the 
 key effector cells of the immune system , cytotoxic t lymphocytes 
 ( ctl ) , and natural killer ( nk ) cells eliminate virus - infected and 
 transformed cells principally through the granule exocytosis pathway .
ctls and nk cells contain secretory vesicles ( granules ) that are 
 used to store various cytotoxic proteins , including a group of proapoptotic 
 serine proteases ( granzymes ) and perforin , 
 a pore - forming glycoprotein .
stable conjugation with a target cell results in 
 exocytic delivery of the granule contents into the immune synapse 
 where perforin facilitates entry of the granzymes into the target 
 cell , triggering various apoptotic death mechanisms .
the crystal 
 structure of monomeric murine perforin has been determined 
 to 2.75  resolution and reveals a bent and twisted four - stranded 
 -sheet macpf domain flanked by two clusters of -helices , 
 an epidermal growth factor ( egf ) domain , and a c - terminal c2 domain 
 that mediates initial , calcium - dependent membrane - binding .
the mechanism of pore formation has also been 
 elucidated ; upon exposure of perforin to the neutral , calcium - rich 
 extracellular environment of the immune synapse , deprotonation of 
 key aspartate residues and subsequent coordination of calcium takes 
 place .
this process triggers conformational changes that result in 
 the assembly of highly ordered oligomers of 1924 subunits 
 that form a pore with an internal diameter of 130200  . 

site directed mutagenesis has established that direct ionic interactions 
 between the opposing faces of adjacent perforin monomers ( arg213 and 
 glu343 in particular ) serve to assist pore self - assembly and stabilization 
 of the resulting pore .
perforin is encoded on a single - copy gene in 
 both mice and humans , and while many of 
 the granule components possess 
 at least some degree of redundancy , perforin is absolutely essential 
 for protective immunosurveillance .
perforin knockout mice show a reduced 
 ability to reject many tumor xenografts , and when backcrossed with 
 nonobese diabetic mice , the incidence of spontaneous diabetes is reduced 
 from 77% in a perforin + /+ control to 16% in perforin - deficient mice , 
 with onset of disease markedly delayed .
this is because perforin is 
 critical to deliver an autoimmune  lethal hit  against 
 the insulin - producing pancreatic  cells .
perforin - deficient mice also demonstrate increased susceptibility 
 and failure to clear many viruses and other intracellular pathogens , 
 and highly aggressive disseminated b cell lymphomas occur in the majority 
 of animals over the age of 12 months . in humans ,
complete loss of perforin function results in familial 
 hemophagocytic lymphohistiocytosis ( fhl ) syndrome , a severe immunoregulatory 
 disorder usually diagnosed in infancy and characterized by severe 
 anemia and hepatosplenomegaly , fever , and thrombocytopenia .
there is abundant evidence to implicate inappropriate perforin 
 activity in a number of human pathologies , including cerebral malaria , 
 insulin - dependent diabetes , juvenile idiopathic arthritis , and postviral 
 myocarditis , as well as therapy - induced conditions such as allograft 
 rejection and graft - versus - host disease .
immunosuppressive 
 agents used to treat these diseases are generally associated with 
 a wide range of side effects , many of which arise from the unintended 
 impact of the drug treatment on nontherapeutic targets .
as perforin is expressed only by  killer lymphocytes  , 
 a selective inhibitor should result in fewer off - target effects , meaning 
 a small molecule inhibitor of perforin is of great interest as a new 
 class of highly specific immunosuppressive agents . in our initial work in this area , we explored two 
 series of compounds 
 ( 1 and 2 , figure 1 ) that showed appreciable 
 perforin - inhibitory activity but possessed limitations such as poor 
 aqueous solubility or reduced activity in serum , precluding further 
 development .
we have substantially overcome 
 these issues in our current series of 5-arylidene-2-thioxoimidazolidin-4-one - based 
 inhibitors ( 3 , 4 ) and now seek to maximize inhibitory activity and optimize  druglike  
 properties .
this class of rhodanine - related heterocycle has recently 
 become the subject of considerable debate in the medicinal chemistry 
 community because of several publications classifying such substructures 
 as either  privileged scaffolds  with potentially valuable 
 biomolecular binding properties or 
frequent / promiscuous 
 hitters  and pan - assay interference compounds ( pains ) which 
 should not be pursued further . while it is clear that these 
 chemotypes should be carefully monitored in development , recent literature 
 contains many examples of selective inhibitors of various target proteins 
 containing such substructures .
in addition , many of these moieties 
 also occur in drugs that are clinically useful , providing templates 
 with valuable structural features favoring protein binding which may 
 be exploited in an advantageous way .
nevertheless , our current lead 
 compound was subjected to and passed the pains filters ( professor jonathan baell , personal correspondence ) possibly because of the presence of an extra 
 nitrogen in the core five - membered ring .
herein we will show that we have identified an exciting new 
 class 
 of inhibitors that unambiguously target perforin , offering up a novel 
 mode of action and the potential to be further developed as immunosuppressive 
 agents for the effective treatment of transplant rejection and selected 
 autoimmune diseases .
lead compound 3 was selected from a small number of 
 hits that showed reproducible perforin - inhibitory activity in a high 
 throughput screen of approximately 100  000 compounds sourced 
 from commercial libraries .
compound 3 can be readily disconnected into three subunits , a 2-thioxoimidazolidin-4-one 
 ( a ) , a furan ( b ) , and a benzofuranone ( c ) , lending itself to a study 
 design where one unit can be independently varied in the presence 
 of two other fixed subunits .
we have reported previously our study 
 of the structure  activity relationships ( sar ) resulting from 
 variation of the a- and b - subunits , and 
 here we focus on the effect of introducing a wide range of c - subunits . 

since thiophene 4 was one of the more potent compounds 
 in our initial work , the current study explores the effect of replacing 
 the benzofuranone c - subunit on a fixed 2-thioxoimidazolidin-4-one / thiophene 
 scaffold . the requirement for substitution on the c - subunit aryl ring 

is explored , followed by a more detailed investigation focusing on 
 compounds containing a bicyclic moiety .
the compounds of tables 1 , 2 , and 3 were prepared by heating a 
 range of substituted aldehydes 
 with 2-thioxoimidazolidin-4-one in acetic acid in the presence of 
 -alanine ( schemes 15 ) . as a consequence , the majority of the chemistry 
 that is described here involves the preparation of key aldehyde intermediates . 
 for the compounds of table 1 ,
a small number 
 of protected aldehydes ( 68 ) were 
 prepared by suzuki reaction of 2-(5-bromothiophen-2-yl)-1,3-dioxolane 
 ( 5 ) with substituted phenylboronic acids , followed by 
 deprotection ( scheme 1 ) .
the remaining aldehydes 
 were commercially available with the exception of amides 3442 , which were prepared from carboxylic acid 28 using standard amide coupling conditions .
table 2 contains compounds where the benzofuranone 
 of 3 has been replaced with a variety of substituted 
 isoindolinones ( 118135 , 165 , 166 ) and 3,4-dihydroisoquinolin-1(2h)-ones ( 167170 ) .
the isoindolinone 
 targets were prepared by bromination of methyl ester 76 , followed by ring closure onto a variety of amines to give intermediate 
 iodides 7781 ( scheme 2 ) .
these iodides were 
 then employed in a palladium - catalyzed reaction using an agno3/kf activator system , as originally described by mori et al .
coupling of the iodides with either 2-(thiophen-2-yl)-1,3-dioxolane 
 or 2-(dimethoxymethyl)thiophene at the vacant 5-position of the thiophene 
 gave the desired protected aldehydes 8286 .
compound 82 was further modified through 
 nah / rx alkylations of the isoindolinone nh to afford 8792 .
the nme derivative 87 was alkylated 
 a second time using lda and methyl iodide to give methylation on the 
 isoindolinone ch2 ( 93 ) in 65% yield .
deprotections 
 of 8293 were carried out under acidic 
 conditions to give the required aldehydes 94106 which were then reacted with 2-thioxoimidazolidin-4-one 
 to give final compounds 118130 and 135 . likewise 136 ( table 3 ) was obtained by reaction of aldehyde 82 with imidazolidine-2,4-dione . 

a small set of amino - substituted compounds ( 131134 ) were also prepared from the c3 alcohol 101 .
then the alcohol converted 
 to the mesylate 108 using mesyl chloride and tea in thf . 

the mesylate was exchanged for iodide by heating with a large excess 
 of nai in acetone .
then the iodide of 109 was displaced 
 with various amines to give intermediates 110113 .
deprotection as described above gave the key aldehydes 114117 which were reacted with 2-thioxoimidazolidin-4-one 
 to give final compounds 131134 .
scheme 3 describes 
 the preparation of  reverse  and five- and six - membered 
 lactams 165170 ( table 2 ) .
the five - membered lactam boronates 147 and 148 were prepared from the corresponding bromides 137 and 138 by pd - catalyzed reaction with bis(pinacolato)diboron . 

the four six - membered lactam boronates 149152 were prepared in the same manner from the bromides 143146 which were in turn synthesized 
 from the carbamates 139142 according 
 to literature procedures .
the boronates were 
 then each employed in a suzuki reaction with bromide 5 to give compounds 153158 which 
 were deprotected and reacted with 2-thioxoimidazolidin-4-one to afford 
 final compounds 165170 . for comparison 
 with the isoindolinones , a single indole - based compound , 3-acetylindole 178 , was also prepared ( scheme 4 ) .
5-bromoindole 171 was protected 
 and acetylated according to literature procedures , followed by displacement of the benzenesulfonyl protecting group 
 to give the corresponding n - methylindole 174 .
subsequent suzuki coupling , deprotection , and reaction with 2-thioxoimidazolidin-4-one 
 gave target compound 178 .
for the compounds of table 3 , protected 
 bromothiophene- ( 179 ) , bromobenzene- ( 180 , 181 ) , bromopyridine- ( 182 ) , bromoindole- 
 ( 183 , 184 ) , or bromoquinolone- ( 185 ) carboxaldehydes were reacted with either commercially available 
 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-1-one or 
 boronate 149 as described above , deprotected to aldehydes 195203 , and converted to final products 204212 ( scheme 5 ) . in the first 
 instance , the isobenzofuran-1(3h)-one 
 c - subunit was replaced with a large range of substituted - benzene derivatives 
 ( table 1 ) .
substitution on this ring is clearly 
 required , with phenyl ( 43 ) possessing no detectable activity . 

each position was then surveyed with a range of substituents , and 
 while methyl and methoxy proved to be inactive ( see supporting information ) , a variety of electron - withdrawing 
 substituents ( halogen , cf3 , cyano , hydroxyl , carboxyl ) 
 did produce activity .
the results for a series of chloro ( 4446 ) , fluoro ( 47 , 48 ) , and cf3 ( 50 , 51 ) compounds 
 show a clear preference for substitution at the 4-position .
carboxy - based 
 examples ( 5961 ) were among the most 
 potent , although in this instance the 3-carboxamide ( 60 , ic50 = 0.79 m ) was slightly better than its 4-isomer 
 ( 61 , ic50 = 1.56 m ) .
methylation of 
 the carboxamide nh of 61 resulted in a reduction of activity 
 ( 67 , 68 ) , while amide - linked weakly basic 
 side chains ( 6971 ) were generally 
 poor . the corresponding amide - linked hydroxylated side chains ( 7275 ) , however , exhibited significant 
 activity ( ic50 = 2.444.35 m ) but were still 
 not as potent as the primary amide 61 .
other compounds 
 with good activity were the 4-br ( 55 , 1.69 m ) , 
 4-sme ( 56 , 1.15 m ) , and 4-acetyl ( 57 , 1.67 m )
 analogue of 4 , compound 62 , showed a 4-fold reduction in activity 
 over its cyclic counterpart ( ic50 = 3.03 m compared 
 to 0.78 m ) .
testing was carried out over a range 
 of concentrations , with the ic50 being equal to the concentration 
 at which 50% inhibition of the lysis of jurkat cells by purified recombinant 
 perforin was observed , as measured by cr release .
although the above study ( table 1 ) produced 
 some potent compounds , none were an improvement on lead compound 4 .
an alternative approach where the entire isobenzofuran-1(3h)-one unit was replaced with an isoindolin-1-one was therefore 
 implemented ( table 2 ) , resulting in a series 
 of compounds considered less susceptible to hydrolysis ( amide versus 
 ester ) and where the nitrogen atom provides an additional position 
 at which a side chain can be appended .
isoindolin-1-one 118 showed a reduction of activity 
 over lactone 4 ( from ic50 = 0.78 m 
 to ic50 = 2.55 m ) .
activity was restored through 
 substitution on the nitrogen atom , with both methyl - substituted ( 119 , ic50 = 0.51 m ) and ethyl - substituted 
 ( 120 , ic50 = 0.60 m ) compounds more 
 potent than the lead compound 4 .
a more bulky substituent 
 such as isopropyl ( 123 , ic50 = 4.42 m ) 
 was less acceptable , but the acetate 128 and carbamate 129 both showed excellent activity ( ic50 = 0.93 
 and 0.55 m , respectively ) . in this series it also appears possible 
 to introduce various side chains off the isoindolinone and
maintain 
 activity , with the two alcohols 125 and 126 among the most potent compounds of the class ( ic50 = 
 0.78 and 0.53 m ) .
diol 127 and o - acetate 124 also demonstrated good activity , but when 
 a basic amino substituent was introduced in an attempt to improve 
 solubility , activity was virtually abolished with only morpholine 
 ( 130 , not reproducible ) , piperidine 132 , 
 and pyrrolidine 134 ( ic50 = 11.78 and 8.93 
 m ) showing any evidence of activity
. introduction of a methyl 
 substituent on the lactam ring ( 135 ) resulted in poorer 
 activity than the corresponding unsubstituted analogue ( 135 ic50 = 3.21 m vs 119 ic50 = 0.51 m ) .
since the 3-carboxamide 60 was 
 2-fold more potent 
 than 4-carboxamide 61 in the substituted benzene series 
 ( table 1 ) , the effect of reversing the orientation 
 of the lactam ring was investigated ( 165 ) .
the same result 
 was observed ; the ic50 for the compound with the lactam 
 amide in the 3-position 165 ( relative to the thiophene ) 
 was around 2-fold better than the corresponding lactam amide in the 
 4-position ( 118 ) ( ic50 = 1.38 m compared 
 to 2.55 m ) .
expansion from a five - membered lactam ring ( 118 ) to a six - membered lactam ring ( 167 ) also 
 resulted in a significant increase in activity ( from ic50 = 2.55 to ic50 = 1.14 m ) and improved a further 
 2-fold ( to 0.64 m ) through introduction of the  reverse  
 3-lactam amide ( 168 ) .
n - methylation of 165 , 167 , and 168 to give 166 , 169 , and 170 , respectively , did not result 
 in the same increase in activity seen when 118 was alkylated 
 to give the analogous n - methyl compound 119 .
testing 
 was carried out over a range 
 of concentrations , with the ic50 being equal to the concentration 
 at which 50% inhibition of the lysis of jurkat cells by purified recombinant 
 perforin was observed , as measured by cr release .
compound 178 contains 
 a 3-acetylindole c - subunit , and its synthesis is described in scheme 4 .
compounds 136 and 204212 ( table 3 ) combine those b - subunits which 
 were previously identified as having excellent activity in the isobenzofuranone - based 
 series , with selected isoindolinones 
 and 3,4-dihydroisoquinolin-1(2h)-ones from the current 
 series .
compound 136 contains a imidazolidine-2,4-dione 
 a - subunit and is the only example to show an improvement , being 3-fold 
 better than the analogous isobenzofuranone reported in our initial 
 paper ( ic50 = 0.40 m compared to 1.19 m ) . 

all other compounds ( 204212 ) contain 
 a 2-thioxoimidazolidin-4-one a - subunit and are 1.5- to 4-fold poorer 
 than the corresponding isobenzofuranones ( see figure 1 in supporting information ) .
testing was carried out over a range 
 of concentrations , with the ic50 being equal to the concentration 
 at which 50% inhibition of the lysis of jurkat cells by purified recombinant 
 perforin was observed , as measured by cr release .
the 2-thioxoimidazolidin-4-one 
 a - subunit 
 is replaced with a imidazolidine-2,4-dione . those compounds that showed good inhibition of the 
 purified protein 

were then also evaluated for their ability to inhibit perforin produced 
 by an intact nk cell line ( khyg-1 ; see experimental 
 section for details ) ; this assay is a better model for in vivo 
 studies , as the perforin is delivered by a functional nk cell . to 
 confirm that inhibition of nk cell function was due to blocking the 
 action of perforin and not nonspecific toxicity against the effector 
 cell , viability
was also measured 24 h after the nk cells were exposed 
 to the compounds ( 4 h , 37 c ) , then washed and replated in culture . 

elimination of any nonspecific toxicity is important because ctls 
 and nk cells play a key role in the overall immunological response , 
 meaning that any potential perforin - targeted treatment must allow 
 rapid recovery of these cytotoxic effector cells .
for the purposes 
 of this study , compounds were classified as toxic if nk cell viability 
 fell below 70% at 24 h. the data contained in table 4 were used 
 to identify compounds suitable for advancement into in vivo pharmacokinetic 
 studies , as a prelude to in vivo efficacy testing . in the absence 
 of serum , the majority of compounds possessed similar or improved 
 inhibitory activity compared to previous lead 4 , notably 118 , 135 , and 167 , which all showed 
 excellent activity in this more demanding system ( 56% , 90% , 83% compared 
 to 53% ) .
as reported with previous series , a number of compounds were adversely affected in the presence of 
 serum , precluding their further consideration ( 125 , 126 , 129 , 168 , 207 ) . 

the solubility of all compounds was substantially improved by their 
 conversion to sodium salts , resulting in an enhanced drug profile , 
 although some examples ( 118 , 119 , 129 , 136 , 168 ) still proved too 
 insoluble in this form for administration in vivo .
compounds 119 and 167 also showed some evidence of toxicity 
 toward the nk cells ( 28% and 38% survival at 20 m , respectively ) . 

the compound with the best overall profile for an in vivo candidate 
 was 135 , combining superior activity in the presence 
 and absence of serum ( 90% and 67% , respectively ) with good viability 
 ( 74% ) and aqueous solubility ( 1408 g / ml ) .
in addition , when 167 was tested at a lower concentration of 5 m , the 
 resultant lack of nk toxicity suggested a possible therapeutic window , 
 meaning this compound was also selected as an in vivo candidate .
inhibition by compounds ( 20 m ) 
 of the perforin - induced lysis of k562 target cells when co - incubated 
 with khyg-1 human nk cells ( see experimental section ) .
percent inhibition calculated compared to untreated control . as for footnote a , but in the 
 presence 
 of 10% mouse serum .
viability 
 of khyg-1 nk cells after 
 24 h by trypan blue exclusion assay ( see experimental 
 section ) .
compound 167 was also 
 tested at 5 m to test for activity under less toxic conditions .
blood samples were collected at multiple time - points 
 after dosing at 5 mg / kg in a solution of 20% hydroxypropyl--cyclodextrin 
 by intravenous injection .
both compounds were determined to have acceptable 
 drug exposures ( auc = 1151 and 955 hng / ml , respectively ) , despite 
 high clearance and volume of distribution values ( table 5 ) . on this basis ,
135 and 167 were 
 progressed to maximum tolerated dose ( mtd ) studies by single or multiple 
 doses ( intraperitoneally ) to establish an appropriate level for efficacy 
 studies .
both compounds were found to be well tolerated after a single 
 dose of 80 and 60 mg / kg , respectively , while when administered twice 
 daily over 3 days , 60 and 40 mg / kg were found to be suitable for multiple 
 dosing .
the results were processed using a bolus 
 iv dose and two - compartmental model with phoenix winnonlin 6.2 ( pharsight 
 corporation , st .
the derived parameters are maximum plasma 
 concentration ( cmax ) , the area under the 
 curve ( auc ) , plasma half - life ( t1/2 ) , 
 volume of distribution ( v ) , and clearance ( cl ) .
while several series of potent inhibitors 
 have now been identified , little is known about the mechanism by which the 
 cytolytic activity 
 of perforin action is blocked . in order to find out more about the 
 nature and degree of inhibitor binding ,
recombinant purified mouse 
 perforin was immobilized on a surface plasmon resonance ( spr ) s - series 
 cm5 sensor chip using standard amide coupling methodology .
then solutions 
 of in vivo candidates 135 and 167 in buffer 
 were passed over the covalently bound protein , and relative binding 
 affinities were determined using a biacore t200 spr machine .
inhibitors 135 and 167 were found to bind 
 reversibly to the immobilized perforin , and during injection a steady - state 
 response was achieved within seconds ( figures 2 and 3 in supporting information ) . at the end of the injection 
 the compounds dissociated rapidly with an almost immediate return 
 to baseline resonance response .
any nonspecific binding to the sensor 
 chip surface was accounted for through the response in the reference 
 flow cell .
because of the relative insolubility of the compounds in 
 the running buffer , low analyte concentrations were required ( 2.5 
 m ) and the relative resonance responses at steady state were 
 typically below 10 response units ( ru ) .
however , although the absolute 
 response values were low , it is clear that 135 and 167 can bind to perforin even at low concentrations .
these 
 data demonstrate for the first time that these inhibitors possess 
 the ability to bind reversibly to perforin monomers . prior to this 

experiment it was also not known whether immobilization of perforin 
 monomers to the sensor chip surface via ( random ) amide couplings could 
 render potential binding sites for inhibitors inaccessible .
combined , 
 these findings show that spr biosensor analysis can be a useful technique 
 to determine perforin protein  drug binding interactions . in 
 order to investigate whether 167 inhibited perforin 
 directly in the context of the physiological immune synapse ,
we also 
 used real - time microscopy to analyze the functional interaction between 
 primary human natural killer cells and their cognate model targets , 
 hela cells .
when the killer cell engages with its target cell , a cascade 
 of downstream signaling events leads to the influx of extracellular 
 ca ( as detected by the calcium ionophore fluo-4 ) . as 
 shown recently , ca influx 
 directly precedes degranulation and the release of perforin into the 
 synaptic cleft .
perforin then forms pores in the target cell membrane , 
 which can be detected as an influx of extracellular propidium iodide 
 ( pi ) , a dye that gains fluorescence upon binding to the cytosolic 
 rna .
calcium influx into the killer cell 
 and pi uptake by the target cell thus serve as indicators of a functional 
 immune synapse . in the dmso - treated control cells , every natural killer 
 cell that engaged with its target cell ( n = 50 cells ) 
 delivered functional perforin to the target ( figure 2 ) .
by contrast , in the presence of 20 m 167 , only 60% of killer cells delivered functional perforin to the target . 

formation of the perforin pore was blocked in the remaining 40% of 
 synapses , despite effective target cell engagement ( figure 2 ) .
these data demonstrate that 167 directly 
 inhibits perforin - induced lysis through reduction of cell membrane 
 binding and/or prevention of transmembrane pore formation , thus preventing 
 target cell death .
the 
 current study has resulted in further optimization of a novel 
 new series of small - molecule inhibitors of the pore - forming protein 
 perforin . by building on our previous studies
, we have designed compounds that possess enhanced druglike 
 properties compared to earlier structures . we also report new mechanistic 
 evidence that reveals a specificity for the granule exocytosis pathway , 
 of which perforin is an integral component . structure 
activity 
 relationships for variation of the c - subunit 
 on a 2-thioxoimidazolidin-4-one / thiophene scaffold showed a need for 
 substitution , especially at the 4-position , for simple substituted - benzene 
 derivatives ( table 1 ) . in this series
the 3- 
 and 4-carboxamides 60 and 61 proved to be 
 the most potent , although this was limited to primary amides , as the 
 introduction of n - substitution and extended hydroxyalkyl or aminoalkyl 
 side chains ( 6775 ) resulted in a 
 loss of activity .
the  acyclic  analogue of the lead 
 compound ( 62 ) also showed an almost 4-fold reduction 
 in activity , suggesting retention of a bicyclic c - subunit to be the 
 best approach .
the isobenzofuranone of 4 was therefore 
 replaced with a variety of isomeric isoindolinones and 3,4-dihydroisoquinolin-1(2h)-ones .
n - substitution on the isoindolinone nitrogen was 
 beneficial , with methyl ( 119 ) , ethyl ( 120 ) , hydroxyethyl ( 125 ) , and hydroxypropyl ( 126 ) substituents all demonstrating excellent activity ( ic50 = 0.51 , 0.60 , 0.78 , 0.53 m , respectively ) .
comparable substitution 
 on the 3,4-dihydroisoquinolin-1(2h)-one nitrogen 
 did not improve activity ( comparing 167/169 and 168/170 pairings ) , although these 
 compounds ( 167 , 168 ) still showed potent 
 inhibitory activity in their own right .
lastly , a series of hybrid 
 compounds were prepared ( table 3 ) , combining 
 b - subunits that were previously identified as conferring good activity with the above optimized c - subunits . in this 
 instance , the isoindolinones were all poorer than the corresponding 
 isobenzofuranones ( figure 1 of supporting information ) . a smaller subset of active compounds were then screened 
 for their 
 ability to inhibit perforin - dependent lysis induced by whole nk cells , 
 resulting in the selection of 135 and 167 as suitable candidates for in vivo studies .
problems observed previously , 
 such as poor aqueous solubility , inactivity in serum , and toxicity 
 have been substantially overcome in the present series .
the 
 mechanism of perforin inhibition by these compounds has also 
 been investigated further , and for the first time , compounds with 
 in vitro inhibitory activity have been shown to reversibly bind perforin 
 by spr detection .
target 
 cell interaction demonstrates that the compounds block perforin within 
 the immunological synapse and prevent target cell death but have no 
 effect on the stability of immune synapse formation between the two 
 cells .
these inhibitors almost certainly target perforin after its 
 release into the immune synapse , consistent with the fact that the 
 original screen through which the inhibitors were identified featured 
 the use of purified perforin protein rather than perforin delivered 
 by an intact killer cell .
together , the data we have amassed 
 demonstrate that this series 
 of compounds are selective inhibitors of perforin and not nonspecific 
 promiscuous binders .
we have previously shown that these compounds 
 do not inhibit the mechanistically related pore - forming protein pneumolysin 
 and that we observe a wide range of activities ( 0.36 to > 20 m ) 
 against both isolated perforin and perforin produced by whole nk cells .
furthermore , the membrane - piercing terminal 
 components of the mammalian membrane attack complex of complement 
 ( mac ) , which share significant structural similarity with perforin 
 and alternative , receptor - mediated proapoptoic pathways used by cytotoxic 
 lymphocytes , are not inhibited ( data not shown ) .
these observations 
 are recapitulated in the current report by the clear sar demonstrated 
 by the c - subunit ( ic50 of 0.51 to > 20 m ) , irrespective 
 of the presence of a 5-arylidene-2-thioxoimidazolidin-4-one as the 
 a - subunit .
a range of activities was also observed against perforin 
 released by intact nk cells , a far more rigorous model that requires 
 the compound to block perforin released into the immunological synapse 
 that transiently forms between the killer cell and its target .
our live cell imaging analyses clearly show 
 for the first time that perforin operating within a functional immune 
 synapse , an intricate microenvironment comprising many proteins taking 
 part in various complex signaling pathways , can be selectively targeted 
 to inhibit perforin - dependent apoptosis of the target cell . in summary
, we have taken significant steps toward elucidating 
 the role of perforin in the granule exocytosis pathway , using small - molecule 
 inhibitors to investigate binding affinity and probe the mechanism 
 of membrane interaction and subsequent pore formation .
we have found 
 compounds 135 and 167 to be both potent 
 and soluble inhibitors of perforin in vitro .
these compounds also 
 possess appropriate in vivo pharmacokinetic characteristics and toxicity 
 profiles for their selection as in vivo efficacy candidates in our 
 pursuit of a novel class of perforin inhibitors suitable for the effective 
 treatment of autoimmune - based diseases and transplant rejection .
analyses were performed by the 
 microchemical laboratory , university of otago , dunedin , new zealand . 

nmr spectra were measured on a bruker avance 400 
 spectrometer and referenced to me4si .
mass spectra were 
 recorded either on a varian vg 7070 spectrometer at nominal 5000 resolution 
 or a finnigan mat 900q spectrometer .
full experimental information for all remaining final 
 compounds and intermediates is given in supporting 
 information .
the acetal 5 ( 278 mg , 1.18 mmol ) 
 was then dissolved in toluene ( 12 ml ) , to which was added a suspension 
 of 4-(hydroxymethyl)benzeneboronic acid ( 150 mg , 0.99 mmol ) 
 in etoh ( 4 ml ) . a solution of 2 m na2co3 ( 3 
 ml ) and pd(dppf)cl2 ( 40 mg , 0.05 mmol ) was added , and the 
 entire mixture was heated at reflux under nitrogen for 2 h. when the 
 mixture was cooled , all solvents were removed under reduced pressure 
 and the resulting residue was partitioned between water ( 50 ml ) and 
 ch2cl2 ( 50 ml )
then the combined organic 
 fractions were dried ( na2so4 ) , filtered , and 
 the solvent was removed under reduced pressure to afford a residue 
 which was purified by flash column chromatography on silica gel ( 20% 
 etoac / hexanes as eluant ) to give 6 as a pale yellow solid 
 ( 171 mg , 66% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.60 ( d , j = 8.3 hz , 2 h ) , 7.36 ( d , j = 3.6 hz , 1 h ) , 7.35 ( d , j = 8.4 hz , 
 2 h ) , 7.20 ( d , j = 3.8 hz , 1 h ) , 6.04 ( s , 1 h ) , 5.19 
 ( t , j = 5.6 hz , 1 h ) , 4.51 ( d , j = 5.5 hz , 2 h ) , 4.104.07 ( m , 2 h ) , 3.934.00 ( m , 
 2 h ) . lrms ( apci ) calcd for c14h15o3s 263 ( mh ) , found 263 .
this material contained 
 5% of deprotected aldehyde which was carried into the next 
 step .
compound 6 ( 171 mg , 0.65 mmol ) was dissolved 
 in acetone ( 10 ml ) , to which was added 1 m hcl ( 2 ml ) .
this mixture 
 was stirred at room temperature for 6 h , then concentrated under reduced 
 pressure to afford a pale yellow suspension which was extracted into 
 ch2cl2 ( 2  50 ml ) .
the combined organic 
 fractions were evaporated down to give 24 as a yellow 
 solid ( 142 mg , 100% ) .
h nmr [ 400 mhz , ( cd3)2so ]  9.90 ( s , 1 h ) , 8.03 ( d , j = 3.9 
 hz , 1 h ) , 7.76 ( d , j = 8.3 hz , 2 h ) , 7.72 ( d , j = 4.0 hz , 1 h ) , 7.42 ( d , j = 8.4 hz , 
 2 h ) , 5.26 ( t , j = 5.7 hz , 1 h ) , 4.54 ( d , j = 5.6 hz , 2 h ) .
lrms ( apci ) calcd for c12h11o2s 219 ( mh ) , found 
 219 .
5-(4-(hydroxymethyl)phenyl)thiophene-2-carbaldehyde 
 ( 24 ) ( 140 mg , 0.64 mmol ) , 2-thioxoimidazolidin-4-one 
 ( 89 mg , 0.77 mmol ) , and -alanine ( 69 mg , 0.77 mmol ) were suspended 
 in acoh ( 5 ml ) , and the mixture was heated at reflux for 15 h. when 
 the mixture was cooled , an orange solid crystallized out of solution 
 which was collected by filtration and purified by flash column chromatography 
 on silica gel ( 20% thf / hexanes ) , giving 58 as an orange 
 solid ( 18% ) , mp ( thf / n - pentane ) 230234 c .
h nmr [ 400 mhz , ( cd3)2so ]  12.37 
 ( s , 1 h ) , 11.95 ( s , 1 h ) , 7.83 ( d , j = 4.0 hz , 1 
 h ) , 7.72 ( d , j = 8.3 hz , 2 h ) , 7.65 ( d , j = 4.0 hz , 1 h ) , 7.44 ( d , j = 8.4 hz , 2 h ) , 6.63 
 ( s , 1 h ) , 5.10 ( s , 2 h ) , 2.08 ( s , 3 h ) .
lrms ( apci ) calcd 
 for c17h15n2o3s2 359 ( mh ) , found 359 .
( c17h14n2o3s2 ) c , h , n. 4-(5-formylthiophen-2-yl)benzoic acid ( 28 ) ( 85 mg , 0.37 
 mmol ) was dissolved in thf ( 5 ml ) , to which was added 
 pyridine ( 289 mg , 3.67 mmol ) , followed by pentafluorophenyl trifluoroacetate 
 ( pfp - tfa ) .
this mixture was stirred at room temperature for 15 h. 
 then all solvent was removed under reduced pressure to afford a crude 
 solid which was dissolved in etoac ( 25 ml ) and washed with 1 m hcl 
 ( 2  25 ml ) , water ( 25 ml ) , saturated nahco3 ( 25 ml ) , 
 and brine ( 25 ml ) . the organic layer was then dried ( na2so4 ) , filtered and the solvent removed under reduced pressure 
 to give the crude intermediate ester ( 146 mg , 0.37 mmol ) which was 
 immediately dissolved in thf ( 5 ml ) and treated with 2 m methylamine 
 in meoh ( 1.85 ml , 3.70 mmol ) . after the mixture was stirred for 1 
 h at room temperature , 1 m hcl ( 5 ml ) was added to hydrolyze the undesired 
 imine .
the reaction mixture was then diluted with water ( 20 ml ) and 
 extracted with etoac ( 2  20 ml ) .
then the combined organic fractions 
 were dried ( na2so4 ) , filtered , and evaporated 
 down to give a crude solid .
h nmr [ 400 mhz , 
 ( cd3)2so ]  9.93 ( s , 1 h ) , 8.478.53 
 ( br m , 1 h ) , 8.07 ( d , j = 4.0 hz , 1 h ) , 7.93 ( d , j = 8.7 hz , 2 h ) , 7.89 ( d , j = 8.7 hz , 
 2 h ) , 7.84 ( d , j = 4.0 hz , 1 h ) , 2.80 ( d , j = 4.5 hz , 3 h ) .
lrms ( apci ) calcd for c13h12no2s 246 ( mh ) , found 
 246 .
reaction of 34 with 2-thiohydantoin according to general 
 procedure c gave 67 as a dark orange solid ( 76% ) , mp 
 ( acoh ) > 300 c .
h nmr [ 400 mhz , ( cd3)2so ]  12.37 ( br s , 1 h ) , 11.97 ( br s , 1 h ) , 8.47 
 ( br 
 q , j = 4.5 hz , 1 h ) , 7.90 ( d , j = 
 8.5 hz , 2 h ) , 7.84 ( d , j = 4.0 hz , 1 h ) , 7.80 ( d , j = 8.5 hz , 2 h ) , 7.74 ( d , j = 4.0 hz , 
 1 h ) , 2.79 ( d , j = 4.5 hz , 3 h ) .
lrms ( apci ) calcd for c16h12n3o2s2 342 ( m  h ) , found 342 .
( c16h13n3o2s2 ) c , h , n. 2-thiophenecarboxaldehyde 
 was protected as the cyclic acetal according to a literature procedure .
2-(thiophen-2-yl)-1,3-dioxolane was then reacted 
 with 5-iodoisobenzofuran-1(3h)-one ( 77 ) using a procedure adapted from a literature reference .
the iodide ( 1.23 g , 4.75 mmol ) , pdcl2(pph3)2 ( 333 mg , 0.48 mmol ) , and kf ( 1.10 g , 
 19.0 mmol ) were weighed into a flask and dissolved in dmso ( 35 ml ) . 

( 2.22 g , 14.2 mmol ) and agno3 ( 807 mg , 4.75 mmol ) were 
 added , . then the resulting suspension was stirred for 0.5 h at 100 
 c .
further portions of agno3 ( 3  807 mg ) were 
 added at 0.5 h intervals to give a total reaction time of 2 h. upon 
 cooling , the mixture was filtered through a plug of celite which was 
 washed well with chcl3 .
the resulting chcl3 solution 
 ( 200 ml ) was washed with water ( 3  100 ml ) , dried ( na2so4 ) , filtered and the solvent removed under reduced 
 pressure to give a crude solid which was purified by flash column 
 chromatography on silica gel ( 5% acetone / ch2cl2 as eluant ) .
h nmr [ 400 mhz , ( cd3)2so ]  8.53 ( br s , 1 h ) , 7.85 ( d , j = 0.6 hz , 1 h ) , 7.76 ( dd , j = 7.9 , 1.6 
 hz , 1 h ) , 7.68 ( d , j = 7.9 hz , 1 h ) , 7.53 ( d , j = 3.7 hz , 1 h ) , 7.25 ( d , j = 3.6 hz , 
 1 h ) , 6.07 ( s , 1 h ) , 4.41 ( s , 2 h ) , 4.024.09 ( m , 2 h ) , 3.944.01 
 ( m , 2 h ) . lrms ( apci ) calcd for c15h14no3s 288 ( mh ) , found 288 .
5-(5-(1,3-dioxolan-2-yl)thiophen-2-yl)isoindolin-1-one 
 ( 82 ) ( 150 mg , 0.52 mmol ) was dissolved in dry dmf ( 10 
 ml ) , and the resulting solution was cooled to 0 c ( ice / water ) . 

nah ( 23 mg , 0.58 mmol ) was added and the mixture stirred for 0.5 h 
 at this temperature .
methyl iodide ( 82 mg , 0.58 mmol ) was added dropwise 
 to the solution of anion and the mixture stirred for 0.25 h at 0 c , 
 followed by 1 h at room temperature .
the mixture was diluted with 
 water ( 50 ml ) and extracted with etoac ( 3  50 ml ) .
the combined 
 etoac fractions were washed with water ( 3  50 ml ) , brine ( 50 
 ml ) and dried ( na2so4 ) .
filtration and removal 
 of the solvent under reduced pressure gave a crude yellow solid which 
 was purified by flash column chromatography on silica gel ( 5% acetone / ch2cl2 as eluant ) to give 87 as a yellow 
 solid ( 82% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.87 ( d , j = 0.7 hz , 1 h ) , 7.76 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.67 ( d , j = 8.0 
 hz , 1 h ) , 7.53 ( d , j = 3.8 hz , 1 h ) , 7.25 ( d , j = 3.8 hz , 1 h ) , 6.07 ( s , 1 h ) , 4.49 ( s , 2 h ) , 4.024.09 
 ( m , 2 h ) , 3.943.99 ( m , 2 h ) , 3.08 ( s , 3 h ) . lrms ( apci ) calcd for c16h16no3s 302 
 ( mh ) , found 302 .
deprotection of 87 according 
 to general procedure b gave 95 as a yellow solid ( 89% ) .
h nmr [ 400 mhz , ( cd3)2so ]  9.94 
 ( s , 1 h ) , 8.08 ( d , j = 4.0 hz , 1 h ) , 8.04 ( d , j = 0.8 hz , 1 h ) , 7.91 ( dd , j = 7.9 , 1.6 
 hz , 1 h ) , 7.86 ( d , j = 4.0 hz , 1 h ) , 7.74 ( d , j = 7.9 hz , 1 h ) , 4.53 ( s , 2 h ) , 3.09 ( s , 3 h ) .
lrms ( apci ) calcd for c14h12no2s 258 
 ( mh ) , found 258 .
reaction of 95 with 
 2-thiohydantoin according to general procedure c gave 119 as an orange solid ( 92% ) , mp ( acoh ) > 300 c .
h 
 nmr [ 400 mhz , ( cd3)2so ; observe e- and z- isomers separately ) ]  11.9012.38 
 ( m , 2 h ) , 7.92 ( br d , j = 0.7 hz , 1 h ) , 7.82 ( br 
 d , j = 4.0 hz , 0.8 h ) , 7.807.84 ( m , 1 h ) , 
 7.757.78 ( m , 1 h ) , 7.677.73 ( m , 1.2 h ) , 6.82 ( s , 0.2 
 h ) , 6.65 ( s , 0.8 h ) , 4.51 ( s , 2 h ) , 3.09 ( s , 3 h ) .
lrms ( apci ) calcd for c17h12n3o2s2 354 ( m  h ) , found 354 .
methyl 4-iodo-2-methylbenzoate ( 76 ) ( 860 mg , 3.12 mmol ) was dissolved 
 in benzene ( 10 ml ) , to which were added n - bromosuccinimide 
 ( nbs ) ( 666 mg , 3.74 mmol ) and 2,2-azobis(2-methylpropionitrile ) 
 ( 51 mg , 0.31 mmol ) .
this mixture was heated at reflux temperature 
 for 6 h. the mixture was allowed to cool , filtered and the resulting 
 filtrate diluted with et2o ( 100 ml ) .
this solution was 
 washed with saturated sodium metabisufite ( 50 ml ) and brine ( 50 ml ) , 
 dried ( na2so4 ) , and filtered .
removal of the 
 solvent under reduced pressure gave a transparent oil which was purified 
 by filtration through a plug of silica gel ( 5% etoac / hexanes as eluant ) . 

the resulting oil solidified under high vacuum to afford a white solid 
 ( 1.08 g ) , shown to be 93% desired monobromide and 7% unreacted starting 
 material by h nmr .
this solid was used directly in the 
 next step . the bromide ( 1.08 mg , 3.04 mmol )
was dissolved in 
 thf ( 10 ml ) , and 2-aminoethanol ( 928 mg , 15.2 mmol ) was added .
the 
 reaction mixture was stirred at room temperature for 15 h. then all 
 solvent was removed under reduced pressure .
the resulting residue 
 was purified by flash column chromatography on silica gel ( 10% acetone / ch2cl2 as eluant ) to give 78 as a crystalline 
 white solid ( 562 mg , 59% over two steps ) .
h nmr [ 400 mhz , 
 ( cd3)2so ]  8.02 ( d , j = 0.7 hz , 1 h ) , 7.84 ( dd , j = 7.9 , 1.4 hz , 1 h ) , 
 7.45 ( d , j = 7.9 hz , 1 h ) , 4.82 ( t , j = 5.4 hz , 1 h ) , 4.52 ( s , 2 h ) , 3.583.64 ( m , 2 h ) , 3.323.57 
 ( m , 2 h ) . lrms ( apci ) calcd for c10h11ino2 304 ( mh ) , found 304 .
2-thiophenecarboxaldehyde 
 was protected as the dimethyl acetal according to a literature procedure , then reacted with 78 according 
 to general procedure e to give 83 .
compound 83 ( 430 mg , 1.29 mmol ) was dissolved in acetone ( 32 ml ) , to which were 
 added water ( 8 ml ) and p - toluenesulfonic acid ( 100 
 mg ) .
this mixture was stirred at 50 c for 5 h. upon cooling , 
 the reaction mixture was diluted with water ( 100 ml ) and extracted 
 with ch2cl2 ( 3  50 ml ) .
purification by 
 flash column chromatography on silica gel ( 5% meoh / ch2cl2 as eluant ) gave 100 as a yellow solid ( 378 mg , 
 71% over two steps ) .
h nmr [ 400 mhz , ( cd3)2so ]  9.94 ( s , 1 h ) , 8.08 ( d , j = 4.0 
 hz , 1 h ) , 8.04 ( d , j = 0.8 hz , 1 h ) , 7.92 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.85 ( d , j = 4.0 
 hz , 1 h ) , 7.75 ( d , j = 8.0 hz , 1 h ) , 4.83 ( t , j = 5.4 hz , 1 h ) , 4.62 ( s , 2 h ) , 3.563.67 ( m , 4 
 h ) .
reaction of 100 with 2-thiohydantoin according to general 
 procedure c gave 124 as a red powder ( 57% ) , mp ( acoh ) 
 261264 c .
h nmr [ 400 mhz , ( cd3)2so ]  12.39 ( s , 1 h ) , 11.98 ( s , 1 h ) , 7.927.96 
 ( m , 1 h ) , 7.87 ( d , j = 4.0 hz , 1 h ) , 7.84 ( dd , j = 8.0 , 1.5 hz , 1 h ) , 7.77 ( d , j = 4.0 
 hz , 1 h ) , 7.73 ( d , j = 7.9 hz , 1 h ) , 6.65 ( s , 1 h ) , 
 4.59 ( s , 2 h ) , 4.26 ( t , j = 5.4 , hz , 2 h ) , 3.78 ( t , j = 5.3 , hz , 2 h ) , 2.00 ( s , 3 h ) .
hydrolysis 
 of 124 ( 161 mg , 0.42 mmol ) was carried out by removing 
 the previous reaction solvent ( acoh , 4 ml ) from the reaction mixture 
 under reduced pressure and treating the crude o - acetate 
 with k2co3 ( 289 mg , 2.09 mmol ) in a mixture 
 of meoh ( 10 ml ) and water ( 2 ml ) at room temperature for 1 h. the 
 meoh was removed under reduced pressure to give an orange - black oil 
 which was cooled to 0 c ( ice / water ) and acidified with 1 m hcl . 

the resulting solid was collected by filtration , allowed to air - dry , 
 then stirred in warm meoh until a homogeneous suspension was obtained . 

this was collected by filtration and dried under vacuum to give 125 as an orange solid ( 78 mg , 48% over two steps ) , mp ( acoh ) 
 285 c ( dec ) .
h nmr [ 400 mhz , ( cd3)2so ; observe e- and z- isomers 
 separately ]  11.9812.39 ( m , 2 h ) , 7.93 ( br s , 1 h ) , 
 7.87 ( d , j = 4.0 hz , 1 h ) , 7.83 ( dd , j = 8.0 , 1.5 hz , 0.9 h ) , 7.77 ( d , j = 4.0 hz , 1 h ) , 
 7.72 ( d , j = 8.0 hz , 1 h ) , 7.68 ( d , j = 4.0 hz , 0.1 h ) , 6.84 ( s , 0.1 h ) , 6.65 ( s , 0.9 h ) , 4.82 ( t , j = 5.2 hz , 1 h ) , 4.60 ( s , 2 h ) , 3.623.67 ( m , 2 
 h ) , 3.563.61 ( m , 2 h ) .
lrms ( apci ) calcd 
 for c18h14n3o3s2 384 ( m  h ) , found 384 .
in this case ( and others where 
 the target compound contained an amine ) the acoh solvent was removed 
 under reduced pressure to afford an orange - black oil which gave an 
 orange solid when suspended and stirred in a mixture of acetone ( 5 
 ml ) and saturated nahco3 ( 10 ml ) .
this solid was collected 
 by filtration , dried under vacuum , and triturated with meoh to afford 130 as an orange solid ( 72% ) , mp ( meoh ) 261264 c .
h nmr [ 400 mhz , ( cd3)2so ; observe e- and z- isomers separately ]  11.9712.29 
 ( m , 2 h ) , 7.94 ( s , 1 h ) , 7.807.87 ( m , 2 h ) , 7.757.79 
 ( m , 1 h ) , 7.72 ( d , j = 8.0 hz , 0.85 h ) , 7.68 ( d , j = 3.9 hz , 0.15 h ) , 6.84 ( s , 0.15 h ) , 6.64 ( s , 0.15 h ) , 
 4.61 ( s , 2 h ) , 3.66 ( t , j = 6.2 hz , 2 h ) , 3.55 ( t , j = 4.5 hz , 4 h ) , 2.57 ( t , j = 6.2 hz , 
 2 h ) , 2.43 ( br s , 4 h ) . lrms ( apci ) calcd for c22h23n4o3s2 455 ( m + h ) , 
 found 455 . hrms ( esi ) calcd for c22h23n4o3s2 455.1206 ( mh ) , 
 found 455.1201 . compound 101 ( 1.20 g , 3.98 mmol ) was 
 dissolved in a mixture of meoh ( 50 ml ) and trimethyl orthoformate 
 ( 5 ml ) .
p - toluenesulfonic acid ( 100 mg ) was added , 
 along with 4  molecular sieves ( 5.0 g ) .
then the reaction mixture 
 was heated at reflux for 15 h. upon cooling , the reaction mixture 
 was diluted with ch2cl2 ( 100 ml ) and filtered 
 through celite which was washed well with ch2cl2 .
the solvent was removed from the combined filtrate under reduced 
 pressure to give an oil which was dissolved in etoac ( 150 ml ) and 
 washed with saturated nahco3 ( 2  100 ml ) , water ( 100 
 ml ) , and brine ( 100 ml ) .
the organic phase was dried ( na2so4 ) , filtered and the solvent removed to afford 107 as a waxy , pale yellow solid ( 100% ) .
h nmr 
 [ 400 mhz , ( cd3)2so ]  7.86 ( d , j = 0.8 hz , 1 h ) , 7.75 ( dd , j = 8.0 , 1.5 
 hz , 1 h ) , 7.67 ( d , j = 7.9 hz , 1 h ) , 7.54 ( d , j = 3.7 hz , 1 h ) , 7.02 ( dd , j = 3.8 , 0.8 
 hz , 1 h ) , 5.66 ( d , j = 0.7 hz , 1 h ) , 4.51 ( s , 2 h ) , 
 4.49 ( t , j = 5.1 hz , 1 h ) , 3.57 ( t , j = 7.2 hz , 2 h ) , 3.45 ( q , j = 5.9 hz , 2 h ) , 3.32 
 ( s , 6 h ) , 1.76 ( pentet , j = 6.7 hz , 2 h ) . lrms ( apci ) calcd for c18h22no4s 348 
 ( mh ) , found 348 . alcohol 107 ( 1.29 g , 
 3.72 mmol ) was dissolved in dry thf ( 40 ml ) .
triethylamine ( 3.76 g , 
 37.2 mmol ) and methanesulfonyl chloride ( 1.70 g , 14.9 mmol ) were added , 
 and the reaction mixture was stirred at room temperature for 3.5 h. 
 the mixture was diluted with etoac ( 200 ml ) , which was washed with 
 water ( 100 ml ) , saturated nahco3 ( 100 ml ) , and brine ( 100 
 ml ) . the organic layer was dried ( na2so4 ) , filtered 
 and the solvent removed under reduced pressure to give a residue which 
 was purified by flash column chromatography on silica gel ( 5% acetone / ch2cl2 as eluent ) , giving 108 as a waxy 
 yellow solid ( 1.58 g , 100% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.87 ( d , j = 0.7 hz , 1 
 h ) , 7.76 ( dd , j = 7.9 , 1.5 hz , 1 h ) , 7.69 ( d , j = 7.6 hz , 1 h ) , 7.55 ( d , j = 3.7 hz , 
 1 h ) , 7.12 ( dd , j = 3.7 , 0.8 hz , 1 h ) , 5.66 ( d , j = 0.6 hz , 1 h ) , 4.53 ( s , 2 h ) , 4.25 ( t , j = 6.2 hz , 2 h ) , 3.63 ( t , j = 6.9 hz , 2 h ) , 3.32 
 ( s , 6 h ) , 3.18 ( s , 3 h ) , 2.04 ( pentet , j = 6.6 hz , 
 2 h ) . lrms ( apci ) calcd for c19h24no6s2 426 ( mh ) , found 426 .
mesylate 108 ( 624 mg , 1.47 mmol ) was 
 dissolved in acetone ( 30 ml ) at 70 c .
stirring was continued at this temperature for 1 
 h. then the mixture was allowed to cool and was filtered through celite 
 which was washed well with acetone .
the solvent was removed from the 
 combined filtrate under reduced pressure to give a residue which was 
 purified by flash column chromatography on silica gel ( 10% acetone / ch2cl2 as eluant ) , giving 109 as a waxy 
 yellow solid ( 660 mg , 96% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.86 ( d , j = 0.8 hz , 1 
 h ) , 7.76 ( dd , j = 7.9 , 1.6 hz , 1 h ) , 7.68 ( d , j = 7.9 hz , 1 h ) , 7.55 ( d , j = 3.7 hz , 
 1 h ) , 7.12 ( dd , j = 3.7 , 0.8 hz , 1 h ) , 5.66 ( d , j = 0.7 hz , 1 h ) , 4.53 ( s , 2 h ) , 3.58 ( t , j = 6.9 hz , 2 h ) , 3.32 ( s , 6 h ) , 3.25 ( t , j = 6.9 
 hz , 2 h ) , 2.14 ( pentet , j = 6.9 hz , 2 h ) . lrms ( apci ) calcd for c18h21ino3s 458 
 ( mh ) , found 458 .
iodide 109 ( 330 mg , 0.71 mmol ) was dissolved in dimethylacetamide 
 ( 10 ml ) , to which was added dimethylamine ( 3.53 ml of a 2 m solution 
 in thf ) , and the resulting mixture was stirred for 15 h at room temperature . 

all solvent was removed under reduced pressure to give a viscous oil 
 which was dissolved in etoac ( 100 ml ) .
this solution was washed with 
 water ( 3  100 ml ) , brine ( 100 ml ) and dried ( na2so4 ) .
filtration and removal of the solvent under reduced pressure 
 gave the crude dimethyl acetal protected product ( 110 ) as a yellow oil .
this product was then deprotected directly to 
 the desired aldehyde according to general procedure h. trituration 
 with et2o gave 114 as a yellow solid ( 140 
 mg , 60% ) .
h nmr [ 400 mhz , ( cd3)2so ]  9.94 ( s , 1 h ) , 8.08 ( d , j = 4.0 hz , 
 1 h ) , 8.03 ( d , j = 0.7 hz , 1 h ) , 7.92 ( dd , j = 8.0 , 1.6 hz , 1 h ) , 7.86 ( d , j = 4.0 
 hz , 1 h ) , 7.75 ( d , j = 7.9 hz , 1 h ) , 4.55 ( s , 2 h ) , 
 3.56 ( t , j = 7.2 hz , 2 h ) , 2.24 ( t , j = 7.1 hz , 2 h ) , 2.13 ( s , 6 h ) , 1.74 ( pentet , j = 
 7.2 hz , 2 h ) . lrms ( apci ) calcd for c18h21n2o2s 329 ( mh ) , found 329 .
reaction of 114 with 2-thiohydantoin according to 
 general procedure c , followed by isolation according to general procedure 
 j , gave 131 as a dark orange solid ( 51% ) , mp ( meoh ) 212 
 c ( dec ) .
h nmr [ 400 mhz , ( cd3)2so ]  11.47 ( v br s , 2 h ) , 7.91 ( br s , 1 h ) , 7.82 ( dd , j = 8.0 , 1.4 hz , 1 h ) , 7.677.73 ( m , 3 h ) , 6.52 ( s , 
 1 h ) , 4.53 ( s , 2 h ) , 3.56 ( t , j = 7.0 hz , 2 h ) , 2.402.47 
 ( m , 2 h ) , 2.33 ( s , 6 h ) , 1.81 ( pentet , j = 7.2 hz , 
 2 h ) .
lrms ( apci ) calcd for c21h23n4o2s2 427 ( mh ) , found 
 427 .
compound 87 was dissolved 
 in dry thf ( 10 ml ) and cooled to 78 c under n2 .
lithium diisopropylamide in cyclohexane ( 0.40 ml of a 1.5 m solution , 
 0.59 mmol ) was added dropwise .
methyl iodide ( 84 mg , 0.59 mmol ) 
 was added dropwise with stirring continued at 78 c for 
 0.5 h , at which point the mixture was allowed to warm to room temperature . 

the mixture was diluted with saturated nh4cl ( 50 ml ) and 
 extracted with etoac ( 3  50 ml ) .
the combined etoac fractions 
 were washed with brine ( 50 ml ) and dried ( na2so4 ) .
filtration and removal of the solvent under reduced pressure gave 
 a brown oil which was purified by flash column chromatography on silica 
 gel ( 5% acetone / ch2cl2 as eluant ) to give 93 as a yellow oil ( 65% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.92 ( t , j = 0.7 
 hz , 1 h ) , 7.75 ( dd , j = 7.9 , 1.4 hz , 1 h ) , 7.66 ( d , j = 7.9 hz , 1 h ) , 7.55 ( d , j = 3.7 hz , 
 1 h ) , 7.26 ( d , j = 3.7 hz , 1 h ) , 6.07 ( s , 1 h ) , 4.59 
 ( q , j = 6.7 hz , 1 h ) , 4.024.09 ( m , 2 h ) , 
 3.944.02 ( m , 2 h ) , 3.01 ( s , 3 h ) , 1.47 ( d , j = 6.7 hz , 3 h ) .
lrms ( apci ) calcd for c17h18no3s 316 ( m + h ) , found 316 .
deprotection of 93 was carried out according to general procedure b to give 106 as a beige solid ( 96% ) .
h nmr [ 400 mhz , ( cd3)2so ]  9.94 ( s , 1 h ) , 8.078.10 ( m , 2 h ) , 
 7.91 ( dd , j = 8.0 , 1.4 hz , 1 h ) , 7.88 ( d , j = 4.0 hz , 1 h ) , 7.73 ( d , j = 7.9 hz , 
 1 h ) , 4.63 ( q , j = 6.7 hz , 1 h ) , 3.02 ( s , 3 h ) , 1.49 
 ( d , j = 6.7 hz , 3 h ) .
lrms ( apci ) calcd 
 for c15h14no2s 272 ( m + h ) , found 
 272 .
reaction of 106 with 
 2-thiohydantoin according to general procedure c gave 135 as an orange solid ( 57% ) , mp ( acoh ) 286 c ( dec ) .
z- isomers separately ]  11.9212.38 
 ( m , 2 h ) , 7.957.99 ( m , 1 h ) , 7.87 ( d , j = 
 4.0 hz , 0.75 h ) , 7.727.82 ( m , 2 h ) , 7.687.72 ( m , 1.25 
 h ) , 6.84 ( s , 0.25 h ) , 6.64 ( s , 0.75 h ) , 4.61 ( q , j = 6.7 hz , 1 h ) , 3.02 ( s , 3 h ) , 1.48 ( d , j = 6.7 
 hz , 3 h ) . hrms ( esi ) calcd for c18h14n3o2s2 368.0533 ( m  
 h ) , found 368.0526 .
6-bromoisoindolin-1-one ( 137 ) was alkylated 
 with methyl 
 iodide and nah according to general procedure f to give 138 as a pale yellow solid ( 68% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.747.79 ( m , 2 h ) , 7.56 ( dd , j = 7.9 , 0.7 hz , 1 h ) , 4.44 ( s , 2 h ) , 3.07 ( s , 3 h ) . lrms 
 ( apci ) calcd for c9h9brno 226 , 228 
 ( mh ) , found 226 , 228 .
6-bromo-2-methylisoindolin-1-one ( 138 ) ( 723 mg , 3.41 mmol ) , bis(pinacolato)diboron ( 1.04 g , 
 4.09 mmol ) , potassium acetate ( 1.00 g , 10.2 mmol ) , and pd(dppf)cl2 catalyst ( 139 mg , 0.17 mmol ) were weighed into a flask that 
 was sealed under n2 .
dmso ( 15 ml ) was added , and the entire 
 mixture was stirred at 90 c for 5 h. upon cooling , the reaction 
 mixture was diluted with water ( 250 ml ) and extracted with ch2cl2 ( 5  50 ml ) .
the combined ch2cl2 fractions were in turn washed with water ( 2  
 100 ml ) , brine ( 100 ml ) , dried ( na2so4 ) , filtered 
 and the solvent was removed under reduced pressure to yield the crude 
 product .
purification was carried out by flash column chromatography 
 on silica gel ( 20% thf / ch2cl2 as eluant ) to 
 give 148 as a crystalline beige solid ( 262 mg , 28% ) .
h nmr [ 400 mhz , ( cd3)2so ]  7.91 
 ( br s , 1 h ) , 7.85 ( dd , j = 7.6 , 1.0 hz , 1 h ) , 7.59 
 ( dd , j = 7.5 , 0.6 hz , 1 h ) , 4.49 ( s , 2 h ) , 3.07 ( s , 
 3 h ) , 1.32 ( s , 12 h ) . lrms ( apci ) calcd for c15h21bno3 274 ( mh ) , found 274 .
reaction of 148 with 2-(5-bromothiophen-2-yl)-1,3-dioxolane 
 ( 5 ) according to general procedure a followed by deprotection 
 of 154 directly to the aldehyde according to general 
 procedure b gave 160 as a pale yellow solid ( 91% ) .
h nmr [ 400 mhz , ( cd3)2so ]  9.93 
 ( s , 1 h ) , 8.06 ( d , j = 4.0 hz , 1 h ) , 7.998.03 
 ( m , 2 h ) , 7.88 ( d , j = 4.0 hz , 1 h ) , 7.70 ( dd , j = 7.8 , 0.8 hz , 1 h ) , 4.52 ( s , 2 h ) , 3.10 ( s , 3 h ) . lrms 
 ( apci ) calcd for c14h12no2s 258 ( mh ) , found 258 .
reaction of 160 with 2-thiohydantoin 
 according to general procedure c gave 166 as an orange 
 solid ( 89% ) , mp ( acoh ) > 310 c .
h nmr [ 400 mhz , 
 ( cd3)2so ; observe e- and z - isomers separately ]  11.9712.34 ( m , 2 h ) , 
 7.917.96 
 ( m , 2 h ) , 7.82 ( d , j = 4.0 hz , 0.85 h ) , 7.757.78 
 ( m , 1 h ) , 7.69 ( d , j = 4.0 hz , 0.15 h ) , 7.66 ( d , j = 8.6 hz , 1 h ) , 6.84 ( s , 0.15 h ) , 6.66 ( s , 0.85 h ) , 4.40 
 ( s , 2 h ) , 3.10 ( s , 3 h ) . lrms ( apci ) calcd for 
 c17h12n3o3s2 354 ( m  h ) , found 354 . as reported previously , compound 3
was identified from screening a commercial 
 library of 100  000 compounds for the ability to reproducibly 
 inhibit perforin - mediated lysis of srbc at a compound concentration 
 of 100 m .
the ability of the compounds to inhibit the 
 lysis of nucleated ( jurkat 
 t lymphoma ) cells in the presence of 0.1% bsa was measured by release 
 of cr label .
jurkat target cells were labeled by incubation 
 in medium with 100 ci cr for 1 h. the cells were 
 then washed three times to remove unincorporated isotope and resuspended 
 at 1  10 cells per milliliter in rpmi buffer supplemented 
 with 0.1% bsa .
each test compound was preincubated to concentrations 
 of 20 , 10 , 5 , 2.5 , and 1.25 m with recombinant perforin for 
 30 min with dmso as a negative control .
cr labeled jurkat 
 cells were then added , and the cells were incubated at 37 c 
 for 4 h. the supernatant was collected and assessed for its radioactive 
 content on a  counter ( wallac wizard 1470 automatic  
 counter ) .
each data point was performed in triplicate , and an ic50 was calculated from the range of concentrations described 
 above .
compounds with ic50 < 1 m were titrated 
 to lower concentrations in the same manner as above , to determine 
 an accurate ic50 .
khyg-1 cells were washed 
 and resuspended in rpmi + 0.1% bsa at 4  10 cells / ml , 
 and an amount of 50 l of khyg-1 cells was dispensed to each 
 well of a 96-well v - bottom plate .
rpmi ( 50 l ) + 0.1% bsa or 
 10% ( final concentration ) of serum was added to each well .
then test 
 compounds were added to khyg-1 cells at various concentrations up 
 to 20 m and incubated at room temperature for 20 min . 1  
 10 k562 target cells were labeled with 75 ci cr in 200 l of rpmi for 90 min at 37 c .
cells 
 were washed as described above and resuspended in 5 ml of rpmi + 0.1% 
 bsa .
an amount of 50 l of cr labeled k562 leukemia 
 target cells was added to each well of the khyg-1 plate ( effector / target 
 2:1 ) and incubated at 37 c for 4 h. cr release 
 was assayed using a skatron harvesting press and radioactivity estimated 
 on a wallac wizard 1470 automatic  counter ( turku , finland ) . 

the percentage of specific cytotoxicity was calculated using the following 
 formula : and expressed as the mean 
 of triplicate assays 
  standard error of the mean .
the toxicity assay was 
 carried out in exactly the same manner as the killing assay above , 
 but instead of adding the labeled k562 target cells , 100 l 
 of rpmi 0.1% bsa was added .
cells were incubated for 4 h at 37 c 
 and then washed 3 in rpmi + 0.1% bsa .
cells were then resuspended 
 in 200 l of complete medium and incubated for 1824 
 h at 37 c .
viable ( clear ) 
 cells and total ( clear + blue ) cells were counted , and the percentage 
 of viable cells was calculated compared to dmso treated cell control 
 ( % viability ) .
pharmacokinetic 
 studies were carried out in healthy cd-1 male mice ( n = 3 at each time point ) using intravenous administration of 135 and 167 at a concentration of 5 mg / kg in 
 20% 2-hydroxypropyl--cyclodextrin ( sigma - aldrich ) .
blood was 
 collected by cardiac puncture at 5 and 30 min and 1 , 2 , 4 , and 6 h 
 postdose in ice - cold k2-edta tubes .
plasma samples 
 were separated by centrifuging at 4000 g for 8 min 
 and stored at 80 c until analysis .
then the sample ( 10 l ) 
 was transferred to a clean microcentrifuge tube and mixed with three 
 volumes of ice - cold acetonitrile / methanol ( 3:1 v / v ) [ containing 213 as internal standard ( table 6 , supporting 
 information ) ] to precipitate the plasma proteins .
the tubes 
 were centrifuged at 15000 g for 10 min to obtain the 
 clear supernatant which was mixed with 0.01% formic acid 
water 
 ( 1:1 ) , and concentration of the parent drug was measured through injection 
 of 1020 l into an lc 
ms / ms ( agilent 6410 series 
 triple quadrupole mass spectrometry detector system ) . a zorbax sb - c18 , 
 50 mm  2.1 mm 5 m column was used at a 0.5 ml / min flow 
 rate , and elution was with a gradient of organic phase ( a ) , acetonitrile 
 in water ( 80:20 v / v ) containing 0.01% formic acid and aqueous phase 

parent drug was 
 quantified against a standard curve 0250 ng / ml in control 
 mouse plasma .
plasma samples from 5 min , 30 min , and 1 h were diluted 
 with control mouse plasma ( 1:20 for 5 min , 1:10 for 30 min and 1 h 
 samples ) to fit within this range .
the resultant concentration vs 
 time data were fitted ( using phoenix winnonlin 6.2 ; pharsight corporation , 
 st .
louis , mo ) to calculate pharmacokinetic parameters including half - life 
 ( t1/2 ) , maximum concentration ( cmax ) , area under the curve ( auc0 ) , volume of distribution ( v ) , and clearance ( cl ) .
 3 intraperitoneal 
 administration through a classical dose escalation design in c57bl/6 
 mice and defined as a dose level that was tolerated by mice ( n = 23 mice / group ) with a body weight loss of < 10% 
 and no death or detectable sickness .
all animal experiments were approved by the animal ethics 
 committee of the university of auckland .
binding experiments 
 with surface immobilized wild - type perforin monomers on a s - series 
 cm5 sensor chip ( ge healthcare life sciences ) were carried out using 
 a biacore t200 spr machine ( ge healthcare life sciences ) .
perforin 
 monomer solution ( 25 g / ml in 10 mm sodium acetate , ph 5.5 ) 
 was injected over the sensor chip surface under standard amide coupling 
 conditions to obtain covalent binding of the protein ( density = 4257 
 ru ) .
the initial and running buffers were made from a 10 stock 
 solution of hepes buffered saline ( hbs ) .
10 hbs ( 50 ml ) was 
 diluted to 500 ml with water and filtered through a 0.22 m 
 filter ( initial buffer ) .
this initial buffer ( 380 ml ) was added to 
 dmso ( 20 ml ) to prepare the 5% dmso running buffer .
these solutions were 
 diluted in hepes initial buffer to 5% dmso ( 125 m ) and then 
 subsequently diluted to the required concentrations using hepes running 
 buffer containing 5% dmso to maintain a constant dmso concentration . 
 a solvent correction was run before and after the injections to account 
 for differences in dmso concentrations between buffer and analyte 
 solutions .
time - lapse 
 microscopy and image analysis were performed as described previously . in brief , primary human natural killer cells 
 were isolated from the peripheral blood of four unrelated donors using 
 negative selection , as described in the above reference .
natural killer 
 cells were labeled with 1 m fluo-4am for 20 min at 37 c/5% 
 co2 , and then cells were washed and preincubated for 10 
 min in rpmi culture medium containing 0.2% ( v / v ) dmso or 20 m 167/0.2% ( v / v ) dmso at room temperature before combining with 
 hela cell targets preseeded in chamber slides .
the final mixture contained 
 complete rpmi-1640 culture medium supplemented 
 with 100 m propidium iodide , 0.2% ( v / v ) dmso or 20 m 167/0.2% ( v / v ) dmso .
 a series of novel 5-arylidene-2-thioxoimidazolidin-4-ones 
 were 
 investigated as inhibitors of the lymphocyte - expressed pore - forming 
 protein perforin . 
 structure  
 activity relationships were explored 
 through variation of an isoindolinone or 3,4-dihydroisoquinolinone 
 subunit on a fixed 2-thioxoimidazolidin-4-one / thiophene core . the 
 ability of the resulting compounds to inhibit the lytic activity of 
 both isolated perforin protein and perforin delivered in situ by natural 
 killer cells was determined . 
 a number of compounds showed excellent 
 activity at concentrations that were nontoxic to the killer cells , 
 and several were a significant improvement on previous classes of 
 inhibitors , being substantially more potent and soluble . 
 representative 
 examples showed rapid and reversible binding to immobilized mouse 
 perforin at low concentrations ( 2.5 m ) by surface plasmon 
 resonance and prevented formation of perforin pores in target cells 
 despite effective target cell engagement , as determined by calcium 
 influx studies . 
 mouse pk studies of two analogues showed t1/2 values of 1.11.2 h ( dose of 5 mg / kg iv ) and 
 mtds of 6080 mg / kg ( ip ) . 
Introduction
Results and Discussion
Conclusions
Experimental Section