , 10.7 . to a stirred solution of alcohol e ( 425 mg , 1.0 mmol ) in ch2cl2 ( 8 ml ) was added 
 thionyl chloride ( 0.78 ml , 10.0 mmol ) slowly at 0 c .
the mixture 
 was allowed to warm to 23 c and stirred for 1 h. after concentration 
 in vacuo to remove residual solvent , the resulting crude residue was 
 used directly for next step without any further purification because 
 of the instability of chloride f. a mixture of crude chloride f from the previous step , 4,6-diamino-2-mercaptopyrimidine 
 ( 625 mg , 4.0 mmol ) , and k2co3 ( 552 mg , 4.0 mmol ) 
 in dmf ( 7 ml ) was stirred at 70 c for 1 h. the solution was 
 cooled , concentrated in vacuo , and purified by flash column chromatography 
 over silica gel ( 25:1 dichloromethane / methanol ) to give the desired 
 product ( )-9 ( 357 mg , 0.65 mmol , 65% in two steps ) 
 as a white solid .
h nmr ( 500 mhz , cdcl3 )  
 7.49 ( d , j = 2.0 hz , 1h ) , 7.35 ( dd , j = 8.5 , 2.0 hz , 1h ) , 6.90 ( d , j = 8.5 hz , 1h ) , 5.24 
 ( s , 1h ) , 5.02 ( q , j = 7.0 hz , 1h ) , 4.58 ( s , 4h ) , 
 4.224.18 ( m , 2h ) , 4.15 ( q , j = 7.0 hz , 2h ) , 
 3.913.87 ( m , 2h ) , 3.783.75 ( m , 2h ) , 3.693.63 
 ( m , 4h ) , 3.563.53 ( m , 2h ) , 3.37 ( s , 3h ) , 2.50 ( s , 3h ) , 1.81 
 ( d , j = 7.0 hz , 3h ) , 1.46 ( t , j = 
 7.0 hz , 3h ) ; c nmr ( 125 mhz , cdcl3 )  
 170.7 , 163.8 , 163.2 ( 2 ) , 153.3 , 149.9 , 149.1 , 127.9 , 126.8 , 119.4 , 
 114.0 , 111.3 , 80.6 , 71.9 , 70.9 , 70.7 , 70.6 , 69.7 , 68.9 , 64.7 , 59.1 , 
 37.7 , 22.0 , 14.8 , 11.6 ; hrms - esi ( m / z ) [ m + h ] calcd for c25h35n5o5s2h , 550.2158 ; found 550.2169 . to a mixture of 3-hydroxy-4-methoxybenzonitrile a ( 3.00 g , 20.11 mmol ) in pyridine ( 30 ml ) and triethylamine 
 ( 3 ml )
was added ammonium sulfide solution ( 20 wt % in h2o , 20.7 ml , 60.3 mmol ) .
the organic layer was dried over anhydrous 
 na2so4 , concentrated in vacuo , and purified 
 by flash column chromatography over silica gel ( 3:1 ethyl acetate / hexanes ) 
 to yield b ( 3.13 g , 17.1 mmol , 85% ) as a yellow solid .
h nmr ( 500 mhz , acetone - d6 )  
 8.77 ( br s , 1h ) , 8.65 ( br s , 1h ) , 7.85 ( s , 1h ) , 7.59 ( d , j = 2.5 hz , 1h ) , 7.56 ( dd , j = 8.5 , 2.3 hz , 1h ) , 
 6.94 ( d , j = 8.5 hz , 1h ) , 3.88 ( s , 3h ) ; c nmr ( 125 mhz , acetone - d6 )  200.7 , 
 150.5 , 145.7 , 132.4 , 119.5 , 114.8 , 110.2 , 55.5 . a mixture of thioamide b ( 2.75 
 g , 15.0 mmol ) and 4-bromopentane-2,3-dione ( 4.03 g , 22.5 mmol ) in 
 ethanol ( 70 ml ) was stirred under refluxing conditions for 4 h. the 
 resulting mixture was cooled and concentrated in vacuo to remove residual 
 solvent .
the crude residue was purified by flash column chromatography 
 over silica gel ( 10:3 hexanes / ethyl acetate ) to yield the desired 
 thiazole intermediate c ( 3.79 g , 14.4 mmol , 96% ) as a 
 white solid .
h nmr ( 500 mhz , dmso - d6 )  9.53 ( br s , 1h ) , 7.34 ( d , j = 2.0 
 hz , 1h ) , 7.26 ( dd , j = 8.5 , 2.0 hz , 1h ) , 6.98 ( d , j = 8.5 hz , 1h ) , 3.80 ( s , 3h ) , 2.66 ( s , 3h ) , 2.57 ( s , 3h ) ; c nmr ( 125 mhz , dmso - d6 )  195.2 , 
 162.5 , 150.1 , 148.5 , 147.1 , 142.7 , 125.6 , 118.2 , 112.9 , 112.5 , 55.9 , 
 29.4 , 13.2 . to a solution of thiazole intermediate c ( 1.58 g , 6.0 mmol ) in dmf ( 35 ml ) were added cs2co3 ( 3.13 g , 9.6 mmol ) and n-(2-bromoethyl)methanesulfonamide 
 ( 2.18 g , 10.8 mmol ) .
after concentration to remove residual solvent , the resulting residue 
 was washed with brine and extracted with ethyl acetate . the organic 
 layer was washed with water three times , dried over anhydrous na2so4 , and concentrated in vacuo , and the crude residue 
 was purified by flash column chromatography over silica gel ( 3:2 ethyl 
 acetate / hexanes ) to yield desired ketone d ( 1.89 g , 4.9 
 mmol , 82% ) as a white solid .
h nmr ( 500 mhz , cdcl3 )  8.00 ( s , 1h ) , 7.51 ( d , j = 2.0 
 hz , 1h ) , 7.46 ( dd , j = 8.5 , 2.0 hz , 1h ) , 6.92 ( d , j = 8.5 hz , 1h ) , 4.254.20 ( m , 2h ) , 3.90 ( s , 3h ) , 
 3.603.55 ( m , 2h ) , 3.03 ( s , 3h ) , 2.76 ( s , 3h ) , 2.70 ( s , 3h ) ; c nmr ( 125 mhz , cdcl3 )  195.8 , 162.5 , 151.5 , 
 148.9 , 147.8 , 143.1 , 126.4 , 121.1 , 112.4 , 111.7 , 69.1 , 55.9 , 42.7 , 
 40.6 , 29.4 , 13.4 . to a stirred solution of
( r)-(+)-2-methyl - cbs - oxazaborolidine ( 6.7 ml of a 1.0 m solution in 
 toluene , 6.7 mmol ) in thf ( 26 ml ) at 78 c under ar was 
 added borane  tetrahydrofuran complex ( 4.4 ml of a 1.0 m solution 
 in thf , 4.4 mmol ) followed by a solution of d ( 284 mg , 
 0.74 mmol ) in thf ( 14 ml ) .
after addition of the d solution 
 with syringe pump for 6 h , the reaction mixture was stirred for another 
 20 min at 78 c .
h2o ( 10 ml ) and meoh ( 5 ml ) 
 were added , and the mixture was allowed to warm to room temperature . 

after concentration to remove residual solvent , the resulting residue 
 was washed with brine and extracted with ethyl acetate .
the organic 
 layer was washed with water three times , dried over anhydrous na2so4 , and concentrated in vacuo , and the crude residue 
 was purified by flash column chromatography twice over silica gel 
 with 3:2 ethyl acetate / hexanes and 40:1 dichloromethane / methanol as 
 washing system separately to yield alcohol e ( 221 mg 
 0.57 mmol , 77% , 96% ee ) as a white solid .
h nmr ( 500 mhz , 
 acetone - d6 )  7.57 ( d , j = 2.0 hz , 1h ) , 7.46 ( dd , j = 8.5 , 2.0 hz , 1h ) , 
 7.05 ( d , j = 8.5 hz , 1h ) , 6.26 ( br s , 1h ) , 5.024.95 
 ( m , 1h ) , 4.21 ( t , j = 5.5 hz , 2h ) , 3.88 ( s , 3h ) , 
 3.57 ( dt , j = 5.5 , 5.5 hz , 2h ) , 3.04 ( s , 3h ) , 2.48 
 ( s , 3h ) , 1.50 ( d , j = 6.0 hz , 3h ) ; c 
 nmr ( 125 mhz , acetone - d6 )  162.9 , 
 156.1 , 151.3 , 148.4 , 127.1 , 126.8 , 119.7 , 112.1 , 111.4 , 68.6 , 64.1 , 
 55.3 , 42.6 , 39.6 , 23.0 , 10.0 . to a stirred solution 
 of alcohol e ( 221 mg , 0.57 mmol ) in ch2cl2
( 13 ml ) was added trifluoroacetic anhydride ( 0.66 ml , 2.9 
 mmol ) slowly at 0 c . after being stirred at 0 c for 30 
 min ,
the mixture was allowed to warm to 23 c and stirred for 
 another 30 min .
after concentration in vacuo to remove residual solvent , 
 the resulting crude residue was used directly for next step without 
 any further purification because of the instability of the desired 
 trifluoroacetate f. a mixture of crude chloride f from the previous step and 4,6-diamino-2-mercaptopyrimidine ( 112 
 mg , 0.86 mmol ) in dmf ( 5 ml ) was stirred at 80 c for 1 h. the 
 solution was cooled , concentrated in vacuo , and purified by flash 
 column chromatography over silica gel ( 25:1 dichloromethane / methanol ) 
 to give the couple of enantiomers 12r and 12s ( 178 mg , 0.35 mmol , 40% ee of 12r , 61% total yield 
 in two steps ) as a white solid .
recrystallization of the enantiomers 
 with meoh / acetone solvent system gave the 12r with > 93% 
 ee .
h nmr ( 500 mhz , acetone - d6 )  7.55 ( d , j = 2.0 hz , 1h ) , 7.48 ( dd , j = 8.5 , 2.0 hz , 1h ) , 7.06 ( d , j = 8.5 
 hz , 1h ) , 6.26 ( br s , 1h ) , 5.605.55 ( m , 4h ) , 5.37 ( s , 1h ) , 
 5.30 ( q , j = 7.0 hz , 1h ) , 4.23 ( t , j = 5.5 hz , 2 h ) , 3.89 ( s , 3h ) , 3.58 ( dt , j = 5.5 , 
 5.5 hz , 2h ) , 3.05 ( s , 3h ) , 2.52 ( s , 3h ) , 1.74 ( d , j = 7.0 hz , 3h ) ; c nmr ( 125 mhz , dmso - d6 )  168.0 , 163.5 ( 2 ) , 162.9 , 153.6 , 150.6 , 147.8 , 
 126.6 , 126.2 , 119.5 , 112.3 , 110.4 , 79.0 , 67.9 , 55.7 , 41.9 , 36.1 , 30.7 , 
 22.2 , 11.2 ; hrms - esi ( m / z )
[ m + 
 h ] calcd for c20h26n6o4s3h , 511.1256 ; found 511.1259 ; 12r [ ]d + 340.0 ( c 0.12 , 
 acetone ) ( 93% ee ) .
, we used the s74e - c4s - dck variant , which 
 is the human dck protein where four solvent - exposed cysteines are 
 mutated into serines ( c4s ) .
we showed that the c4s mutant generates 
 better quality crystals without altering the three - dimensional conformation 
 of the enzyme or its enzymatic activity .
additionally , the enzyme contained the mutation of ser74 to glutamic 
 acid ( s74e ) ; this mutation serves to mimic the phosphorylated state 
 of this residue .
when we refer to dck in this report , we mean the 
 c4s - s74e - dck variant .
dck was expressed in escherichia coli bl21 c41(de3 ) cells using a pet-14b vector ; the cells were grown 
 in 2xyt medium and induced with 0.1 mm iptg for 4 h at 310 k. the 
 cells were harvested , and the pellet was lysed by sonication .
the 
 lysate was cleared by centrifugation at 30  000 rev / min for 
 1 h at 277 k , and the supernatant was loaded onto a 5 ml histrap nickel - affinity 
 column ( ge healthcare ) .
the column was washed with 300 ml of a buffer 
 composed of 25 mm tris - hcl , ph 7.5 , 500 mm nacl , 30 mm imidazole . 

the bound protein was eluted with the same buffer but containing 250 
 mm imidazole and was further purified by gel filtration using an s-200 
 column in a buffer consisting of 25 mm hepes , ph 7.5 , 200 mm sodium 
 citrate , 2 mm edta , 3 mm dtt .
the protein fractions were pooled , concentrated , 
 aliquoted , flash - frozen in liquid nitrogen , and stored at 193 k until 
 use .
the phosphorylation activity of dck was 
 determined using a spectroscopic nadh - dependent enzyme - coupled assay .
all measurements were taken in triplicate at 310 k in a buffer consisting 
 of 100 mm tris , ph 7.5 , 200 mm kcl , 5 mm mgcl2 , 0.5 mm 
 edta , 0.8 mm phosphoenolpyruvate , 0.4 mm nadh with 50 nm dck , and 
 1 mm atp . ic50 and ki were determined as described by us , and all data were fitted using the kaleidagraph 
 software .
these were performed 
 in ccrf - cem acute lymphoblastic leukemia cells as previously described .
pet studies to determine % inhibition of 
 dck activity in vivo were performed as previously described .
these assays were 
 performed by cyprotex ( watertown , ma ) according to standard operating 
 protocols .
briefly , c57bl/6 female mice were treated 
 with the dck inhibitors via intraperitoneal injection .
the drugs were 
 administered in 50% polyethylene glycol ( peg 400)/50 mm tris - hcl , 
 ph 7.5 .
five minutes after drug injection , whole blood ( 75 
 l ) was obtained at various time points from the retro - orbital 
 sinus using hematocrit capillary tubes .
g for 5 min , and the supernatant ( 5 
 l ) was transferred into a clean tube .
calibration standards 
 were prepared by spiking various amounts of 11 and 12 in 5 l of supernatant from the plasma of untreated 
 mice to obtain final concentrations between 0.001 to 100 pmol/l . 

samples and the calibration standards were mixed with 500 l 
 ice - cold acetonitrile / water ( 50/50 , v / v ) containing an internal standard 
 ( 1 ) .
the residue was reconstituted in 100 l 
 of acetonitrile / water ( 50/50 , v / v ) .
samples ( 5 l ) were injected 
 onto a reverse phase column ( agilent zorbax rapid resolution high 
 definition eclipse plus c18 , 2.1 mm  50 mm , 1.8 m ) equilibrated 
 in water acetonitrile / formic acid , 95/5/0.1 , and eluted ( 200 l / min ) 
 with an increasing concentration of solvent b ( acetonitrile / formic 
 acid 100/0.1 , v / v : min/% acetonitrile ; 0/5 , 2/5 , 8/80 , 9/80 , 10/5 , 
 12/5 ) .
the effluent from the column was directed to an electrospray 
 ion source ( agilent jet stream ) connected to a triple quadrupole mass 
 spectrometer ( agilent 6460 qqq ) operating in the positive ion mrm 
 mode .
the ion transitions for 1 , 11 , and 12 are 476.2334.5 , 550.2408.2 , and 511.1369.1 
 respectively .
the peak areas for 11 and 12 were normalized to the peak area of the internal standard , and the 
 plasma concentrations were computed using the standard curves generated 
 by calibration standards spiked in plasma from untreated mice .
approximated 
 values of the area under the curve ( auc ) , half - life ( t1/2 ) , maximum concentration in the plasma ( cmax ) , and time to reach the maximum concentration ( tmax ) were calculated using boomer / multi - forte 
 pk functions from microsoft excel .
crystals of human dck 
 in complex with inhibitors and udp were grown 
 at 285 k using the hanging - drop vapor - diffusion method .
all dck - inhibitor 
 complexes were prepared as follows : 1 l of dck protein at 1017 
 mg / ml in complex with a 2.5-fold molar excess of inhibitor , and 2 
 mm udp and 5 mm mgcl2 were mixed with 1 l of reservoir 
 buffer solution .
the reservoir solution consisted of 0.91.5 
 m trisodium citrate dehydrate and 25 mm hepes , ph 7.5 .
prior to data 
 collection , crystals were soaked in mineral oil for cryoprotection . 

diffraction data for dck in complex with compounds 48 were collected on the life sciences collaborative access 
 team ( ls - cat ) beamline 21-id - g .
data for all other complexes ( compounds 912 ) were collected using the in - house 
 x - ray source ( rigaku ru-200 rotating anode ) with a r - axis iv++ image 
 plate detector .
structures were determined by molecular replacement 
 with molrep using the dck structure ( pdb 
 entry 4jln  ) as a search model .
refinement was conducted 
 using refmac , and model building was 
 conducted using coot .
all data sets were perfectly twinned , and iterative 
 refinements were carried out using refmac with the twin option active . 

structural figures were prepared using the pymol 
 molecular graphics system ( version 1.6.0 , schrdinger ) .
the s - isomer in position 
 1 and the r - isomer in position 2 were generated by 
 flipping the chirality of the linker carbon using maestro , version 
 9.1 , schrdinger , llc , 2010 .
this program was also used to 
 generate the torsion scans around the bond connecting the chiral linker 
 carbon and the thiazole ring ( torsion angle defined by cac  cbc 
equilibration simulations were performed using the mcpro 2.0 software 
 package with the opls - aa force field .
the protein 
 backbone and all bond lengths within the protein were held fixed . 

angles and torsions within 11  of the center of the bound molecule 
 were allowed to vary .
equilibration began with 5  10 configurations 
 of solvent - only moves , followed by 10  10 configurations 
 in which the protein and bound molecule were sampled , with additional 
 solvent sampling at every tenth configuration .
equilibrations were 
 performed using metropolis monte carlo in the npt ensemble at 1 atm and 25 c . for the unbound structures ,
implicit solvent was simulated with 
 the generalized born / surface area ( gb / sa ) method .
 recently , 
 we have shown that small molecule dck inhibitors in combination 
 with pharmacological perturbations of de novo dntp biosynthetic pathways 
 could eliminate acute lymphoblastic leukemia cells in animal models . 
 however , our previous lead compound had a short half - life in vivo . 
 therefore , we set out to develop dck inhibitors with favorable pharmacokinetic 
 properties . 
 we delineated the sites of the inhibitor for modification , 
 guided by crystal structures of dck in complex with the lead compound 
 and with derivatives . 
 crystal structure of the complex between dck 
 and the racemic mixture of our new lead compound indicated that the r - isomer is responsible for kinase inhibition . 
 this was 
 corroborated by kinetic analysis of the purified enantiomers , which 
 showed that the r - isomer has > 60-fold higher affinity 
 than the s - isomer for dck . 
 this new lead compound 
 has significantly improved metabolic stability , making it a prime 
 candidate for dck - inhibitor based therapies against hematological 
 malignancies and , potentially , other cancers . 
Introduction
Results and Discussion
Conclusion
Experimental Section