the heat shock protein 70 ( hsp70 ) is a 
 molecular chaperone which 
 plays an important function in protein homeostasis as well as in cell 
 signaling and survival .
some of its functions include 
 folding newly synthesized peptides , refolding misfolded proteins , 
 assembling multiprotein complexes , and transporting proteins across 
 cellular membranes .
in addition to these housekeeping functions , hsp70 
 is an important regulator of malignant transformation , both through 
 its role as a powerful antiapoptotic protein and as a cochaperone 
 of heat shock protein 90 ( hsp90 ) . as a cochaperone of hsp90 , 
 hsp70
is thought to load client proteins onto the hsp90 machinery 
 through the action of another cochaperone , heat shock organizing protein 
 ( hop ) .
the hsp90 machinery is an important mechanism 
 by which cancer cells regulate the function of several cancer - driving 
 proteins , such as those involved in altered signaling , the cell cycle , 
 and transcriptional regulation .
indeed , it is primarily for this reason 
 that hsp90 has been actively pursued as an anticancer target . as an antiapoptotic molecule , hsp70 acts at multiple points in the 
 apoptotic pathway to prevent cell death . due to these functions , 

it is not surprising that hsp70 is frequently overexpressed in cancer , 
 where the elevated expression is furthermore believed to be a cause 
 of or to lead to resistance to chemotherapy and other treatments .
these dual roles of hsp70 in cancer , i.e. , cochaperone 
 of hsp90 and antiapoptotic molecule , suggest that inhibition of hsp70 
 may offer a valuable anticancer strategy , as supported by hsp70 knockdown 
 studies .
indeed , hsp70 is an important 
 and highly sought after cancer target , and as such it is of no surprise that the discovery 
 and development of hsp70 inhibitors is currently a hot topic .
to identify druglike hsp70 inhibitors , we took a structure - based 
 approach . in the first preceding paper in this issue , we described 
 the development of inhibitors that target an allosteric pocket of 
 hsp70 located in the n - terminal domain of the protein .
this pocket , not evident in the available crystal 
 structures of hsp70 , has been recently identified by us through computational 
 analyses .
thus , in the absence of an 
 appropriate x - ray structure of human hsp70 , we used this homology 
 model to design ligands that could bind to the hsp70 allosteric pocket . 
 because the pocket also harbors a potentially reactive cysteine residue , 
 the initially designed inhibitors , all built around the 2,5-thiodipyrimidine 
 and 5-(phenylthio)pyrimidine scaffolds , also incorporated an acrylamide 
 moiety suitably positioned to interact with this amino acid upon insertion 
 into the binding site ( figure 1 ) .
this body 
 of work led to the identification of low micromolar inhibitors of 
 hsp70 with a good cell permeability profile and potent and selective 
 biological activity in several cancer cells through an hsp70-mediated 
 mechanism of action .
our data indicated a good fit for these molecules 
 inside the hsp70 pocket , suggesting that enthalpy played an important 
 role in their interaction with the protein .
chemical 
 structure of acrylamide - containing 2,5-thiodipyrimidine 
 and 5-(phenylthio)pyrimidine scaffold hsp70 inhibitors that were designed 
 to insert into the hsp70 allosteric pocket and form a covalent bond 
 with cys267 upon binding .
the yellow surface shows the geometry of 
 the allosteric pocket as determined by sitemap ( schrodinger llc , new 
 york ) .
in addition to being good leads , 
 these compounds were also useful 
 in demonstrating the therapeutic relevance of inhibiting the novel 
 allosteric hsp70 pocket as a potential anticancer approach .
specifically , by inserting into the allosteric 
 pocket , these inhibitors alter the oncogenic hsp70hsp90client 
 complexes , resulting in degradation of hsp90hsp70onco - client 
 proteins and inhibition of cell growth and induction of apoptosis . 

they do so without activating a feedback heat shock response , a mechanism believed to be responsible for limiting 
 the anticancer activity of hsp90 inhibitors .
the hsp90hsp70 machinery is also a known repressor of heat 
 shock factor 1 ( hsf-1 ) .
inhibition of 
 hsp90 , but not depletion of the hsp90 cochaperones hsp70 , hop , hip , 
 p23 , and cyp40 , led to hsf-1 activation , possibly because while these 
 cochaperones participate with hsp90 in the regulation of hsf-1 , only 
 hsp90 plays a nonredundant role in repressing its heat shock activation 
 ability .
activation of hsf-1 has a protective 
 effect on the cancer cell as it leads to the upregulation of antiapoptotic 
 molecules .
thus , hsp70 allosteric inhibitors 
 by differentiating between the regulatory activity of the hsp90hsp70 
 machinery on onco - clients and on hsf-1 may result in more robust apoptosis 
 in cancer cells when compared to hsp90 inhibitors . here
we focus 
 on the identification of inhibitors that act on hsp70 
 through reversible binding into the allosteric pocket .
although covalent 
 inhibition may offer certain advantages ( i.e. , maintaining activity 
 against mutations and the ability to inhibit the target in the presence 
 of millimolar concentrations of atp within the cell ) and can be desirable 
 in some instances , it may come with some drawbacks .
because of the presence of a reactive moiety , covalent 
 inhibitors have a greater potential for off - target effects , such as 
 inherent reactivity toward nonspecific thiols such as glutathione . therefore , we desired molecules that could also 
 act in this pocket in a reversible manner . at this early point in 
 hsp70 drug discovery ,
it is not clear which mode is best ; therefore , 
 we thought it wise to pursue both , and in this paper we show our initial 
 efforts in the elaboration of a covalent inhibitor targeting the allosteric 
 site into a reversible noncompetitive inhibitor of low micromolar 
 potency .
the design of reversible ligands for the allosteric 
 hsp70 pocket 
 took advantage of our knowledge on the geometry of the allosteric 
 pocket and the structure 
activity 
 relationship ( sar ) we have learned from previous studies in the irreversible 
 ligand series ( figure 2 ) .
our tactic was to first take a localized approach in an 
 attempt to phase out the covalent binding potential and to maximize 
 noncovalent binding in this immediate region ( modification on x4 ) .
then we took a global molecular perspective to probe and 
 to enhance interaction in other parts of the molecule ( modification 
 on x3 , rings a and b , x5 , and x6 ) . 

specifically , our modifications focused on replacing the acrylamide 
 ( x4 ) with a favorable noncovalent modifying functionality 
 and on altering the nature of rings a and b ( figure 2a , highlighted in blue ) .
they then aimed to gain additional 
 favorable enthalpy by filling out a hydrophobic pocket now only occupied 
 by small x5,6 groups such as methoxy ( figure 2a , highlighted in blue , and figure 2b , red circle ) .
favorable modifications , such as methylpiperazine 
 at position x7 , were left mostly unchanged ( figure 2a ) .
design of the reversible hsp70 inhibitors and their proposed 
 mode 
 of interaction with the hsp70 pocket .
( a ) general strategy that indicates 
 modifications that are explored here in the design of reversible inhibitors 
 ( highlighted in blue ) .
( b ) step - by - step process that highlights key 
 modifications that led to 27c , a reversible hsp70 binder 
 of activity rivaling that of the most potent irreversible inhibitor 1e .
thus , our overall design 
 strategy entailed ( 1 ) determination of 
 the extent of dependence of covalent modification for activity by 
 replacing the acrylamide with groups that could provide favorable 
 but noncovalent interaction with cysteine , ( 2 ) exploration of the 
 sar to maximize noncovalent interactions within the pocket occupied 
 by acrylamide , and ( 3 ) exploration of the sar in other parts of the 
 molecule to maximize binding affinity .
modifying the acrylamide 
 was based on several observations . as mentioned above , our modeling 
 studies indicated that the region of hsp70 occupied by x4 also contained leu237 , val238 , arg264 , asp234 , and lys271 in addition 
 to the reactive cys267 ( figure 2a ) . while the 
 ability to form a covalent bond with the cysteine added significantly 
 to the potency of the irreversible inhibitors ,
modeling studies indicated 
 that the alkene moiety of the acrylamide group was positioned to also 
 form hydrophobic interactions with adjacent protein residues , including 
 leu237 and val238 , while its carbonyl was poised to form electrostatic 
 interactions with arg264 ( figure 2a ) .
therefore , 
 we reasoned that suitable groups substituted at x4 would 
 be capable of interacting with these residues in a wholly noncovalent 
 manner . in our initial design of reversible ligands
, we kept the amide 
 functionality of x4 but modified the alkene with a variety 
 of groups ( r , figure 2a ) incapable of interacting 
 with cysteine covalently .
these included alkyl groups of various sizes 
 as well as aryl groups ( r = methyl , ethyl , cyclopropyl , cyclobutyl , 
 cyclohexyl , n - heptyl , allyl , phenyl , and furanyl ) 
 to probe the extent of modifications allowed by the pocket at this 
 position .
we also replaced the acrylamide with substituents that could 
 potentially establish an ionic pair interaction with the s of cys267 ( i.e. , ionizable amines such as in 7f ( r 
 = aminomethyl ) and 9 ( r = 2-(dimethylamino)ethyl ) ) ( tables 1 and 2 ) . inhibition of growth measured in 
 kasumi-1 acute myeloid leukemia cells .
ring b 
 is predicted to occupy the lower part 
 of the binding site that also contains arg264 .
thus , the ligand s 
 interaction with the pocket could potentially be stabilized by cation 
 interactions between the aromatic ring of ring b and the guanidine 
 group of arginine ( figure 2a ) .
cation interactions play an important role 
 in protein structure and molecular recognition and can contribute significantly 
 to the binding energy of a ligand .
such 
 interactions are more likely to occur between an electron - rich  
 system ( i.e. , phenyl , ethylene , acetylene ) and a neighboring cation 
 and furthermore are generally favored with heterocycles that incorporate 
 lone pair electrons into the aromatic system ( i.e. , indole , pyrrole ) 
 and become deactivated when the lone pair does not contribute to aromaticity 
 ( i.e. , pyridine , pyrimidine ) . in the 
 irreversible series , pyrimidine was favored for ring b ; the two nitrogen 
 atoms of the pyrimidine ring rendered the ring more electron deficient , 
 thus activating the acrylamide s michael acceptor capability . in the reversible series , on the other hand , 
 electron - rich b rings should be favored over electron - deficient ones 
 because of their higher propensity toward cation interactions 
 with arg264 ( figure 2a ) . to test the hypothesis , 
 we made compounds in which ring b was either phenyl ( i.e. , activating ) 
 or pyrimidine ( i.e. , deactivating ) ( tables 1 and 2 ) . ring a and 
 its substituents x5 and x6 are placed midpocket . 

our initial investigations have mainly explored small substituents 
 at this position such as methoxy , ethoxy , and methyl .
it is lined by amino acids that could provide 
 hydrophobic ( val59 , tyr41 , phe68 , trp90 ) and electrostatic ( arg261 ) 
 interactions with the ligand ( figure 2a ) .
therefore , 
 we explored here the effects of larger substituents on ring a , especially 
 those poised for interactions within the lipophilic pocket formed 
 by tyr41 , phe68 , and trp90 , such as derivatives in which x5 contains an aryl ( red circle in figure 2b 
 and table 3 ) .
we also disrupted the symmetry 
 of these molecules around ring a by probing monosubstitution of the 
 ring ( x5  x6 = h , figure 2b ) .
ring b orients its x3 substituent 
 toward several important pocket residues such 
 as asp234 and glu268 ( x3 is nh2 in 1a , figure 2a ) .
nh2 at this position 
 is poised to form hydrogen bonds with their carboxylate groups , and 
 we retained this functionality from the irreversible series in the 
 design of new derivatives .
the introduction of an nh2 at this position was synthetically challenging when ring b was phenyl , 
 and for these , hydrogen was introduced at x3 .
thus , all 
 compounds of this series contain either an nh2 or a h at 
 position x3 .
our previous 
 sar also showed 
 that n - methylpiperazine was a preferred substituent 
 on ring a at position x7 , presumably because of a proper 
 fit in the minor groove located at the exit of the binding site and 
 because it could form a hydrogen bond interaction with the backbone 
 carbonyl of his89 ( figure 2a for 1a ) . as a result ,
the 
 synthesis of all designed compounds evaluated in this study 
 is shown in schemes 13 .
target compounds 3a  d and 4a  e were prepared by reaction of 2a  d with the appropriate acid chloride 
 ( scheme 1 ) .
similarly , 
 target compounds 7a  h were prepared 
 by reaction of 6a , b with the appropriate 
 acid chloride ( scheme 1 ) .
thioethers 6a and 6b were prepared by cui / neocuproine - catalyzed 
 coupling of iodo derivative 5a or 5b , respectively , 
 with 3-aminothiophenol ( scheme 1 ) .
9 was prepared by michael addition of dimethylamine to 8 with dbu ( scheme 1 ) .
bromopyridine 10 was reacted with n - methylpiperazine 
 at 130 c for 16 h to give 11 in 88% yield .
12 was obtained in 95% yield 
 following reaction of 11 with nis at rt .
12 was coupled to 4-amino-2-mercaptopyrimidine with cui / neocuproine 
 to give thioether 13 in 60% yield .
acylation of 13 with propionyl chloride or cyclopropanecarbonyl chloride 
 resulted in 14a or 14b , respectively .
coupling 
 of 12 with 3-aminothiophenol resulted in 15 , which was acylated with various acid chlorides to give target compounds 16a  d .
17 was obtained from 
 demethylation of 11 by refluxing in 48% hbr(aq ) along 
 with a catalytic amount of tetrabutylammonium bromide .
following pmb 
 protection and reaction with nis , iodo derivative 19 was 
 obtained , which was coupled with 3-aminothiophenol to give thioether 20 .
reaction of 20 with boc - glycine and subsequent 
 deprotection with tfa resulted in 21 .
first , 2,4-dichloropyrimidine ( 22 ) was reacted with 
 benzyl alcohol under ptc conditions to result in a regioisomeric mixture 
 of the desired 4-(benzyloxy)-2-chloropyrimidine ( 23a ) 
 along with 2-(benzyloxy)-4-chloropyrimidine in a ratio of 75:25 , respectively . 

this mixture was used directly in the next step and reacted with n - methylpiperazine to give 24a , which was obtained 
 pure following chromatography .
after iodination with nis to yield 25a and further coupling with 3-aminobenzenethiol , 26a was obtained .
27a  f was then obtained 
 by reaction of the appropriate acid chloride with 26a .
phenyl - substituted analogue 27 g was obtained by a 
 similar route used for compounds 27a  f ; however , the first step in the synthesis utilized a suzuki coupling 
 to install a phenyl group selectively onto the 4-position of 22 to yield 23b ( scheme 3 ) .
our target - derived biological 
 investigation was based on a specific 
 battery of assays that we designed to probe hsp70-derived mechanisms 
 in a cancer cell .
these include biochemical 
 and phenotypic assays that probe biological effects that stem from 
 alteration of the hsp70hop 
these biological 
 tests were paralleled by computational studies , and each derivative 
 was docked into the allosteric pocket of hsp70 to understand the contribution 
 of each chemical modification to the observed biological activity .
our first step toward the potential realization of reversible inhibitors 
 involved removing the reactive acrylamide moiety .
rewardingly , elimination 
 of the covalent mode of binding by isosteric replacement of the acrylamide 
 to ethylamide diminished the biological activity by only 1 log ( i.e. , 1a vs 3a , 1b vs 4b , 1c vs 7a , and 1d vs 4c , tables 1 and 4 ) while 
 retaining an hsp70-driven mechanism of action ( table 1 and figure 3 ) . specifically , when 
 cancer cells were incubated with select derivatives ( i.e. , 1a and 3a , acrylamide- and ethylamide - containing derivatives , 
 respectively ) , we observed dissociation of hsp70hop complexes 
 at concentrations at which we also noted degradation of the hsp70 
 onco - client protein kinase , her2 ( figure 3 ) . 

as previously mentioned , hop is a cochaperone that bridges hsp70 and 
 hsp90 to form the megachaperone complex that regulates the stability 
 of several onco - client proteins , such as her2 and raf-1 .
when these 
 complexes become disrupted , the client proteins ( i.e. , her2 and raf-1 ) 
 become unstable and are cleared through the proteasome pathway . as mentioned above
, the pocket occupied by the 
 x4 substituent contains leu237 , val238 , and cys267 , and 
 the ethyl group of 3a would be well positioned to form 
 hydrophobic interactions with these residues .
her2 and raf-1 steady - state levels , 
 parp cleavage and inhibition of growth measured in skbr3 breast cancer 
 cells .
binding to skbr3 
 cell extracts tested 
 at the maximum concentration allowed by solubility .
skbr3 cells were treated for 
 24 h with vehicle 
 or indicated concentrations of 1a or 3a . 

dissociation : upon cell lysing , hsp70 complexes were 
 isolated with an anti - hsp70 antibody ( ip bb70 ) and analyzed by western 
 blotting ( wb ) .
her2 degradation : proteins in the lysate were analyzed by immonoblotting 
 with an anti - her2 antibody .
gels were quantified by densitometry , values normalized 
 to the control ( vehicle only treated cells ) , and data graphed against 
 the hsp70 inhibitor concentration .
we wanted to explore the further potential of increasing 
 potency 
 by exploring this pocket with r groups of various sizes and to take 
 advantage of potential hydrophobic interactions within the pocket . 
 substituting ethyl with methyl ( i.e. , 3c and 4a ) diminished activity , while cyclopropyl at this position slightly 
 increased activity ( i.e. , 4c vs 4e and 3a vs 7b , table 1 , x1,2 = n ) .
substitution of ethyl with groups such as allyl or 
 cyclobutyl ( i.e. , 7a vs 7 g and 7c ) led to no significant change in activity , suggesting that such 
 groups can be accommodated in this site , but that no additional favorable 
 interactions were possible .
much larger hydrophobic substituents at 
 this position abolished both activity ( i.e. , n - heptyl 
 in 3d , steric clashes with asn235 , leu237 , val238 , and 
 val260 ) and solubility ( i.e. , cyclohexyl 
 in 7d ) , which in the latter case precluded biological 
 evaluation .
we also found that flat , conformationally restricted 
 aromatic groups 
 were well tolerated at this position and in fact significantly improved 
 activity . specifically , the 2-furanyl derivative ( i.e. , 4d ) was 23-fold more potent than the corresponding ethyl and 
 cyclopropyl derivatives ( i.e. , 4c and 4e , respectively ) and only 5-fold less potent than the corresponding 
 acrylamide derivative ( i.e. , 1d ) ( table 1 ) .
even more favored was a phenyl at this position ( i.e. , 7e ) , this compound being 810-fold more active than 
 the corresponding ethyl derivative ( i.e. , 7a ) ( tables 1 and 4 ) .
such an increase 
 in potency for hydrophobic and -electron - containing r groups 
 may be explained by enhanced hydrophobic interactions with leu237 , 
 val238 , leu263 , and phe241 or a potential s interaction 
 between the sulfur of cys267 and the r group .
such favorable interactions 
 between sulfur and  aromatic systems were first suggested by 
 morgan et al . , and a number of stable 
 orientations are possible , including one where the sulfur is positioned 
 3.54.0  above the plane of the aromatic ring and another 
 where the sulfur is positioned slightly above ( 2.5  ) 
 and to the edge ( 4.56.0  ) of the aromatic ring .
both orientations have been found to occur with 
 cysteine residues in proteins , and recently a s 
h/ interaction 
 was proposed to rationalize the binding affinity of an flt3 kinase 
 inhibitor .
our second approach 
 toward enhancing activity within the pocket 
 was to take advantage of the native cysteine residue and its potential 
 to form ionic interactions through its side chain thiolate ( s ) .
we attempted to take advantage of such an interaction 
 by incorporating an amine group at the same position as the -carbon 
 of the michael acceptor acrylamide in 1a .
an increase in potency was observed for r substituents that could 
 potentially be positively ionized and form an ionic pair with s of cysteine ( 2-aminomethyl in 7f and 16d ) .
indeed , 2-aminomethyl appears to favorably position 
 its amine , which is likely protonated at physiological ph , to interact 
 with the sulfur of cysteine ( i.e. , 7f in table 1 and 16d in table 2 ) .
these derivatives have an activity that is comparable to 
 that of the corresponding phenyl and/or furanyl compounds ( 7f vs 7e and 4d , table 1 , and 16d vs 16c , table 2 ) .
positioning the amine one carbon away , such as in 9 ( r = 2-(dimethylamino)ethyl , table 1 ) , resulted in activity comparable to that of 7f in 
 the growth inhibition and caspase-3,7 activation assays . in this case
, 9 has the potential to undergo bioconversion in cells via 
 -elimination to the corresponding alkene , and therefore , in addition to possible interactions described 
 above , it may also interact with cys267 covalently .
having determined 
 these two potential strategies that replace the 
 acrylamide with minimal loss of activity , i.e. , a -electron- 
 or positively ionizable functionality - containing r group , we went 
 on to explore other sites on the molecule that would provide a gain 
 in binding affinity .
as mentioned in our design , changing ring b from 
 pyrimidine to phenyl is expected to increase potency by enhancing 
 the interaction of the ring with arg264 .
indeed , unlike in the irreversible 
 inhibitor series , where pyrimidine was favored over phenyl because 
 it increased the reactivity of acrylamide for cysteine , in the reversible inhibitor series , we found 
 the opposite to be true ( i.e. , 7a vs 4b and 16b vs 14b , tables 1 and 2 , and additional examples in table 1 ) .
arg264 has its guanidinium group positioned atop 
 ring b , thus favorably aligned for cation interactions 
 to occur ( figure 2a ) .
thus , an electron - rich aromatic ring , such as phenyl , may form a 
 stronger stacking interaction than pyrimidine with this residue . in 
 heterocycles , where the lone pair does not contribute to aromaticity , 
 the electronegativity of the heteroatom weakens the cation 
 binding ability .
we next explored modifications to ring a. having 
 ring a either 
 pyrimidine or pyridine could increase the chemistry feasibility for 
 exploring distinct x5 and x6 combinations on 
 the molecules , and thus , we first explored the effect on activity 
 by changing the nature of the ring . rewardingly , a change from pyrimidine 
 to pyridine led to no substantial change in activity ( i.e. , 7e vs 16c and 7f vs 16d , tables 1 and 2 ) . 
 on the other hand , disrupting the aromaticity of ring a completely 
 abolished activity ( 21 , table 2 ) .
2-hydroxypyridines undergo tautomerism to give pyridones which 
 can behave like amides and exist mainly as the  amide  
 tautomer in most solvents .
the dramatic loss of activity observed 
 upon replacing the methoxy group with a hydroxy group at x5 hinted at the importance of this position for binding energy and 
 demonstrates that this group makes significant interactions that are 
 essential for the potency of these compounds .
thus , having the 
 ability to use both pyridine and pyrimidine as 
 ring a increased our chemical versatility , and we next explored distinct 
 x5,6 combinations .
results with the pyridine series ( table 2 ) showed that disubstitution of x5 and 
 x6 was not necessarily required as having a single methoxy 
 group on the molecule performed as well as having two .
this was also 
 observed within the pyrimidine series , as 7h was of potency 
 similar to that of its disubstituted analogue 7f ( table 1 ) .
we therefore sought to explore this position 
 further , especially by adding aryl substituents that , as indicated 
 above , would be poised to fill the hydrophobic pocket currently occupied 
 by methoxy ( figure 2b , red circle ) .
substitution 
 of x5 with benzyloxy led to a remarkable 1 log increase 
 in potency ( compare 27a to 7a ) .
in fact , 
 this derivative was almost as potent as the corresponding acrylamide - containing 
 derivative ( compare 27a to 1c ) .
we further 
 increased the activity of this compound once the methyl ( r on x4 ; see figure 2a ) was substituted with 
 a phenyl or aminomethyl ( 27b and 27c , respectively , 
 table 3 ) , both favored x4 substituents 
 ( tables 1 and 2 ) . with 
 aminomethyl
being favored at this position , we also probed whether 
 adding hydrophobic bulk to this functionality could lead to a gain 
 in affinity .
as indicated above , leu237 and val238 are in the vicinity 
 of cys267 , and thus , there is potential for minor affinity gain by 
 increasing hydrophobicity . replacing the h in 27c with 
 a methyl ( 27d ) or isopropyl ( 27e ) or switching 
 the aminomethyl to 2-pyrrolidinyl ( 27f ) resulted in a 
 minor increase in activity or had no measurable effect on activity 
 ( table 3 ) . changing x5 from 
 benzyloxy to the less flexible phenyl 
 substituent directly attached to the pyrimidine a ring resulted in 
 a significant 10-fold loss of activity ( 27 g , table 3 ) .
furthermore , substituting the phenyl ring of 27c with the relatively more polar pyridine in 27h also decreased affinity 5-fold .
these results are very much concordant 
 with the proposed mode of binding for these ligands .
docking analysis 
 showed that the benzyloxy group can be accommodated in the left - side 
 hydrophobic subpocket ( red circle , figure 2b ) and form hydrophobic interactions with tyr41 , phe68 , and trp90 . 
 for 27 g
, the phenyl substituent is not able to orient 
 properly in this cavity and , due to its inflexibility , rather orients 
 toward asp69 and glu231 , providing an explanation for its loss of 
 activity .
similarly , replacing the phenyl in 27c with 
 pyridine as in 27h increases the polarity and thus its 
 ability to favorably interact with the hydrophobic residue environment . altogether , and as seen in the irreversible series , our tests demonstrated 
 a good correlation between the predicted binding mode ( figure 2 ) and the observed biological activity ( tables 14 ) of the designed 
 ligands , further consolidating that the biological effect of these 
 molecules in cancer cells is majorly hsp70 mediated .
no effect was 
 noted for these compounds on hsp90 at concentrations as high as 500 
 m ( table 4 ) .
this ligand 
 combines individual features that this study 
 identifies as most favorable at each evaluated position ; its x4 is glycine , ring b is phenyl , and x5 is benzyloxy 
 ( figure 2b ) .
this analogue has an activity 
 in cells comparable to that of the most active reported irreversible 
 inhibitors of this class , namely , low 
 micromolar activity in cancer cells ( figure 4 ) .
we found that 27c interfered with the formation of 
 functional hsp70hop  hsp90 machinery as indicated by 
 its ability to dose - dependently alter the megacomplex components and 
 to destabilize an hsp70hsp90 machinery client , raf-1 ( figure 4a ) .
hsp90 in concert with hsp70 maintains the transforming 
 capacity of several oncoproteins , including her2 , akt , raf-1 , igf - ir , 
 and hif-1 .
when this chaperone complex becomes pharmacologically 
 inhibited , these oncoproteins become destabilized and are degraded 
 mainly by the proteasomal pathway . indeed
, we found that the steady - state 
 levels and/or the activity of several oncoproteins involved in increased 
 signaling through a pathogenic pathway were sensitive to hsp70 inhibition 
 by 27c ( figure 4b ) .
these signaling 
 oncoproteins include her2 and raf-1 in the her2-overexpressing skbr3 
 breast cancer cells , stat3 and raf-1 in the triple - negative breast 
 cancer mda - mb-468 cells , stat3 , raf-1 , and akt in the miapaca2 pancreatic 
 cancer cells , and mutant flt3 and stat5 in molm13 acute myeloid leukemia 
 cells .
27c also resulted in induction of apoptosis in 
 these cancer cells , as indicated by substantial parp cleavage ( cparp ; 
 figure 4b ) .
allosteric ligands with a reversible mode 
 of binding mimic the 
 cellular phenotype observed with the irreversible hsp70 inhibitors . 

addition of 27c to cancer cells dose - dependently alters 
 the formation of the hsp70hop complex , a phenomenon associated 
 with their destabilization and reduction in half - life ( a ) .
it is associated 
 with degradation and/or inhibition of hsp90hsp70 oncoproteins 
 and induction of apoptosis ( b ) .
collectively , these data indicate 
 that its mechanism of action is mediated by interaction with the hsp70 
 allosteric pocket in a fashion resembling the interaction of 1e .
next , we investigated whether binding of 27c to hsp70 
 interfered with its main biochemical activities , specifically refolding 
 of a denatured client protein ( figure 5 ) .
activities are stimulated by hsp40 proteins and nucleotide exchange 
 factors , such as hsp110 .
humans have several cytosolic hsp40 s , including hdj1 , dja1 , 
 dja2 , and dja4 , and it has recently been reported that dja2 is most 
 efficient in promoting the refolding of an hsp70 polypeptide substrate , 
 firefly luciferase . in cells ,
the refolding of heat - denatured luciferase 
 by endogenous as well as transfected hsp70 was inhibited by 27c .
the nonspecific capacity of transfected hsp70 to maintain 
 substrate solubility after heat shock was not greatly affected , indicating 
 that 27c , in a manner we reported for 1e , targeted the specific substrate folding 
 activity of hsp70 ( figure 5 ) .
the cells were treated with cycloheximide and either 
 vehicle or 27c at 10 m , incubated at 45 c 
 for 1 h , and allowed to recover at 37 c for 2 h ( left ) .
cells 
 were lysed during and after refolding , and soluble ha - tagged luciferase 
 and chaperones were detected in the lysates ; exogenous transfected 
 flag - tagged hsp70 is visible as a band above endogenous hsp70 ( middle ) . 

luciferase enzymatic activities in the lysates were measured at 2 
 h of refolding , unless otherwise indicated , and are represented as 
 percentages of the initial activity before heat shock ( right ) .
27c significantly inhibited endogenous hsp70 and transfected 
 hsp70 in multiple experiments ( n  3 ) .
we have shown that significant 
 biological activity may be retained 
 through reversible hsp70 inhibitors targeting an allosteric pocket 
 located at the n - terminal domain .
because of the appropriate fit , 
 and thus good enthalpy of binding of the irreversible inhibitors , 
 simple modifications such as replacement of the covalent linkage with 
 an ionic bridge ( yellow , figure 2b ) and filling 
 of the hydrophobic pocket occupied by methoxy in 1e with 
 a benzyloxy ( red , figure 2b ) have led to ligands 
 of reversible mode of binding that not only mimic the phenotype observed 
 with 1e , but do so with similar potency ( figure 4 ) .
our data show that the acrylamide group 
 could be eliminated altogether 
 by improving the enthalpy of the binding and indicate that significant 
 binding energy can be attained through additional hydrophobic interactions 
 of x5 substituents with the tyr41 , phe68 , and trp90 residues . 

this interaction weighs majorly toward the binding of these ligands 
 to the allosteric pocket of hsp70 .
combined , the sar data from 
 this and the accompanying paper validate 
 the homology model and the proposed 
 binding of these ligands to the allosteric pocket .
first , the model 
 has allowed for the rational design of a ligand that , when incubated 
 with the thousands of proteins expressed in a cancer cell , affinity 
 purified one , hsp70 .
second , the correctness of the binding mode analyses 
 has allowed the rational design of specific ligands with a tractable 
 sar .
some of these specific ligands as we show have a reversible mode 
 of binding whose potency rivals that of the irreversible ligands , 
 indicating that favorable enthalpy drives the binding of these compounds 
 to hsp70 .
third , the concordance in the observed biochemical and phenotypic 
 effects observed with these agents in cancer cells , such as exemplified 
 for 27c , suggests that , in the tested concentration range , 
 the biological activity of these agents is majorly and selectively 
 channeled through an hsp70-binding mechanism .
in addition to 
 providing both a novel pharmacophore and medicinal 
 chemistry for its assembly , we describe in our papers a testing battery 
 for assessing hsp70-mediated mechanisms in cancer cells and for evaluating 
 specific ligand action in cancer cells through hsp70 inhibition .
therefore , 
 we provide a novel blueprint for a cancer - oriented development of 
 hsp70-directed ligands . in conclusion ,
our findings propose 
 the allosteric hsp70 inhibitors 
 as important leads toward the development of novel targeted anticancer 
 therapeutics .
27c serves as a molecule for further development 
 that can potentially be elaborated into more potent molecules with 
 in vivo efficacy .
we are currently working to further optimize this 
 class of compounds for potency and in vivo activity and will disclose 
 our results in due course .
all reagents were purchased from either aldrich 
 or acros organics and used without purification .
nmr spectra were recorded on a bruker 
 av - iii-500 or 600 mhz nmr spectrometer .
chemical shifts are reported 
 in  values in parts per million downfield from tms as the internal 
 standard .
h data are reported as follows : chemical shift , 
 multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , 
 br = broad , m = multiplet ) , coupling constant ( hz ) , integration .
c chemical shifts are reported in  values in parts 
 per million downfield from tms as the internal standard .
low - resolution 
 mass spectra were obtained on a waters acquity ultra performance lc 
 instrument with electrospray ionization and an sq detector .
analytical 
 hplc was performed on a waters autopurification system with pda , micromass 
 zq , and elsd detectors .
the purity of the title compounds used in 
 pharmacology testing was verified by hplc  ms using the following 
 method : 1012 min gradient on a waters2525 binary gradient 
 pump of increasing concentrations of acetonitrile in water ( 5%  
 95% ) containing 0.1% formic acid with a flow rate of 1.2 ml / min and 
 uv detection at  = 220 and 254 nm on an xbridge c18 150 mm 
  4.6 mm , 5 m column .
analytical thin - layer chromatography was 
 performed 
 on 250 m silica gel f254 plates .
preparative thin - layer 
 chromatography was performed on 1000 m silica gel f254 plates .
the syntheses of 1a  d and 8 are described 
 elsewhere . to a solution of 2a ( 20 mg , 
 0.049 mmol ) and et3n ( 49 mg , 0.49 mmol ) in 1 ml of anhydrous 
 dioxane was added propionyl chloride ( 45 mg , 0.49 mmol ) .
the resulting 
 mixture was stirred at rt for 12 h. solvent was removed under reduced 
 pressure , and the residue was purified by preparatory tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 15 mg ( 66% ) 
 of 3a .
h nmr ( 500 mhz , cdcl3 ) : 
  8.17 ( br s , 1h ) , 6.95 ( s , 1h ) , 4.84 ( br s , 2h ) , 4.35 ( q , j = 7.5 , 4h ) , 3.82 ( m , 4h ) , 2.44 ( m , 4h ) , 2.34 ( s , 3h ) , 
 2.34 ( q , j = 7.5 hz , 2h ) , 1.28 ( t , j = 7.5 hz , 6h ) , 1.15 ( t , j = 7.5 hz , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  173.3 , 170.6 , 170.5 , 164.2 , 
 160.0 , 156.7 , 87.7 , 80.3 , 62.4 , 54.9 , 46.3 , 43.7 , 30.6 , 14.5 , 9.1 . 
 hrms ( m / z ) : [ m + h ] calcd 
 for c20h31n8o3s , 463.2240 ; 
 found , 463.2253 .
to a solution of 2a ( 20 mg , 
 0.049 mmol ) and et3n ( 49 mg , 0.49 mmol ) in 1 ml of anhydrous 
 dioxane was added cyclopropanecarbonyl chloride ( 62 mg , 0.49 mmol ) . 

the resulting mixture was stirred at rt for 12 h. solvent was removed 
 under reduced pressure , and the residue was purified by preparatory 
 tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 
 23 mg ( 62% ) of 3b .
h nmr ( 500 mhz , cdcl3 ) :  8.48 ( br s , 1h ) , 6.91 ( s , 1h ) , 4.83 ( br s , 2h ) , 
 4.35 ( q , j = 7.1 hz , 4h ) , 3.82 ( m , 4h ) , 2.44 ( m , 
 4h ) , 2.34 ( s , 3h ) , 1.52 ( m , 1h ) , 1.27 ( t , j = 7.1 
 hz , 6h ) , 1.03 ( m , 2h ) , 0.84 ( m , 2h ) .
c nmr ( 125 mhz , 
 cdcl3 ) :  173.3 , 170.6 , 170.5 , 164.2 , 160.0 , 156.7 , 
 87.8 , 80.4 , 62.5 , 54.9 , 46.3 , 43.7 , 15.9 , 14.5 , 8.7 . hrms ( m / z ) : [ m + h ] calcd for c21h31n8o3s , 475.2240 ; found , 
 475.2237 .
to a solution of 2b ( 20 mg , 
 0.049 mmol ) and et3n ( 49 mg , 0.49 mmol ) in 1 ml of anhydrous 
 dioxane was added acetyl chloride ( 38 mg , 0.49 mmol ) .
the resulting 
 mixture was stirred at rt for 12 h. solvent was removed under reduced 
 pressure , and the residue was purified by preparatory tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 21 mg ( 61% ) 
 of 3c .
h nmr ( 500 mhz , cdcl3 ) : 
  7.99 ( br s , 1h ) , 6.94 ( s , 1h ) , 4.81 ( br s , 2h ) , 3.88 ( m , 10h ) , 
 2.47 ( m , 4h ) , 2.36 ( s , 3h ) , 2.11 ( s , 3h ) .
c nmr ( 125 
 mhz , cdcl3 ) :  171.1 , 170.5 , 169.3 , 164.3 , 160.1 , 
 156.6 , 87.8 , 80.1 , 54.9 , 54.2 , 46.2 , 43.7 , 24.8 . hrms ( m / z ) : [ m + h ] calcd for c17h25n8o3s , 421.1770 ; found , 421.1765 .
to a solution of 2b ( 20 mg , 
 0.049 mmol ) and et3n ( 49 mg , 0.49 mmol ) in 1 ml of anhydrous 
 dioxane was added octanoyl chloride ( 80 mg , 0.49 mmol ) .
the resulting 
 mixture was stirred at rt for 12 h. solvent was removed under reduced 
 pressure , and the residue was purified by preparatory tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 17 mg ( 71% ) 
 of 3d .
h nmr ( 500 mhz , cdcl3 ) : 
  8.01 ( br s , 1h ) , 6.97 ( s , 1h ) , 4.86 ( br s , 2h ) , 3.89 ( s , 6h ) , 
 3.86 ( m , 4h ) , 2.46 ( m , 4h ) , 2.35 ( s , 3h ) , 2.30 ( t , j = 7.4 hz , 2h ) , 1.62 ( m , 2h ) , 1.201.30 ( m , 8h ) , 0.87 ( t , j = 6.9 hz , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  172.7 , 171.1 , 170.4 , 164.3 , 160.0 , 156.7 , 87.9 , 80.1 , 
 54.9 , 54.2 , 46.3 , 43.7 , 37.7 , 31.6 , 29.1 , 29.0 , 25.2 , 22.6 , 14.1 . 

hrms ( m / z ) : [ m + h ] calcd 
 for c23h37n8o3s , 505.2709 ; 
 found , 505.2701 .
to a solution of 2c ( 50 mg , 
 0.138 mmol ) and et3n ( 139 mg , 1.38 mmol ) in 2 ml of anhydrous 
 dioxane was added acetyl chloride ( 108 mg , 1.38 mmol ) dropwise .
the 
 resulting mixture was stirred at rt for 12 h. solvent was removed 
 under reduced pressure , and the residue was purified by preparatory 
 tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 
 56 mg ( 73% ) of 4a .
h nmr ( 500 mhz , cdcl3 ) :  8.34 ( d , j = 6.2 hz , 1h ) , 8.09 
 ( s , 1h ) , 7.75 ( d , j = 6.2 hz , 1h ) , 3.89 ( br s , 10h ) , 
 2.50 ( m , 4h ) , 2.38 ( s , 3h ) , 2.18 ( s , 3h ) .
c nmr ( 125 
 mhz , cdcl3 ) :  171.4 , 171.1 , 169.3 , 160.1 , 158.9 , 
 157.0 , 105.4 , 79.4 , 54.8 , 54.2 , 46.2 , 43.6 , 24.8 . hrms ( m / z ) : [ m + h ] calcd for c17h24n7o3s , 406.1661 ; found , 406.1661 .
to a solution of 2c ( 50 mg , 
 0.138 mmol ) and et3n ( 139 mg , 1.38 mmol ) in 2 ml of anhydrous 
 dioxane was added propionyl chloride ( 128 mg , 1.38 mmol ) dropwise . 

the resulting mixture was stirred at rt for 12 h. solvent was removed 
 under reduced pressure , and the residue was purified by preparatory 
 tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 
 58 mg ( 81% ) of 4b.h nmr ( 500 
 mhz , cdcl3 ) :  8.33 ( d , j = 5.6 
 hz , 1h ) , 8.05 ( s , 1h ) , 7.77 ( d , j = 5.6 hz , 1h ) , 
 3.96 ( m , 4h ) , 3.89 ( s , 6h ) , 2.59 ( m , 4h ) , 2.42 ( s , 3h ) , 2.41 ( q , j = 7.6 hz , 2h ) , 1.20 ( t , j = 7.6 hz , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  173.0 , 171.2 , 
 171.1 , 160.1 , 158.9 , 157.0 , 105.5 , 77.0 , 54.6 , 54.3 , 45.7 , 43.1 , 30.8 , 
 9.0 .
hrms ( m / z ) : [ m + h ] calcd for c18h26n7o3s , 420.1818 ; found , 420.1838 . to a solution of 2d ( 50 mg , 
 0.151 mmol ) and et3n ( 152 mg , 1.51 mmol ) in 2 ml of anhydrous 
 dioxane was added propionyl chloride ( 140 mg , 1.51 mmol ) dropwise . 

the resulting mixture was stirred at rt for 12 h. solvent was removed 
 under reduced pressure , and the residue was purified by preparatory 
 tlc ( chcl3/meoh  nh3
( 7 n ) , 10:1 ) to afford 
 59 mg ( 83% ) of 4c .
h nmr ( 500 mhz , cdcl3 ) :  8.35 ( d , j = 5.7 hz , 1h ) , 7.84 
 ( s , 1h ) , 7.81 ( d , j = 5.7 hz , 1h ) , 3.92 ( s , 4h ) , 
 2.48 ( m , 4h ) , 2.44 ( s , 6h ) , 2.40 ( q , j = 7.5 hz , 
 2h ) , 2.35 ( s , 3h ) , 1.21 ( t , j = 7.5 hz , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  173.0 , 171.9 , 
 170.7 , 160.4 , 159.1 , 157.3 , 108.7 , 105.7 , 55.0 , 46.2 , 43.5 , 30.8 , 
 23.9 , 8.9 .
hrms ( m / z ) : [ m + h ] calcd for c18h26n7os , 388.1920 ; 
 found , 388.1921 .
to a solution of 2d ( 50 mg , 
 0.151 mmol ) and et3n ( 152 mg , 1.51 mmol ) in 2 ml of anhydrous 
 dioxane was added 2-furoyl chloride ( 197 mg , 1.51 mmol ) dropwise . 

the resulting mixture was stirred at rt for 12 h. solvent was removed 
 under reduced pressure , and the residue was purified by preparatory 
 tlc ( chcl3/meoh  nh3 ( 7 n ) , 10:1 ) to afford 
 56 mg ( 87% ) of 4d .
h nmr ( 600 mhz , cdcl3 ) :  8.58 ( br s , 1h ) , 8.40 ( d , j = 
 5.6 hz , 1h ) , 7.94 ( d , j = 5.6 hz , 1h ) , 7.567.58 
 ( m , 1h ) , 7.31 ( d , j = 3.5 hz , 1h ) , 6.60 ( dd , j = 3.5 , 1.7 hz , 1h ) , 3.903.97 ( m , 4h ) , 2.49 ( m , 
 4h ) , 2.46 ( s , 6h ) , 2.36 ( s , 3h ) .
c nmr ( 150 mhz , cdcl3 ) :  171.9 , 170.9 , 160.5 , 159.1 , 157.1 , 156.3 , 146.4 , 
 145.4 , 117.2 , 113.0 , 108.5 , 106.0 , 55.0 , 46.3 , 43.5 , 23.9 . hrms ( m / z ) : [ m + h ] calcd for c20h24n7o2s , 426.1712 ; found , 
 426.1727 .
to a solution of 2d ( 50 mg , 
 0.151 mmol ) and et3n ( 152 mg , 1.51 mmol ) in 2 ml of anhydrous 
 dioxane was added cyclopropanecarbonyl chloride ( 158 mg , 1.51 mmol ) 
 dropwise .
the resulting mixture was stirred at rt for 12 h. solvent 
 was removed under reduced pressure , and the residue was purified by 
 preparatory tlc ( chcl3/meoh  nh3 ( 7 n ) , 
 10:1 ) to afford 48 mg ( 79% ) of 4e . h nmr 
 ( 500 mhz , cdcl3 ) :  8.35 ( d , j = 
 5.6 hz , 1h ) , 8.20 ( s , 1h ) , 7.78 ( d , j = 5.6 hz , 1h ) , 
 3.92 ( m , 4h ) , 2.49 ( m , 4h ) , 2.45 ( s , 6h ) , 2.36 ( s , 3h ) , 1.54 ( m , 1h ) , 
 1.10 ( m , 2h ) , 0.93 ( m , 2h ) .
c nmr ( 125 mhz , cdcl3 ) :  173.1 , 172.0 , 170.6 , 160.4 , 159.0 , 157.3 , 108.8 , 
 105.7 , 55.0 , 46.2 , 43.5 , 23.8 , 16.0 , 9.1 . hrms ( m / z ) : [ m + h ] calcd for c19h26n7os , 400.1920 ; found , 400.1913 .
a mixture of 5a ( 0.200 g , 
 0.549 mmol ) , 3-aminothiophenol ( 70 l , 0.082 g , 0.659 mmol ) , 
 neocuproine ( 0.023 g , 0.110 mmol ) , cui ( 0.021 , 0.110 mmol ) , and k2co3 ( 0.152 g , 1.10 mmol ) in dmf ( 7 ml ) was heated 
 at 120 c for 24 h. solvent was removed under reduced pressure , 
 and the residue was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 200:1 to 40:1 ) to 
 afford 0.133 g ( 67% ) of 6a .
h nmr ( 500 mhz , 
 cdcl3 ) :  6.97 ( t , j = 8.0 hz , 1h ) , 
 6.466.51 ( m , 1h ) , 6.366.40 ( m , 2h ) , 3.884.03 
 ( m , 10h ) , 3.58 ( br s , 2h ) , 2.52 ( m , 4h ) , 2.38 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  171.8 , 160.1 , 146.9 , 139.5 , 
 129.6 , 116.2 , 112.2 , 112.1 , 81.1 , 55.0 , 54.6 , 46.3 , 43.8 . hrms
( m / z ) : [ m + h ] calcd for c17h24n5o2s , 362.1651 ; found , 
 362.1649 . to a solution of 6a ( 10 mg , 
 0.027 mmol ) and et3n ( 50 l , 36 mg , 0.36 mmol ) in 
 1 ml of ch2cl2
was added propionyl chloride 
 ( 22 l , 24.4 mg , 0.27 mmol ) .
the reaction was stirred at rt 
 for 12 h and then quenched by adding cold meoh .
solvent was evaporated 
 under reduced pressure , and the residue was purified by preparatory 
 tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 
 20:1 ) to afford 7.3 mg ( 65% ) of 7a .
h nmr 
 ( 500 mhz , cdcl3 )  7.44 ( d , j = 
 7.5 hz , 1h ) , 7.13 ( m , 2h ) , 7.04 ( s , 1h ) , 6.78 ( d , j = 7.5 hz , 1h ) , 3.93 ( m , 4h ) , 3.90 ( s , 6h ) , 2.55 ( m , 4h ) , 2.36 ( s , 
 3h ) , 2.33 ( q , j = 7.2 hz , 2h ) , 1.21 ( t , j = 7.2 hz , 3h ) . hrms ( m / z ) : [ m 
 + h ] calcd for c20h28n5o3s , 418.1913 ; found , 418.1910 . to a solution of 6a ( 10.9 
 mg , 0.0302 mmol ) and et3n ( 21 l , 15.3 mg , 0.151 
 mmol ) in ch2cl2 ( 1 ml ) was added cyclopropanecarbonyl 
 chloride ( 14 l , 15.8 mg , 0.1508 mmol ) , and the resulting solution 
 was stirred at rt for 2 h. solvent was removed under reduced pressure , 
 and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 20:1 ) to afford 7.9 mg 
 ( 61% ) of 7b .
h nmr ( 500 mhz , cdcl3 ) :  7.49 ( m , 1h ) , 7.29 ( s , 1h ) , 7.13 ( t , j = 7.8 hz , 1h ) , 6.97 ( s , 1h ) , 6.78 ( d , j = 7.6 hz , 
 1h ) , 3.883.95 ( m , 10h ) , 2.48 ( m , 4h ) , 2.36 ( s , 3h ) , 1.051.13 
 ( m , 1h ) , 0.740.96 ( m , 4h ) .
hrms ( m / z ) : [ m + h ] calcd for c21h28n5o3s , 430.1913 ; found , 430.1897 .
6a ( 10 mg , 0.028 mmol ) , et3n ( 19.5 l , 14.2 mg , 0.14 mmol ) , and cyclobutanecarbonyl 
 chloride ( 9.6 l , 10.0 mg , 0.084 mmol ) in ch2cl2 ( 1 ml ) were stirred at rt for 2 h. solvent was removed under 
 reduced pressure , and the residue was purified by preparatory tlc 
 ( etoac / meoh  nh3 ( 7 n ) , 20:1 ) to afford 4.1 mg ( 33% ) 
 of 7c .
h nmr ( 500 mhz , cdcl3 ) : 
  7.50 ( d , j = 7.8 hz , 1h ) , 7.14 ( t , j = 7.9 hz , 1h ) , 7.00 ( s , 1h ) , 6.94 ( br s , 1h ) , 6.77 ( d , j = 7.9 hz , 1h ) , 3.90 ( br s , 10h ) , 3.07 ( m , 1h ) , 2.50 ( m , 
 14h ) , 2.142.42 ( m , 7h ) , 1.822.02 ( m , 2h ) . hrms ( m / z ) : [ m + h ] calcd for c22h30n5o3s , 444.2069 ; found , 
 444.2052 .
6a ( 10 mg , 0.028 mmol ) , et3n ( 19.5 l , 14.2 mg , 0.14 mmol ) , and cyclohexanecarbonyl 
 chloride ( 11.4 l , 12.3 mg , 0.084 mmol ) in ch2cl2 ( 1 ml ) were stirred at rt for 3 h. solvent was removed under 
 reduced pressure , and the residue was purified by preparatory tlc 
 ( etoac / meoh  nh3 ( 7 n ) , 20:1 ) to afford 12 mg ( 91% ) 
 of 7d .
h nmr ( 500 mhz , cdcl3 ) : 
  7.50 ( d , j = 7.9 hz , 1h ) , 7.18 ( s , 1h ) , 7.13 
 ( t , j = 7.9 hz , 1h ) , 7.02 ( s , 1h ) , 6.76 ( d , j = 7.7 hz , 1h ) , 3.90 ( br s , 10h ) , 2.51 ( m , 4h ) , 2.38 ( s , 
 3h ) , 2.112.22 ( m , 1h ) , 1.191.96 ( m , 10h ) .
6a ( 10 mg , 0.028 mmol ) , et3n ( 19.5 l , 14.2 mg , 0.14 mmol ) , and benzoyl chloride 
 ( 9.8 l , 11.8 mg , 0.084 mmol ) in ch2cl2 ( 1 ml ) were stirred at rt for 3 h. solvent was removed under reduced 
 pressure , and the residue was purified by preparatory tlc ( etoac / meoh  nh3 ( 7 n ) , 20:1 ) to afford 8.6 mg ( 66% ) of 7e .
h nmr ( 500 mhz , cdcl3 ) :  7.83 ( d , j = 7.7 hz , 2h ) , 7.74 ( br s , 1h ) , 7.59 ( d , j = 7.8 hz , 1h ) , 7.54 ( t , j = 6.4 hz , 1h ) , 7.47 ( t , j = 7.8 hz , 2h ) , 7.20 ( t , j = 8.0 hz , 1h ) , 
 7.13 ( s , 1h ) , 6.86 ( d , j = 7.9 hz , 1h ) , 3.873.93 
 ( m , 10h ) , 2.50 ( m , 4h ) , 2.37 ( s , 3h ) . hrms ( m / z ) : [ m + h ] calcd for c24h28n5o3s , 466.1913 ; found , 466.1919 . to a solution of 6a ( 20 mg , 
 0.0552 mmol ) in thf ( 1 ml ) were added boc - glycine ( 9.7 mg , 0.0552 
 mmol ) and dcc ( 12 mg , 0.058 mmol ) , and the resulting solution was 
 stirred at rt for 5 h. the reaction mixture was concentrated under 
 reduced pressure , and the residue was purified by preparatory tlc 
 ( etoac / meoh  nh3 ( 7 n ) , 20:1 ) to afford a solid which 
 was dissolved in 2 ml of ch2cl2/tfa ( 4:1 ) .
the 
 resulting solution was stirred at rt for 1 h. the reaction mixture 
 was concentrated under reduced pressure , and the residue was purified 
 by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 15:1 ) to afford 13.9 mg ( 60% ) of 7f .
h nmr ( 500 mhz , cdcl3/meoh - d4 ) :  7.38 ( d , j = 8.2 hz , 1h ) , 7.26 
 ( s , 1h ) , 7.15 ( t , j = 7.9 hz , 1h ) , 6.79 ( d , j = 7.6 hz , 1h ) , 3.863.97 ( m , 10h ) , 3.52 ( s , 2h ) , 
 2.58 ( m , 4h ) , 2.41 ( s , 3h ) . hrms ( m / z ) : [ m + h ] calcd for c19h27n6o3s , 419.1865 ; found , 419.1862 . to a solution of 6a ( 10 mg , 
 0.027 mmol ) and et3n ( 50 l , 36 mg , 0.36 mmol ) in 
 1 ml of ch2cl2 was added 3-butenoyl chloride 
 ( 28.2 mg , 0.27 mmol ) .
the reaction was stirred at rt for 12 h and 
 then quenched by adding cold meoh .
solvent was evaporated under reduced 
 pressure , and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 20:1 ) to 
 afford 8.3 mg ( 72% ) of 7 g .
h nmr ( 500 mhz , 
 cdcl3 ) :  7.35 ( d , j = 8.1 hz , 1h ) , 
 7.29 ( br s , 1h ) , 7.107.19 ( m , 2h ) , 6.79 ( d , j = 7.5 hz , 1h ) , 5.976.00 ( m , 1h ) , 5.255.35 ( m , 2h ) , 
 3.923.96 ( m , 4h ) , 3.90 ( s , 6h ) , 3.14 ( d , j = 7.1 hz , 2h ) , 2.542.60 ( m , 4h ) , 2.42 ( s , 3h ) .
hrms ( m / z ) : [ m + h ] calcd for c21h28n5o3s , 430.1913 ; found , 
 430.1911 . a mixture of 5b ( 0.200 g , 
 0.600 mmol ) and k2co3 ( 0.166 g , 1.20 mmol ) in 
 dmf ( 6 ml ) was evacuated and backfilled with argon three times .
copper(i ) 
 thiophene-2-carboxylate ( 0.034 g , 0.180 mmol ) was added , and the resulting 
 mixure was evacuated and backfilled with argon two times .
3-aminothiophenol 
 ( 76 l , 0.090 g , 0.72 mmol ) was added , and the reaction mixture 
 was heated at 120 c for 24 h. solvent was removed under reduced 
 pressure , and the residue was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 200:1 to 
 40:1 ) to afford 0.147 g ( 74% ) of 6b .
h nmr 
 ( 500 mhz , cdcl3 ) :  8.22 ( s , 1h ) , 7.00 ( t , j = 7.8 hz , 1h ) , 6.52 ( d , j = 7.7 hz , 1h ) , 
 6.386.45 ( m , 2h ) , 3.91 ( s , 3h ) , 3.89 ( m , 4h ) , 3.60 ( br s , 
 2h ) , 2.49 ( m , 4h ) , 2.36 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  169.4 , 164.9 , 161.6 , 146.9 , 138.8 , 129.6 , 116.9 , 
 112.9 , 112.5 , 99.2 , 54.9 , 53.9 , 46.2 , 43.8 . hrms ( esi ) m / z [ m + h ] calcd for c16h22n5os , 332.1545 ; found , 332.1532 . to a solution of 6b ( 15.5 
 mg , 0.047 mmol ) in thf ( 1 ml ) were added boc - glycine ( 9.1 mg , 0.052 
 mmol ) and dcc ( 10.7 mg , 0.052 mmol ) . after the resulting solution 
 was stirred overnight at rt , thf was evaporated and 1 ml of ch2cl2/tfa ( 4:1 ) was added .
the solution was stirred 
 for 45 min and then concentrated to dryness under reduced pressure 
 to give a residue which was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 15:1 ) to afford 
 13.4 mg ( 73% ) of 7h .
h nmr ( 500 mhz , cdcl3 ) :  9.29 ( br s , 1h ) , 8.23 ( s , 1h ) , 7.457.50 
 ( m , 1h ) , 7.307.34 ( m , 1h ) , 7.17 ( t , j = 8.0 
 hz , 1h ) , 6.816.87 ( m , 1h ) , 3.91 ( s , 3h ) , 3.863.90 
 ( m , 4h ) , 3.45 ( s , 2h ) , 2.462.52 ( m , 4h ) , 2.35 ( s , 3h ) .
ms 
 ( m / z ) : [ m + h ] 389.3 . to a solution of 8 ( 23 mg , 
 0.053 mmol ) in ch3cn ( 1 ml ) were added dimethylamine ( 2 
 m , thf ; 53 l , 0.106 mmol ) and dbu ( 4 mg , 0.026 mmol ) at rt , 
 and the resulting solution was stirred for 6 h. the reaction mixture 
 was concentrated under reduced pressure , and the residue was purified 
 by preparatory tlc ( ch2cl2/meoh  nh3
( 7 n ) , 10:1 ) to afford 13.5 mg ( 53% ) of 9 .
h nmr ( 600 mhz , cdcl3 ) :  11.1 ( br s , 1h ) , 
 6.91 ( s , 1h ) , 4.79 ( br s , 2h ) , 3.843.91 ( m , 10h ) , 2.56 ( t , j = 5.8 hz , 2h ) , 2.452.51 ( m , 4h ) , 2.41 ( t , j = 5.8 hz , 2h ) , 2.37 ( s , 3h ) , 2.22 ( s , 6h ) .
c nmr ( 150 mhz , cdcl3 ) :  171.9 , 171.2 , 170.2 , 164.3 , 
 160.0 , 157.1 , 88.4 , 80.8 , 54.9 , 54.4 , 54.1 , 46.2 , 44.1 , 43.6 , 33.6 . 
 hrms ( m / z ) : [ m + h ] calcd 
 for c20h32n9o3s , 478.2349 ; 
 found , 478.2343 .
to a solution of 2-bromo-6-methoxypyridine 
 ( 10 ) ( 150 mg , 0.8 mmol ) and 1-methylpiperazine ( 240 mg , 
 2.4 mmol ) in 
 2 ml of dmf was added of k2co3 ( 220 mg , 1.6 
 mmol ) , and the resulting mixture was heated to 130 c for 16 
 h. solvent was evaporated under reduced pressure , and the residue 
 was purified by column chromatography ( 510% meoh in ch2cl2 ) to afford 145 mg ( 88% ) of 11 .
h nmr ( 500 mhz , cdcl3 ) :  7.41 ( t , j = 8.0 hz , 1h ) , 6.16 ( d , j = 8.0 hz , 1h ) , 
 6.08 ( d , j = 8.0 hz , 1h ) , 3.87 ( s , 3h ) , 3.54 ( m , 
 4h ) , 2.51 ( m , 4h ) , 2.54 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  163.1 , 158.3 , 140.1 , 98.2 , 98.1 , 54.8 , 52.9 , 46.2 , 
 45.1 . ms ( m / z ) : [ m + h ] 208.4 . to a solution of 11 ( 124 mg , 
 0.6 mmol ) in 
 5 ml of acetonitrile was added n - iodosuccinimide 
 ( 203 mg , 0.9 mmol ) , and the resulting mixture was stirred at rt for 
 2 h. the solvent was evaporated under reduced pressure , and the residue 
 was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 85:15 ) to afford 190 mg 
 ( 95% ) of 12 .
h nmr ( 500 mhz , cdcl3 ) :  7.70 ( d , j = 8.0 hz , 1h ) , 6.02 ( d , j = 8.0 hz , 1h ) , 3.90 ( s , 3h ) , 3.60 ( m , 4h ) , 2.62 ( m , 4h ) , 
 2.39 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  
 177.7 , 160.5 , 157.8 , 148.6 , 100.7 , 61.7 , 54.2 , 45.5 , 44.5 .
ms ( m / z ) : [ m + h ] 334.1 . a mixture of 12 ( 100 mg , 0.3 
 mmol ) , 4-aminopyrimidine-2-thiol ( 39 mg , 0.3 mmol ) , k2co3 ( 83 mg , 0.6 mmol ) , neocuproine ( 11 mg , 0.05 mmol ) , and cui 
 ( 10 mg , 0.05 mmol ) in dmf ( 3 ml ) was heated to 130 c for 16 
 h. solvent was removed under reduced pressure , and the residue was 
 purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 85:15 ) to afford 60 mg ( 60% ) of 13 .
h nmr ( 500 mhz , cdcl3 ) :  7.95 ( d , j = 8.0 hz , 1h ) , 7.59 ( d , j = 8.5 hz , 1h ) , 
 6.20 ( d , j = 8.5 hz , 1h ) , 6.05 ( d , j = 8.0 hz , 1h ) , 5.01 ( s , 2h ) , 3.87 ( s , 3h ) , 3.62 ( m , 4h ) , 2.54 ( m , 
 4h ) , 2.36 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) : 
  171.9 , 162.9 , 162.5 , 158.6 , 156.2 , 147.8 , 101.1 , 98.6 , 97.6 , 
 54.6 , 53.6 , 46.0 , 44.6 . ms ( m / z ) : 
 [ m + h ] 333.5 . to a solution of 13 ( 20 mg , 
 0.06 mmol ) in 1.5 ml of ch2cl2 and et3n ( 100 l ) was added propionyl chloride in ch2cl2 dropwise . upon completion
solvent was removed under reduced pressure , and 
 the residue was purified by column chromatography ( meoh  nh3 ( 7 n ) , 210% in ch2cl2 ) to afford 
 18 mg ( 80% ) of 14a .
h nmr ( 500 mhz , cdcl3 ) :  8.53 ( br s , 1h ) , 8.34 ( d , j = 
 8.0 hz , 1h ) , 7.79 ( d , j = 8.0 hz , 1h ) , 7.58 ( d , j = 8.0 hz , 1h ) , 6.21 ( d , j = 8.0 hz , 1h ) , 
 3.86 ( s , 3h ) , 3.63 ( m , 4h ) , 2.52 ( m , 4h ) , 2.41 ( q , j = 7.2 hz , 2h ) , 2.36 ( s , 3h ) , 1.08 ( t , j = 7.2 hz , 
 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  173.4 , 
 172.2 , 171.7 , 162.9 , 158.8 , 157.3 , 147.6 , 105.6 , 98.7 , 96.7 , 54.6 , 
 53.7 , 46.0 , 44.6 , 30.6 , 8.96 . hrms ( m / z ) : [ m + h ] calcd for c18h25n6o2s , 389.1760 ; found , 389.1751 . to a solution of 13 ( 20 mg , 
 0.06 mmol ) in 1.5 ml of ch2cl2 and et3n ( 100 l ) was added cyclopropanecarbonyl chloride in ch2cl2 dropwise . upon completion
solvent was removed under reduced 
 pressure , and the residue was purified by column chromatography ( meoh  nh3 ( 7 n ) , 210% in ch2cl2 ) to afford 
 21 mg ( 90% ) of 14b .
h nmr ( 500 mhz , cdcl3 ) :  8.87 ( br s , 1h ) , 8.31 ( d , j = 
 8.0 hz , 1h ) , 7.75 ( d , j = 8.0 hz , 1h ) , 7.57 ( d , j = 8.0 hz , 1h ) , 6.20 ( d , j = 8.0 hz , 1h ) , 
 3.87 ( s , 3h ) , 3.62 ( m , 4h ) , 2.52 ( m , 4h ) , 2.36 ( s , 3h ) , 1.61 ( m , 1h ) , 
 1.08 ( m , 2h ) , 0.88 ( m , 2h ) .
c nmr ( 125 mhz , cdcl3 ) :  176.4 , 173.5 , 171.7 , 162.9 , 158.8 , 157.2 , 147.6 , 
 105.7 , 98.6 , 96.7 , 54.7 , 53.6 , 46.1 , 44.6 , 14.0 , 7.7 . hrms ( m / z ) : [ m + h ] calcd for c19h25n6o2s , 401.1760 ; found , 
 401.1767 .
a mixture of 12 ( 100 mg , 0.3 
 mmol ) , 3-aminothiophenol ( 37 mg , 0.3 mmol ) , k2co3 ( 83 mg , 0.6 mmol ) , neocuproine ( 33 mg , 0.15 mmol ) , and cui ( 29 mg , 
 0.15 mmol ) in dmf ( 3 ml ) was heated to 130 c for 16 h. solvent 
 was removed under reduced pressure , and the residue was purified by 
 column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 90:10 ) to afford 60 mg ( 60% ) of 15 .
ms ( m / z ) : [ m + h ] 331.2 . to a solution of 15 ( 20 mg , 
 0.06 mmol ) and
et3n ( 100 l ) in 1.5 ml of ch2cl2 was added propionyl chloride in ch2cl2 dropwise . upon completion
solvent was removed under reduced pressure , 
 and the residue was purified by column chromatography ( meoh  nh3 ( 7 n ) , 210% in ch2cl2 ) to afford 
 15 mg ( 70% ) of 16a .
h nmr ( 500 mhz , cdcl3 ) :  7.55 ( d , j = 8.0 hz , 1h ) , 7.45 
 ( d , j = 8.0 hz , 1h ) , 7.22 ( br s , 1h ) , 7.14 ( t , j = 8.0 hz , 1h ) , 7.08 ( s , 1h ) , 6.81 ( d , j = 8.0 hz , 1h ) , 6.20 ( d , j = 8.0 hz , 1h ) , 3.88 ( s , 
 3h ) , 3.47 ( m , 4h ) , 2.57 ( m , 4h ) , 2.36 ( s , 3h ) , 2.33 ( q , j = 7.2 hz , 2h ) , 1.19 ( t , j = 7.2 hz , 3h ) . hrms ( m / z ) : [ m + h ] calcd for c20h27n4o2s , 387.1855 ; found , 
 387.1855 . to a solution of 15 ( 20 mg , 
 0.06 mmol ) and et3n ( 100 l ) in 1.5 ml of ch2cl2 was added cyclopropanecarbonyl chloride in 
 ch2cl2 dropwise . upon completion
solvent was removed under 
 reduced pressure , and the residue was purified by column chromatography 
 ( meoh  nh3 ( 7 n ) , 210% in ch2cl2 ) to afford 16 mg ( 70% ) of 16b .
h 
 nmr ( 500 mhz , cdcl3 )  7.61 ( s , 1h ) , 7.31 ( d , j = 8.0 hz , 1h ) , 7.20 ( br s , 1h ) , 6.88 ( m , 2h ) , 6.55 ( m , 
 1h ) , 5.95 ( d , j = 8.0 hz , 1h ) , 3.63 ( s , 3h ) , 3.37 
 ( m , 4h ) , 2.29 ( m , 4h ) , 2.09 ( s , 3h ) , 1.25 ( m , 1h ) , 0.78 ( m , 2h ) , 0.57 
 ( m , 2h ) .
c nmr ( 125 mhz , cdcl3 )  176.4 , 
 172.1 , 162.7 , 158.4 , 147.5 , 139.3 , 138.7 , 129.2 , 122.2 , 117.5 , 116.8 , 
 99.0 , 54.6 , 53.6 , 46.1 , 44.7 , 13.9 , 7.9 . hrms ( m / z ) : [ m + h ] calcd for c21h27n4o2s , 399.1855 ; found , 399.1853 . to a solution of 15 ( 20 mg , 
 0.06 mmol ) and et3n ( 100 l ) in 1.5 ml of ch2cl2
solvent was removed under reduced pressure , 
 and the residue was purified by column chromatography ( meoh  nh3 ( 7 n ) , 210% in ch2cl2 ) to afford 
 20 mg ( 75% ) of 16c .
h nmr ( 500 mhz , cdcl3 )  7.84 ( s , 1h ) , 7.80 ( d , j = 7.5 
 hz , 2h ) , 7.387.60 ( m , 5h ) , 7.24 ( m , 1h ) , 7.20 ( t , j = 8.0 hz , 1h ) , 6.88 ( d , j = 7.9 hz , 1h ) , 
 6.19 ( d , j = 8.4 hz , 1h ) , 3.88 ( s , 3h ) , 3.65 ( m , 
 4h ) , 2.61 ( m , 4h ) , 2.41 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 )  165.7 , 162.8 , 158.4 , 147.7 , 139.7 , 138.5 , 135.0 , 
 131.8 , 128.7 , 129.4 , 127.0 , 122.8 , 118.0 , 117.2 , 99.4 , 99.1 , 54.4 , 
 53.7 , 45.7 , 44.4 . hrms ( m / z ) : [ m 
 + h ] calcd for c24h27n4o2s , 435.1855 ; found , 435.1849 . to a solution of 15 ( 20 mg , 
 0.06 mmol ) in ch2cl2 ( 1 ml ) were added boc - glycine 
 ( 10.6 mg , 0.06 mmol ) , dmap ( 1.0 mg ) , et3n ( 10 l ) , 
 and edci ( 11 mg , 0.06 mmol ) .
the resulting solution was stirred at 
 rt for 2 h. solvent was evaporated under reduced pressure , and the 
 residue was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 85:15 ) to afford 
 26 mg ( 90% ) of residue . to this
ch2cl2 , and the resulting solution was stirred at 
 rt for 1 h. solvent was evaporated under reduced pressure , and the 
 residue was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 85:15 ) to afford 
 16 mg ( 85% ) of 16d .
h nmr ( 500 mhz , cdcl3 ) :  9.26 ( br s , 1h ) , 7.55 ( d , j = 
 8.0 hz , 1h ) , 7.47 ( d , j = 8.0 hz , 1h ) , 7.28 ( s,1h ) , 
 7.15 ( t , j = 8.0 hz , 1h ) , 6.82 ( d , j = 8.0 , 1h ) , 6.21 ( d , j = 8.0 hz , 1h ) , 3.89 ( s , 
 3h ) , 3.62 ( m , 4h ) , 3.42 ( s , 2h ) , 2.54 ( m , 4h ) , 2.36 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  170.6 , 162.7 , 
 158.4 , 147.5 , 139.4 , 138.0 , 129.3 , 122.5 , 117.5 , 116.5 , 99.4 , 98.9 , 
 54.6 , 53.6 , 46.0 , 45.1 , 44.7 . hrms ( m / z ) : [ m + h ] calcd for c19h26n5o2s , 388.1807 ; found , 388.1808 .
a mixture of 11 ( 8.0 g , 0.0386 
 mol ) in 50 ml of 48% 
 hbr(aq ) and a catalytic amount of tetrabutylammonium bromide was refluxed 
 overnight .
the reaction mixture was concentrated under reduced pressure 
 and purified by column chromatography to give 5.1 g ( 68% ) of 17 .
h nmr ( 500 mhz , cdcl3 ) :  
 7.287.33 ( m , 1h ) , 5.94 ( d , j = 8.7 hz , 1h ) , 
 5.54 ( d , j = 7.8 hz , 1h ) , 3.38 ( m , 4h ) , 2.55 ( m , 
 4h ) , 2.34 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) : 
  165.2 , 153.6 , 142.6 , 106.7 , 90.6 , 54.4 , 47.3 , 46.0 . ms ( m / z ) : [ m + h ] 194.0 . to a solution of 17 ( 1.2 g , 
 6.22 mmol ) in dmf ( 40 ml ) was added nah ( 0.596 g , 24.8 mmol ) at rt , 
 and the mixture was stirred for 10 min . then ( pmb)cl ( 1.06 g , 6.83 
 mmol ) was added dropwise , and the mixture was stirred at rt for 1 
 h. the reaction mixture was concentrated under reduced pressure and 
 purified by column chromatography to give 1.63 g ( 84% ) of 18 .
ms ( m / z ) : [ m + h ] 314.2 . to a solution of 18 ( 1.16 
 g , 3.7 mmol ) in acetonitrile ( 50 ml ) were added nis ( 1.66 g , 7.4 mmol ) 
 and tfa ( 1.42 ml , 18.5 mmol ) , and the solution was stirred for 1 h 
 at rt .
the reaction mixture was concentrated under reduced pressure 
 and purified by column chromatography to give 1.22 g ( 75% ) of 19 .
a mixture of 19 ( 452 mg , 1.03 
 mmol ) , 3-aminothiophenol ( 137 mg , 1.10 mmol ) , k2co3 ( 552 mg , 4.0 mmol ) , neocuproine ( 45 mg , 0.2 mmol ) , and cui 
 ( 39 mg , 0.2 mmol ) in dmf ( 10 ml ) was heated to 130 c for 16 
 h. solvent was removed under reduced pressure , and the residue was 
 purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 85:15 ) to afford 224 mg ( 50% ) of 20 . ms ( m / z ) : [ m + h ] 437.2 . to a solution of 20 ( 220 mg , 
 0.5 mmol ) in ch2cl2 ( 10 ml ) were added boc - glycine 
 ( 96 mg , 0.55 mmol ) , dmap ( 6.1 mg , 0.05 mmol ) , and edci ( 105 mg , 0.55 
 mmol ) .
the resulting solution was stirred at rt for 2 h. solvent was 
 evaporated under reduced pressure , and the residue was purified by 
 column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 1:0 to 85:15 ) to afford a residue . to this
was added 
 10 ml of 30% tfa / ch2cl2 , and the resulting solution 
 was stirred at rt for 2 h. solvent was evaporated under reduced pressure , 
 and the residue was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7n ) , 1:0 to 85:15 ) to 
 afford 153 mg ( 82% ) of 21 .
h nmr ( 500 mhz , 
 cdcl3 ) :  9.31 ( br s , 1h ) , 7.62 ( d , j = 8.4 hz , 1h ) , 7.44 ( d , j = 8.0 hz , 1h ) , 7.31 ( s , 
 1h ) , 7.19 ( t , j = 8.0 hz , 1h ) , 6.93 ( d , j = 7.8 hz , 1h ) , 5.58 ( d , j = 8.4 hz , 1h ) , 3.34 ( s , 
 2h ) , 3.33 ( m , 4h ) , 2.29 ( m , 4h ) , 2.22 ( s , 3h ) . hrms ( m / z ) : [ m + h ] calcd for c18h24n5o2s , 374.1651 ; found , 374.1646 . to a 
 solution of 2,4-dichloropyrimidine ( 22 ) ( 2.0 g , 
 0.0134 mmol ) in toluene ( 20 ml ) were added benzyl alcohol ( 1.53 ml , 
 1.59 g , 0.0147 mol ) , koh ( 0.82 g , 0.0147 mol ) , and 18-crown-6 ( 0.177 
 g , 0.00067 mol ) , and the resulting solution was stirred at rt for 
 1 h. the reaction mixture was diluted with etoac ( 400 ml ) , washed 
 with water ( 3  50 ml ) , dried over mgso4 , filtered , 
 and concentrated to give a white solid that was chromatographed ( hexane / ch2cl2 , 1:1 to 3:7 ) to afford 2.09 g ( 71% ) of a mixture 
 of 23a with regioisomeric 2-(benzyloxy)-4-chloropyrimidine 
 ( relative ratio 75:25 by h nmr , respectively ) . ms ( m / z ) : [ m + na ] 243.1 . to a solution of 23a ( 2.09 
 g , 0.00947 mol ; 
 contains regioisomer ) in dmf ( 34 ml ) was added 1-methylpiperazine 
 ( 3.15 ml , 2.85 g , 0.0284 mol ) , and the resulting solution was heated 
 at 80 c for 1.75 h. solvent was removed under reduced pressure , 
 and the residue was taken up into etoac ( 350 ml ) and washed with brine 
 ( 3  50 ml ) .
the aqueous layer was extracted with etoac ( 2  
 50 ml ) , and the combined organic layers were dried over mgso4 , filtered , and concentrated to give an oil that was purified by 
 column chromatography ( etoac / meoh  nh3 ( 7 n ) , 1:0 
 to 25:1 ) to afford 1.88 g ( 70% ) of 24a .
h 
 nmr ( 500 mhz , cdcl3 ) :  8.06 ( d , j = 5.6 hz , 1h ) , 7.41 ( d , j = 7.0 hz , 2h ) , 7.35 ( t , j = 7.0 hz , 2h ) , 7.32 ( d , j = 7.0 hz , 1h ) , 
 6.03 ( d , j = 5.6 hz , 1h ) , 5.35 ( s , 2h ) , 3.83 ( m , 
 4h ) , 2.45 ( m , 4h ) , 2.33 ( s , 3h ) . ms ( m / z ) : [ m + h ] 284.9 . to 24a ( 0.937 g , 0.0033 mol ) 
 in 
 acetonitrile ( 16 ml )
were added tfa ( 1.02 ml , 1.51 g , 0.0132 mol ) 
 and n - iodosuccinimide ( 0.965 g , 0.0043 mol ) , and 
 the resulting solution was stirred at rt for 1 h. then 7 ml of 10% 
 na2co3 ( 0.70 g , 0.066 mol ) was added , and the 
 resulting solution was stirred for 2 min .
the reaction mixture was 
 concentrated to dryness , and the residue was taken up into ch2cl2 ( 200 ml ) and washed with 10% na2co3 ( 2  50 ml ) , 10% sodium thiosulfate ( 50 ml ) , 
 and brine ( 50 ml ) .
the organic layer was dried over mgso4 , filtered , and concentrated to give an oil which was purified by 
 column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 50:1 ) to yield 1.31 g ( 97% ) of 25a .
h nmr ( 500 mhz , cdcl3 ) :  8.27 ( s , 1h ) , 7.44 
 ( d , j = 7.4 hz , 2h ) , 7.37 ( t , j = 
 7.2 hz , 2h ) , 7.32 ( d , j = 7.3 hz , 1h ) , 5.40 ( s , 2h ) , 
 3.79 ( m , 4h ) , 2.42 ( m , 4h ) , 2.32 ( s , 3h ) . ms ( m / z ) : [ m + h ] 411.0 .
a mixture of 25a ( 0.985 g , 
 2.40 mmol ) , 3-aminobenzenethiol ( 255 l , 300.5 mg , 2.40 mmol ) , 
 neocuproine ( 150 mg , 0.72 mmol ) , copper iodide ( 137 mg , 0.72 mmol ) , 
 and potassium carbonate ( 0.663 g , 4.80 mmol ) in dmf ( 25 ml ) was stirred 
 at 120 c for 18 h. solvent was removed under reduced pressure , 
 and the residue was purified by column chromatography ( ch2cl2/meoh  nh3 ( 7 n ) , 50:1 to 20:1 ) to 
 afford 0.75 g ( 77% ) of 26a .
h nmr ( 500 mhz , 
 cdcl3 ) :  8.24 ( s , 1h ) , 7.167.30 ( m , 5h ) , 
 6.99 ( t , j = 7.3 hz , 1h ) , 6.56 ( d , j = 7.6 hz , 1h ) , 6.386.46 ( m , 2h ) , 5.38 ( s , 2h ) , 3.84 ( m , 
 4h ) , 3.56 ( br s , 2h ) , 2.45 ( br s , 4h ) , 2.33 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  169.3 , 164.5 , 161.4 , 158.1 , 
 146.9 , 138.5 , 136.8 , 129.6 , 128.3 , 127.7 , 127.4 , 117.6 , 113.6 , 112.6 , 
 67.6 , 54.8 , 46.2 , 43.9 . ms ( m / z ) : 
 [ m + h ] 408.1 . to 26a ( 5.2 mg , 0.013 mmol ) 
 in ch2cl2 ( 0.2 ml ) was added acetic anhydride 
 ( 1.5 l , 1.6 mg , 0.0156 mmol ) , and the resulting solution was 
 stirred at rt for 4 h. the solution was then concentrated to dryness 
 under reduced pressure to give a residue which was purified by preparatory 
 tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 
 20:1 ) to afford 4.5 mg ( 79% ) of 27a .
h nmr 
 ( 500 mhz , cdcl3/meoh - d4 ) :  
 8.24 ( s , 1h ) , 7.54 ( d , j = 7.7 hz , 1h ) , 7.227.27 
 ( m , 3h ) , 7.137.20 ( m , 3h ) , 7.09 ( s , 1h ) , 6.87 ( d , j = 7.7 hz , 1h ) , 5.36 ( s , 2h ) , 3.85 ( m , 4h ) , 2.49 ( m , 4h ) , 
 2.35 ( s , 3h ) , 2.10 ( s , 3h ) . ms ( m / z ) : [ m + h ] 450.1 .
26a ( 14 mg , 0.034 mmol ) , 
 et3n ( 24 l , 17.2 mg , 0.17 mmol ) , and benzoyl chloride 
 ( 12 l , 14.3 mg , 0.102 mmol ) in ch2cl2 ( 1 ml ) were stirred at rt for 2 h. solvent was removed under reduced 
 pressure , and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 25:1 ) to 
 afford 13.6 mg ( 78% ) of 27b .
h nmr ( 500 mhz , 
 cdcl3/meoh - d4 ) :  8.26 
 ( s , 1h ) , 7.84 ( d , j = 7.4 hz , 2h ) , 7.69 ( dd , j = 1.4 , 8.2 hz , 1h ) , 7.54 ( t , j = 7.3 
 hz , 1h ) , 7.47 ( t , j = 7.9 hz , 2h ) , 7.147.27 
 ( m , 7h ) , 6.93 ( dd , j = 0.9 , 7.9 hz , 1h ) , 5.37 ( s , 
 2h ) , 3.85 ( m , 4h ) , 2.48 ( m , 4h ) , 2.35 ( s , 3h ) .
c nmr 
 ( 125 mhz , cdcl3/meoh - d4 ) :  
 168.6 , 166.3 , 164.2 , 161.3 , 138.6 , 138.3 , 136.4 , 134.8 , 131.8 , 129.3 , 
 128.6 , 128.3 , 127.7 , 127.5 , 127.2 , 123.4 , 118.8 , 117.9 , 100.1 , 67.8 , 
 54.6 , 45.9 , 43.6 . ms ( m / z ) : [ m + 
 h ] 512.1 . to 26a ( 30 mg , 0.0736 mmol ) 
 in thf ( 3 ml ) were added boc - glycine ( 14.2 mg , 0.081 mmol ) and dcc 
 ( 16.7 mg , 0.081 mmol ) , and the resulting solution was stirred at rt 
 overnight .
the reaction mixture was concentrated under reduced pressure , 
 the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 25:1 ) to afford an oil 
 which was dissolved in 2.5 ml of ch2cl2/tfa 
 ( 4:1 ) , and the resulting solution was stirred at rt for 45 min .
the 
 reaction mixture was concentrated under reduced pressure , and the 
 residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 15:1 ) to afford 24.6 mg ( 72% ) 
 of 27c .
h nmr ( 500 mhz , cdcl3/meoh - d4 ) :  8.24 ( s , 1h ) , 7.50 ( dd , j = 1.2 , 8.1 hz , 1h ) , 7.31 ( m , 1h ) , 7.227.27 ( m , 
 3h ) , 7.137.20 ( m , 3h ) , 6.86 ( d , j = 7.9 hz , 
 1h ) , 5.37 ( s , 2h ) , 3.85 ( m , 4h ) , 3.39 ( s , 2h ) , 2.49 ( m , 4h ) , 2.35 
 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3/meoh - d4 ) :  171.4 , 168.5 , 164.1 , 161.2 , 138.2 , 
 138.1 , 136.4 , 129.2 , 128.2 , 127.6 , 127.2 , 123.0 , 118.2 , 117.0 , 67.7 , 
 54.5 , 45.8 , 44.6 , 43.5 . ms ( m / z ) : 
 [ m + h ] 465.3 . to 26a ( 8.4 mg , 0.0206 mmol ) 
 in thf ( 0.5 ml ) were added boc - l - alanine ( 5.6 mg , 0.0227 
 mmol ) and dcc ( 4.7 mg , 0.0227 mmol ) , and the resulting solution was 
 stirred at rt overnight .
the reaction mixture was concentrated under 
 reduced pressure , the residue was dissolved in 0.35 ml of ch2cl2/tfa ( 4:1 ) , and the resulting solution was stirred 
 at rt for 45 min .
the reaction mixture was concentrated under reduced 
 pressure , and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 15:1 ) to 
 afford 4.1 mg ( 41% ) of 27d .
h nmr ( 500 mhz , 
 cdcl3 ) :  9.34 ( s , 1h ) , 8.26 ( s , 1h ) , 7.59 ( d , j = 8.2 hz , 1h ) , 7.207.30 ( m , 4h ) , 7.127.19 
 ( m , 3h ) , 6.85 ( m , 1h ) , 5.37 ( s , 2h ) , 3.813.91 ( m , 4h ) , 3.60 
 ( q , j = 7.0 hz , 1h ) , 2.432.52 ( m , 4h ) , 2.35 
 ( s , 3h ) , 1.42 ( d , j = 7.0 hz , 3h ) . ms ( m / z ) : [ m + h ] 479.4 . to 26a ( 6.6 mg , 0.0162 mmol ) 
 in thf ( 0.5 ml ) were added boc - l - valine ( 4.1 mg , 0.0178 mmol ) 
 and dcc ( 3.7 mg , 0.0178 mmol ) , and the resulting solution was stirred 
 at rt overnight .
the reaction mixture was concentrated under reduced 
 pressure , the residue was dissolved in 0.35 ml of ch2cl2/tfa ( 4:1 ) , and the resulting solution was stirred at rt for 
 45 min .
the reaction mixture was concentrated under reduced pressure , 
 and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 15:1 ) to afford 4.4 mg 
 ( 54% ) of 27e .
h nmr ( 500 mhz , cdcl3 ) :  9.39 ( s , 1h ) , 8.26 ( s , 1h ) , 7.60 ( d , j = 8.0 hz , 1h ) , 7.207.34 ( m , 4h ) , 7.127.19 ( m , 3h ) , 
 6.84 ( d , j = 7.8 hz , 1h ) , 5.37 ( s , 2h ) , 3.813.92 
 ( m , 4h ) , 3.36 ( m , 1h ) , 2.392.52 ( m , 5h ) , 2.35 ( s , 3h ) , 1.04 
 ( d , j = 6.8 hz , 3h ) , 0.86 ( d , j = 
 6.7 hz , 3h ) . ms ( m / z ) : [ m + h ] 507.2 . to 26a ( 8.6 mg , 0.0211 mmol ) 
 in thf ( 0.5 ml ) were added boc - l - proline ( 5 mg , 0.0232 mmol ) 
 and dcc ( 5 mg , 0.0232 mmol ) , and the resulting solution was stirred 
 at rt overnight .
the reaction mixture was concentrated under reduced 
 pressure , the residue was dissolved in 0.35 ml of ch2cl2/tfa ( 4:1 ) , and the resulting solution was stirred at rt for 
 45 min .
the reaction mixture was concentrated under reduced pressure , 
 and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 15:1 ) to afford 6.0 mg 
 ( 57% ) of 27f .
h nmr ( 500 mhz , cdcl3 ) :  9.63 ( s , 1h ) , 8.26 ( s , 1h ) , 7.58 ( dd , j = 1.2 , 8.1 hz , 1h ) , 7.31 ( t , j = 1.9 hz , 1h ) , 7.127.17 
 ( m , 3h ) , 7.227.26 ( m , 3h ) , 6.83 ( d , j = 7.9 
 hz , 1h ) , 5.36 ( s , 2h ) , 3.803.91 ( m , 5h ) , 3.043.11 
 ( m , 1h ) , 2.943.00 ( m , 1h ) , 2.47 ( m , 4h ) , 2.35 ( s , 3h ) , 2.152.25 
 ( m , 1h ) , 1.982.06 ( m , 1h ) , 1.701.81 ( m , 1h ) .
ms ( m / z ) : [ m + h ] 505.2 . to a 
 mixture of 22 ( 50 mg , 0.336 mmol ) , phenylboronic acid 
 ( 41 mg , 0.336 mmol ) , sodium carbonate ( 110 mg in 0.5 ml of water ) , 
 and dme ( 2.5 ml ) were added palladium acetate ( 3.8 mg , 0.0168 mmol ) 
 and triphenylphosphine ( 8.8 mg , 0.0336 mmol ) .
the reaction mixture 
 was heated at 95 c for 20 h. solvent was removed under reduced 
 pressure and the residue taken up into dichloromethane ( 20 ml ) , washed 
 with water ( 3  5 ml ) , dried over mgso4 , and concentrated 
 to give a residue which was purified by preparatory tlc ( hexane / etoac , 
 8:2 ) to yield 41 mg ( 64% ) of 23b .
h nmr ( 500 
 mhz , cdcl3 ) :  8.63 ( d , j = 5.2 
 hz , 1h ) , 8.068.11 ( m , 2h ) , 7.64 ( d , j = 5.3 
 hz , 1h ) , 7.477.57 ( m , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  167.2 , 161.9 , 159.8 , 135.1 , 131.9 , 129.1 , 127.4 , 
 115.2 .
to a solution of 23b ( 38 mg , 0.201 mmol ) 
 in 0.5 ml of dmf was added 1-methylpiperazine ( 56 l , 50 mg , 
 0.31 mmol ) , and the resulting solution was heated at 90 c for 
 1.5 h. solvent was removed under reduced pressure , and the residue 
 was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 20:1 ) to yield 49 mg ( 95% ) of 24b .
h nmr ( 500 mhz , cdcl3 ) :  8.36 ( d , j = 5.2 hz , 1h ) , 8.018.09 ( m , 2h ) , 7.437.50 
 ( m , 3h ) , 6.92 ( d , j = 5.2 hz , 1h ) , 3.96 ( m , 4h ) , 
 2.50 ( m , 4h ) , 2.34 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  164.4 , 162.1 , 158.4 , 137.8 , 130.6 , 128.8 , 127.1 , 
 105.8 , 55.2 , 46.4 , 43.9 . ms ( m / z ) : [ m + h ] 255.1 . to 24b ( 49 mg , 0.193 mmol ) 
 in acetonitrile 
 ( 1.4 ml ) were added tfa ( 59 l , 88 mg , 0.772 mmol ) and n - iodosuccinimide ( 43 mg , 0.193 mmol ) , and the resulting 
 solution was stirred at rt for 1 h. solvent was evaporated , and the 
 residue was taken up into dichloromethane ( 15 ml ) , washed with 10% 
 na2co3 ( 2  5 ml ) and water ( 5 ml ) , dried 
 over mgso4 , and concentrated to give a residue which was 
 purified by preparatory tlc ( ch2cl2/meoh , 10:1 ) 
 to yield 67 mg ( 92% ) of 25b .
h nmr ( 500 mhz , 
 cdcl3 ) :  8.60 ( s , 1h ) , 7.657.73 ( m , 2h ) , 
 7.427.49 ( m , 3h ) , 3.86 ( m , 4h ) , 2.45 ( m , 4h ) , 2.33 ( s , 3h ) .
c nmr ( 125 mhz , cdcl3 ) :  167.5 , 165.8 , 
 160.9 , 140.3 , 129.7 , 129.3 , 128.1 , 76.2 , 55.0 , 46.4 , 43.9 . ms ( m / z ) : [ m + h ] 380.9 . a mixture of 25b ( 37.6 mg , 
 0.099 mmol ) ,
3-aminobenzenethiol ( 12 l , 13.6 mg , 0.109 mmol ) , 
 neocuproine ( 6.2 mg , 0.0297 mmol ) , copper iodide ( 5.7 mg , 0.0297 mmol ) , 
 and potassium carbonate ( 42 mg , 0.198 mmol ) in dmf ( 1.4 ml ) was stirred 
 at 110 c for 12 h. solvent was removed under reduced pressure , 
 and the residue was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 10:1 ) to afford 10 mg ( 27% ) 
 of 26b .
h nmr ( 500 mhz , cdcl3 ) : 
  8.45 ( s , 1h ) , 7.71 ( d , j = 6.6 hz , 2h ) , 7.327.42 
 ( m , 3h ) , 6.99 ( t , j = 7.9 hz , 1h ) , 6.47 ( d , j = 7.8 hz , 1h ) , 6.43 ( dd , j = 2.0 , 6.4 
 hz , 1h ) , 6.36 ( d , j = 1.8 hz , 1h ) , 3.97 ( m , 4h ) , 
 3.60 ( br s , 2h ) , 2.52 ( m , 4h ) , 2.37 ( s , 3h ) . ms ( m / z ) : [ m + h ] 378.1 . to a solution of 26b ( 5 mg , 
 0.0133 mmol ) in thf ( 0.5 ml ) were added boc - glycine ( 2.3 mg , 0.0133 
 mmol ) and dcc ( 3 mg , 0.0146 mmol ) .
after the resulting solution was 
 stirred for 2 h at rt , thf was evaporated , and 0.5 ml of ch2cl2/tfa ( 4:1 ) was added .
the solution was stirred for 
 45 min and then concentrated to dryness under reduced pressure to 
 give a residue which was purified by preparatory tlc ( ch2cl2/meoh  nh3 ( 7 n ) , 10:1 ) to afford 
 2 mg ( 35% ) of 27 g .
h nmr ( 500 mhz , cdcl3/meoh - d4 ) :  8.45 ( s , 1h ) , 
 7.69 ( d , j = 8.2 hz , 2h ) , 7.357.45 ( m , 4h ) , 
 7.31 ( m , 1h ) , 7.17 ( t , j = 8.0 hz , 1h ) , 6.76 ( d , j = 7.8 hz , 1h ) , 3.96 ( m , 4h ) , 3.36 ( s , 2h ) , 2.56 ( m , 4h ) , 
 2.38 ( s , 3h ) . ms ( m / z ) : [ m + h ] 435.0 .
neal rosen 
 ( memorial sloan - kettering cancer center , mskcc ) and kasumi-1 and molm-13 
 from dr .
cells were cultured routinely in dme / f12 
 ( skbr3 , mda - mb-468 , mia - paca-2 ) or in rpmi ( kasumi-1 , moml-13 ) supplemented 
 with 10% fetal bovine serum , 1% l - glutamine , 1% penicillin , 
 and streptomycin .
cells were grown 
 to 6070% 
 confluence and treated with inhibitor or dmso vehicle for the indicated 
 times .
protein lysates were prepared in 50 mm tris , ph 7.4 , 150 mm 
 nacl , and 1% np-40 lysis buffer .
protein concentrations were measured 
 using the bca kit ( pierce ) according to the manufacturer s 
 instructions .
page , transferred onto a nitrocellulose membrane , and 
 incubated with the indicated primary antibodies : anti - her2 from rabbit 
 ( 1:250 , 28 - 0004 , zymed ) , anti - raf-1 from rabbit ( 1:500 , sc-133 , santa 
 cruz ) , anti - parp ( p85 fragment ) from rabbit ( 1:500 , g7341 , promega ) , 
 anti--actin from mouse ( 1:2500 , a1978 , sigma - aldrich ) , anti - akt 
 from rabbit ( 1:500 , 9272 , cell signaling ) , anti - p - stat3 ( y705 ) from 
 rabbit ( 1:500 , 9145 , cell signaling ) , anti - p - stat5 ( y694 ) from rabbit 
 ( 1:500 , 9351 , cell signaling ) , anti - flt3 form rabbit ( 1:500 , sc-480 , 
 santa cruz ) , anti - hop from mouse ( 1:500 , sra-1500 , enzo ) , and anti - hsp / c70 
 from mouse ( 1:2000 , smc-106b , stressmarq ) .
membranes were then incubated 
 with a corresponding peroxidase - conjugated secondary antibody ( 1:3000 
 dilution ) . for the competition 
 studies , fluorescence 
 polarization ( fp ) assays
briefly , fp measurements were performed on an 
 analyst gt instrument ( molecular devices , sunnyvale , ca ) .
3650 ) where 
 both the excitation and the emission occurred from the top of the 
 wells . a stock of 10 m gm - cy3b was prepared in dmso and diluted 
 with felts buffer ( 20 mm hepes ( k ) , ph 7.3 , 50 mm kcl , 2 mm dtt , 5 
 mm mgcl2 , 20 mm na2moo4 , and 0.01% 
 np40 with 0.1 mg / ml bgg ) .
to each 96-well plate were added 6 nm fluorescent 
 gm ( gm - cy3b ) , 3 g of skbr3 lysate ( total protein ) , and tested 
 inhibitor ( initial stock in dmso ) in a final volume of 100 l 
 of hfb buffer .
background wells ( buffer only ) , tracer controls ( free , fluorescent 
 gm only ) , and bound gm controls ( fluorescent gm in the presence of 
 skbr3 lysate ) were included on each assay plate .
the assay plate was incubated on a shaker at 4 c 
 for 24 h , and the fp values ( mp ) were measured .
the fraction of tracer 
 bound to hsp90 was correlated to the fp value and plotted against 
 the values of competitor concentrations .
the inhibitor concentration 
 at which 50% of bound gm was displaced was obtained by fitting the 
 data .
all experimental data were analyzed using softmax pro 4.3.1 
 and plotted using prism 4.0 ( graphpad software inc . , san diego , ca ) .
this reagent offers 
 a rapid objective measure of cell viability in cell culture , and it 
 uses the indicator dye resazurin to measure the metabolic capacity 
 of cells , an indicator of cell viability .
briefly , cells were plated 
 on costar 96-well plates . for attached cells ( such as skbr3 ) , 8000 
 cells / well were used . for suspension cells ( such as kasumi-1 ) , 20000 
 cells / well were plated .
cells were allowed to incubate for 24 h at 
 37 c before drug treatment .
drugs were added in triplicate at 
 the indicated concentrations , and the plate was incubated for 72 h. 
 alamar blue ( 50 m ) was added and the plate read 6 h later using 
 the analyst gt ( fluorescence intensity mode , excitation 530 nm , emission 
 580 nm , with a 560 nm dichroic mirror ) .
the percentage cell growth inhibition was 
 calculated by comparing fluorescence readings obtained from treated 
 versus control cells , accounting for the initial cell population ( time 
 zero ) .
molm-13 cells ( 30000 cells / well ) were 
 plated in black 96-well plates 
 ( corning no .
3603 ) in 40 l of rpmi medium and left in an incubator 
 ( 37 c , 5% co2 ) for up to 24 h. cells were treated 
 for 16 h with compounds or dmso ( control ) at the desired concentrations 
 in 50 l of medium .
following exposure of cells to hsp70 inhibitors , 
 50 l of buffer containing 10 mm hepes ( ph 7.5 ) , 2 mm edta , 
 0.1% chaps , and the caspase substrate z - devd - r110 at 25 m was 
 added to each well .
plates were incubated until the signal stabilized , 
 and then the fluorescence signal of each well was measured in an analyst 
 gt microplate reader .
the percentage increase in apoptotic cells was 
 calculated by comparison of the fluorescence reading obtained from 
 treated versus control cells .
skbr3 cells were 
 treated with the indicated concentrations of the inhibitor for 24 
 h. samples were collected and lysed in 20 mm tris , ph 7.4 , 25 mm nacl , 
 0.1% np-40 buffer with protease inhibitors added .
aliquots of 500 
 g of total protein adjusted to 100 l with the lysis 
 buffer were prepared .
samples were incubated with 5 l of bb70 
 antibody ( stressmarq ) or normal igg ( as a negative control ) and 20 
 l of protein g agarose beads ( upstate ) at 4 c overnight . 

samples were washed five times with the lysis buffer and applied to 
 sds  page followed by a standard western blotting procedure 
 to detect levels of hop protein in the hsp / c70 complexes upon treatment .
hek293 cells were transfected with 2 g of luciferase 
 plasmid and 8 g of hsp70 plasmid or control vector per 60 mm 
 dish .
human hsp70 , hsp70-c267s , and hsc70 were n - terminally flag - tagged , 
 and luciferase was c - terminally ha - tagged in pcdna3.1 .
two days after 
 transfection , cells were treated with cycloheximide ( sigma ) at 50 
 g / ml to inhibit protein synthesis , transferred to 45 c 
 for 1 h , and then brought back to 37 c for up to 2 h of recovery . 27c
cell samples , 
 taken before heat shock and at 0 , 1 , and 2 h of recovery , were lysed 
 on ice with pbs containing 1% triton x-100 , and the insoluble material 
 was removed by centrifugation at 20000 g .
total protein 
 amounts in the supernatants were determined using the bca protein 
 assay kit ( pierce ) .
luciferase enzymatic activities in the supernatants 
 were measured using the luciferase reporter assay kit ( promega ) and 
 activity values normalized to total protein amounts .
cells were treated with cycloheximide 
 ( at a final concentration of 100 g / ml ) with added vehicle ( dmso ) 
 or 27c ( 20 m ) for the indicated times .
cells were 
 lysed as indicated above , and the resulting samples were analyzed 
 by western blotting .
 the 
 discovery and development of heat shock protein 70 ( hsp70 ) 
 inhibitors is currently a hot topic in cancer . in the preceding paper 
 in this issue ( 10.1021/jm401551n ) , we have 
 described structure  activity relationship studies in the first 
 hsp70 inhibitor class rationally designed to bind to a novel allosteric 
 pocket located in the n - terminal domain of the protein . 
 these ligands 
 contained an acrylamide to take advantage of an active cysteine embedded 
 in the allosteric pocket and acted as covalent protein modifiers upon 
 binding . here , we perform chemical modifications around the irreversible 
 inhibitor scaffold to demonstrate that covalent modification is not 
 a requirement for activity within this class of compounds . 
 the study 
 identifies derivative 27c , which mimics the biological 
 effects of the irreversible inhibitors at comparable concentrations . 
 
 collectively , the back - to - back manuscripts describe the first pharmacophores 
 that favorably and selectively interact with a never explored pocket 
 in hsp70 and provide a novel blueprint for a cancer - oriented development 
 of hsp70-directed ligands . 