the mitotic kinesin eg5 is a promising 
 potential therapeutic target for small molecule inhibitors and has 
 been validated in preclinical in vivo models of cancer to be essential 
 for the progression of mitosis . eg5 is instrumental in forming the 
 bipolar spindle by separation of the duplicate spindle poles in the 
 early prometaphase stage of mitosis . several eg5 
 and other mitotic kinesin inhibitors are at preclinical and clinical 
 stages ( reviewed in ref ( 3 ) ) , including ispinesib , one of the most advanced eg5 inhibitors currently 
 in multiple phase ii clinical trials . previously , we identified s - trityl - l - cysteine ( stlc ) as a potent and selective 
 inhibitor of human eg5 ( figure 1 , 1 ) . stlc and its derivatives induce mitotic arrest by 
 activation of the mitotic checkpoint , which eventually leads to apoptosis 
 in certain tumor cell lines by both caspase - dependent and -independent 
 pathways . furthermore , the p - methoxy 
 analogue 5 has recently been shown to significantly prolong 
 survival in in vivo experiments using bladder and prostate cancer 
 xenografts in nude mice . as with most other 
 allosteric eg5 specific inhibitors , stlc binds in an allosteric pocket 
 formed by helix 2/loop l5 and helix 3 . initial structure  activity relationship ( sar ) studies performed 
 prior to detailed structural analysis of eg5stlc complexes 
 resulted in the development of analogues with gi50 values 
 in the range of 200 nm ( figure 1 , 2 and 3 ) . recent investigations 
 have identified novel analogues with improved potency in cell - based 
 assays . we have determined previously the structure of 
 eg5 complexed with stlc ( 1 ) and identified the key binding 
 interactions in the inhibitor - binding pocket ( figure 2a and 2b ) . the three phenyl rings of the trityl headgroup are situated in a 
 predominantly hydrophobic core region of the allosteric inhibitor - binding 
 site , in the three pockets denoted p1p3 ( figure 2b ) , while the cysteine tail forms several important hydrogen 
 bond interactions with eg5 , in agreement with those observed in another 
 eg5stlc complex by kim et al . prior sar studies had demonstrated that stlc analogues with a lipophilic 
 substituent on one phenyl ring generally displayed increased potency , which we confirmed structurally 
 by solving the crystal structure of eg5 with an stlc analogue incorporating 
 a p - chlorophenyl substituent in the hydrophobic p3 
 region ( figure 2b ) . crystal structures of other ligands bound to eg5 have revealed that 
 small polar substituents like the m - hydroxy group 
 in ( s)-monastrol and its analogues interact with 
 residues lining the p2 pocket , such as glu118 , arg119 , and ala133 . crystallographic studies on stlc and its analogues have also established 
 that the phenyl ring in p1 -stacked with the tyr 211 residue 
 and exposed its leading edge to bulk solvent near glu215 ( figure 2a ) . these data from both structural observations 
 and previous sar studies provided the foundation to investigate the 
 optimal lipophilic and polar substituents on the trityl system to 
 improve potency against eg5 . eg5inhibitor structures showing important 
 hydrophobic , aromatic , and hydrogen bond ( represented by broken lines ) 
 interactions between the protein and ligands . ( a ) stlc ( 1 ) , colored 
 green , in the eg5 inhibitor - binding pocket . ( b ) surface diagram showing 
 an stlc analogue ( gray ) with a para - substituted chlorine on one phenyl 
 ring , in the inhibitor - binding pocket . ( c ) stereoplot showing 25 ( blue ) 
 in the inhibitor - binding pocket . ( d ) stereoplot showing ( s)-29 ( pink ) and ( r)-29 ( yellow ) in the inhibitor - binding pocket . in any hit - to - lead optimization process , improving 
 potency against the target protein must be pursued in tandem with 
 optimizing the physicochemical properties of the ligand , as physicochemical 
 properties have profound influences on absorption , distribution , and 
 elimination and ultimately determine drug efficacy . our optimization strategy therefore needed to consider 
 the potential metabolic liability of the thioether linkage to the 
 trityl headgroup and the zwitterionic nature of the cysteine amino 
 acid group in 1 . a parallel approach was adopted for 
 sar investigations : we explored a range of substituents on the trityl 
 system by thioetherification of tertiary alcohols to improve potency 
 against eg5 and in the case of polar substituents act as alternatives 
 to the zwitterionic carboxylic acid in the cysteine tail . second , 
 isosteric replacements of the sulfur heteroatom ( n , o , ch2 ) were investigated , with the ultimate aim of transferring the optimal 
 trityl head from the stlc series to the sulfur - isosteric analogues . they display strong in vitro basal eg5 inhibitory activity ( 10 
 nm ) and potently inhibit a number of cancer tumor cell lines ( 100 
 nm ) and improve on comparable stlc derivatives . the in vitro and in 
 vivo pharmacokinetic properties of these analogues are promising and 
 warrant further development , and initial in vivo studies with one compound in nude mice 
 bearing a cell lung cancer explant xenograft lxfs 538 exhibited tumor 
 stasis . o - trityl analogue 6 was prepared by coupling 
 of n-(2-hydroxyethyl)acetamide with trityl chloride 
 followed by acetyl hydrazinolysis ( supporting 
 information , scheme s7 ) . n - trityl analogues 7 and 8 were prepared via substitution of trityl 
 chloride with fmoc - protected diamines ( scheme 1 ) . thioethers were prepared from tertiary alcohols in trifluoroacetic 
 acid ( scheme 2 ) or as previously described with boron trifluoride and acetic acid . intermediate trityl alcohols were furnished 
 by organometallic mediated reduction of substituted benzophenones 
 and methyl esters or alternatively lithiated aryl bromides , which 
 allowed introduction of a wider range of substituents to the trityl 
 headgroup ( scheme 2 ) . reagents and conditions : 
 ( i ) tmscl , ch2cl2/thf ( 5:1 ) , reflux , 2 h ; ( ii ) 
 trcl , net3 , ch2cl2 , rt , 1 h ; ( iii ) 
 piperidine ( 20% in meoh ) , rt , 2 h ; ( iv ) trcl , net3 , dmf , 
 rt , 3h . reagents and conditions : 
 ( i ) n - buli , 78 c , 1 h ; ( ii ) benzophenone , 
 thf , 78 c 46 h , then rt overnight ; ( iii ) cysteamine 
 hydrochloride or l - cysteine , tfa , rt , 3 h. a more elaborate methodology was required for creation 
 of the c - trityl series , exemplified by compounds 9 and 10 . to introduce the carbon scaffold , the 
 lithium alkoxide salt of trityl alcohol was subjected to a mild allylation 
 procedure employing iron trichloride and allyltrimethylsilane ( scheme 3 ) . the alkene 55 was then oxidized by hydroboration  oxidation and 
 subsequent swern oxidation to afford the aldehyde 57 , 
 which was converted into the racemic protected -aminonitrile 
 intermediate 58 in a variation of the strecker synthesis 
 employing montmorillonite ksf clay . after 
 the nitrile was hydrolyzed , a mild ammonium formate based deprotection 
 of the benzylamine 59 afforded c - trityl 
 analogue 9 as a racemic mixture , in an overall yield 
 of 45% . triphenylbutan-1-amine 
 analogues without the carboxylic acid , exemplified by 10 , were synthesized by conversion of the intermediate alcohol 56 to the azide 60 , with consequent staudinger 
 reduction to afford the target compound 10 in an overall 
 yield of 49% ( scheme 4 ) . reagents and conditions : 
 ( i ) n - buli , ch2cl2 , rt , 30 
 min ; ( ii ) allyltrimethylsilane , fecl3 , rt , 6 h ; ( iii ) nabh4 , conc h2so4 in et2o , diglyme , 
 rt , 3.5 h , then 75 c , 1.5 h ; ( iv ) 30% aq h2o2 , 3 m aq naoh , rt , 6.5 h ; ( v ) dess  martin periodinane , 
 ch2cl2 , rt , 3 h ; ( vi ) benzylamine , tmscn , montmorillonite 
 ksf clay , ch2cl2 , rt , 2 h ; ( vii ) 6 m hcl in 
 dioxane , reflux , 48 h ; ( viii ) propylene oxide , etoh , reflux , 30 min ; 
 ( ix ) hcoonh4 , 10% pd / c , meoh , 80 c , 1 h. ( i ) mscl , pyridine , 
 rt , overnight ; ( ii ) nan3 , dmf , microwave , 175 c , 
 10 min ; ( iii ) ph3p , thf / h2o ( 10:1 ) , 60 c , 
 overnight . the racemic mixtures 
 of the c - trityl amino acids rac-9 , rac-29 , and rac-32 were separated by semipreparative chiral hplc , and 
 their absolute stereochemistry was determined by application of a 
 modification of marfey s method . this involved 
 derivatization of the racemic mixture with the chiral agent n-(2,4-dinitro-5-fluorophenyl)-l - valinamide ( fdva ) , followed by analysis of the hplc retention times 
 of the resulting diastereomers ( figure 3b(i ) ) . 
 in these diastereomers , the dinitrobenzene moiety of fdva is in a 
 planar conformation with the diamino substituents adopting a cis- 
 or trans - arrangement relative to this ( figure 3a(ii ) ) . the diastereomer with the two 
 more hydrophobic substituents in a cis - arrangement undergoes stronger 
 interactions with the reverse stationary phase and is retained for 
 longer than the corresponding diastereomer in the trans - configuration , 
 which enables a nonempirical determination of the stereochemistry 
 of amino acids and chiral amines . applying this 
 method to the resolved enantiomers of 32 , the first diastereomer 
 to elute was 32 - 1-fdva . we therefore assigned 32 - 1 as the ( s ) configuration and 32 - 2 as the ( r ) enantiomer . as marfey s method is 
 an indirect method of determining absolute stereochemistry , to ensure 
 the diastereomers 32 - 1-fdva and 32 - 2-fdva eluted in the order that would be expected under the conditions 
 employed , stlc 1 and stdc were evaluated by the same 
 methodology . stlc - fdva was eluted first , as would be expected from 
 their relative stereochemistries . further 
 investigations with d- and l - cysteine and d- and l - phenylalanine also proved consistent with designated 
 assignments ( figure 3b ) . further assignments 
 were based on the results of optical rotation and circular dichroism 
 measurements when compared to the assigned enantiomers . ( a ) ( i ) structure of n-(2,4-dinitro-5-fluorophenyl)-l - valinamide 
 ( fdva ) ; ( ii ) derivatised diastereoisomers 29 - 1-fdva and 29 - 2-fdva ; ( iii ) schematic representations of proposed cis- 
 and trans - like diastereomer conformations . ( b ) retention times of 32 - 1-fdva and 32 - 2-fdva and corresponding standard amino acids . replacements of the 
 cysteine sulfur atom of stlc were investigated in order to establish 
 its overall effect on potency and adme characteristics ( 610 , table 1 ) . a trityl 
 group is often used as a protecting group in organic synthesis that 
 is removed under acidic conditions ; hence , it may be removed from 
 stlc on encountering the acidic environment of the stomach if administered 
 orally . using lc  hrms , we established that stlc was stable 
 at ph 1.0 over 24 h ( supporting information , table s1 and figure s1 ) . on the other hand , corresponding n - trityl derivatives 7 and 8 not 
 only displayed weak inhibition of the basal atpase activity of eg5 
 compared with stlc but also were completely hydrolyzed at ph 1.0 ( t1/2 of 2.9 and 7.0 h , respectively ) . the oxygen analogue 6 was also a weak inhibitor of eg5 
 and decomposed after 16 h at ph 1.0 ( t1/2 = 2.5 h ) . we did not investigate further whether the lower activity 
 of these o - trityl and n - trityl analogues 
 was due to their instability in the assay medium or the reduced pka of the primary amine and its consequent effect 
 on the proportion of molecules ionized to facilitate interaction with 
 the protein . a thioether functionality is a site for metabolism 
 via s - oxidation , and the enzyme phenylalanine monoxygenase has recently 
 been identified as a possible source for the conversion of the mucoregulatory 
 agents s - methyl - l - cysteine and s - carboxymethyl - l - cysteine to the corresponding inactives - oxides . an obvious isostere 
 for sulfur that would not be susceptible to acid hydrolysis or s - oxidation 
 is a methylene group . the carbon analogues 9 and 10 were prepared and shown to be stable for 5 days at ph 1.0 . furthermore , they had activity similar to that of stlc against eg5 , 
 and when 9 was resolved , ( r)-9 was the more potent enantiomer . these compounds also demonstrated 
 a systematic improvement in the k562 human leukemia cell based assays 
 compared to their sulfur counterparts 1 and 2 , and ( r)-9 reproduced its higher activity 
 in the cellular assay compared with its enantiomer . interestingly , 
 in the eg5 assay , the carbon analogue without the carboxylic acid 
 in the tail ( 10 ) showed improved activity over the amphoteric 
 form ( 9 ) , a reversal of the trend observed with sulfur - based 
 analogues 1 and 2 . eg5 structure 
 was the hydrogen bond network between the primary ammonium group of 
 the cysteine tail and the backbone carbonyl of gly117 and the side 
 chain of glu116 . to establish whether this was being reproduced in 
 the c - trityl analogues 9 and 10 , derivatives were prepared in which the length of the amine tail 
 was varied incrementally ( table s2 ) . a 
 20-fold and 8-fold decrease in eg5 inhibitory activity was observed 
 for the propyl-1-amine and pentyl-1-amine analogues , respectively , 
 suggesting that the optimal network is achieved with the four - carbon 
 butyl-1-amine chain ( 10 , table s2 ) . corresponding compounds were also prepared for the n - trityl analogue 8 to ensure that the decreased length 
 of the primary amine tail was not the reason for the weak inhibitory 
 activity observed . in this case , an ethylene unit proximal to the n - trityl unit again proved to be the optimal chain length 
 ( 8 , table s2 ) . these chain 
 length manipulations concur with the earlier observations made for 
 the alkyl chain in stlc . having 
 established that carbon was an appropriate isostere in the tail for 
 sulfur and the optimum length was a butyl chain , we next evaluated 
 triphenylbutan-1-amine analogues featuring functional group replacements 
 and/or modifications to the alkyl chain , including those prepared 
 as intermediates ( 5560 , table s3 ) . replacement of the primary amine with the secondary methylamine resulted 
 in a marked dropoff in activity , reinforcing the importance of a three 
 hydrogen bond network between the ammonium group and the glutamate 
 and glycine residues in the pocket for efficient inhibition ( s50 , table s3 ) . the bulkier secondary 
 benzylamine derivatives rac-58 and rac-59 displayed further reductions in activity . replacement of the primary amine with a primary alcohol ( 56 , table s3 ) abolished activity , which 
 again reinforces the need for three hydrogen bond donors and a role 
 for an ionic interaction between ligand and protein . additional n - trityl analogues incorporating cyclic piperazines in the 
 amino acid tail region displayed no notable activity in comparison 
 to either stlc 1 or 9 ( data not shown ) . the most potent 
 stlc derivatives prepared previously incorporated a lipophilic para - substituent 
 in one phenyl ring of the trityl moiety ( figure 1 ) . we have recently solved the structure of an stlc 
 derivative with a p - chloro on one phenyl ring , which , 
 as expected , occupied the environment of the p3 pocket formed by ile136 , 
 pro137 , leu160 , leu214 , and phe239 ( figure 2b ) . a series of s - trityl 
 thioethers were prepared to investigate this environment further . compounds exhibiting eg5 inhibition ki  100 nm were also evaluated in growth inhibition 
 assays using k562 cells . while the o - chloro 
 substituent of 11 significantly reduced activity against 
 eg5 compared with stlc 1 , a m - chloro 
 ( 13 ) produced an approximately 2-fold improvement in ki for basal inhibition of eg5 
 ( table 2 ) . further thioethers incorporating 
 meta - substituents of varying electronic , hydrophobic , and steric character 
 were synthesized to optimize this interaction , with alkyl groups particularly 
 beneficial for eg5 inhibitory activity . the stepwise improvement in 
 activity from 16 ( me , ki = 80  24 nm ) , 19 ( n - pr , ki = 26.9  11 
 nm ) , 18 ( i - pr , ki = 10.3  3.4 nm ) to 17 ( et , ki = 5.9  2.3 nm ) illustrates 
 that the interaction is very sensitive to steric bulk at this position 
 and most likely reflects an interaction between the proximal lipophilic 
 side chain of leu214 and these alkyl substituents . furthermore , electronic 
 influences on the attached phenyl ring are also important : 20 with a m - cf3group , which can be considered 
 as similar in size to a methyl substituent but electron - withdrawing , 
 is 4-fold less active than 16.in our evaluation of all 
 the crystal structures of stlc and its analogues with eg5 , we observed 
 in the p3 pocket a c interaction between the phenyl group 
 of stlc and the side chain of leu214 ( figure 2a ) . this 
 interaction is mediated via the -electron clouds of the phenyl 
 ring , so changes in its distribution will affect the strength of its interaction with 
 nearby residues . analysis of these results using a craig plot substantiated 
 the optimal meta - substituents as both hydrophobic and electron - donating 
 ( figure 3 ) . in the cellular assay , submicromolar activity was 
 evident for alkyl ( 1618 ) and acetate 
 substituents ( 24 , 25 , 27 ) , 
 which demonstrated an approximate 2-fold improvement in this assay 
 versus the unsubstituted s - trityl benchmarks ( 1 and 2 ) . paradoxically , 19 with 
 an n - pr substituent exhibited a dramatic loss in 
 cellular activity , probably as a result of limited aqueous solubility 
 ( turbidimetric solubility of 3.75 m ) . introduction of a carboxylic 
 acid into the tail of 27 to produce 26 resulted 
 in a 4-fold reduction in eg5 activity , comparable to that seen with 25 and 24 , but produced a much more pronounced 
 reduction in activity in the k562 cell line , which was a reversal 
 of the pattern seen with 1 and 2 . insufficient 
 related compounds were prepared in this series to determine if this 
 trend was consistent . from the comparable s - trityl 
 inhibitors prepared , two pairs with modified trityl moieties had better 
 activity in the eg5 basal assay when prepared without the carboxyl 
 acid ( 24 and 25 ; 26 and 27 ) , while all modified s - trityl analogues 
 with the carboxylic acid moiety exhibited lower activity in the cellular 
 assay than their corresponding counterparts without the carboxylate 
 ( 13 and 14 ; 24 and 25 ; 26 and 27 ) . clearly , further investigations 
 are needed before definitive conclusions can be made regarding the 
 sar role of the carboxylic acid with respect to eg5 inhibitory and 
 cellular activity . the positioning of the p1 ring extending 
 toward the solvent front and the nearby glu215 residue afforded an 
 additional optimization opportunity to introduce more polar substituents 
 ( figure 2a ) . a series of analogues that incorporated 
 polar h - bond acceptor / donors on one phenyl ring was synthesized to 
 target bulk solvent interactions and the proximal acidic residue . it was envisaged that these substituents could attenuate the highly 
 lipophilic nature of the headgroup and produce compounds with physicochemical 
 properties ( e.g. , log  d ) compatible with predicted 
 favorable adme profiles.s - trityl 
 thioethers were again prepared to investigate these potential interactions 
 ( table 3 ) , and only one compound , the m - hydroxy stlc analogue 35 , demonstrated any 
 significant improvement in basal eg5 inhibitory activity over 1 . this is likely due to formation of hydrogen bonds with 
 the main chain nitrogen of leu132 and the side chain of arg119 in 
 the p2 pocket , as observed in the crystal structures for dihydropyrimidine the direct analogue 36 , which differed only by the absence of the carboxylic acid in the 
 tail , while 4-fold less potent in the basal assay , was 5-fold more 
 potent than 35 in the cellular assay . this difference 
 can be attributed to the difference in cell permeability resulting 
 from the presence of the carboxylic acid . evaluation of the passive 
 in vitro cellular permeability by parallel artificial membrane permeability 
 assays ( pampa ) demonstrated 36 to have higher permeability 
 ( papp = ( 16.7  2.6 )  10 cm s ) , with no permeability recorded 
 for 35 . all other compounds in this series displayed 
 only moderate inhibition of the basal activity of eg5 but generally 
 similar cellular activity to 2 , demonstrating a wide 
 tolerability of polar substrates . a modest improvement in k562 cellular 
 activity was observed in the meta - primary and secondary amides 42 and 43 , although their eg5 inhibitory activity 
 was reduced 2-fold and 8-fold , respectively . likewise , the p - ch2oh containing compound 47 had 
 improved cellular activity but reduced eg5 inhibitory activity . this 
 may suggest that their cellular activity is being expressed through 
 an alternative mechanism to eg5 inhibition . the meta - primary amide 41 , which has the carboxylic acid group in the tail , has similar 
 eg5 inhibitory activity , compared to 42 , but significantly 
 reduced cellular activity ( 32-fold less than 42 and 18-fold 
 less than stlc ) , probably due to a very low log  d ( log  d7.4 = 1.13 for 41 ; log  d7.4 = 1.41 for 42 ) . measurement of the cell permeability by pampa assays found 42 to possess high permeability of ( papp = ( 42.8  15.0 )  10 cm s ) , while 41 had no membrane diffusion 
 detected when evaluated by this method . thus , while the meta - primary 
 amide and secondary amides of 42 and 43 in 
 the trityl headgroup may act as a suitable alternatives to the zwitterionic 
 carboxylate in the cysteine tail for enhancing the physicochemical 
 balance of stlc analogues , the dual presence of the trityl amide modification 
 and carboxylate appears to be incompatible with effective cell penetration . 
 to conclude , overall improvements to both basal and cellular activity 
 were associated with hydrophobic rather than hydrophilic para- or 
 meta - substituents . after establishment of the sar of the trityl headgroup 
 in thioethers and the optimal characteristics of the tail group with 
 isosteric butan-1-amine , the appropriate para- or meta - substituted 
 trityl groups were next translated into the c - trityl 
 series ( 2833 , table 2 ) . we found a corresponding systematic increase in activity 
 against the target eg5 protein when evaluated in both basal in vitro 
 and cellular growth inhibition assays ; an approximate 3-fold increase 
 was evident for comparable analogues 14 and 28 and 16 and 30 in the k562 assay . the most 
 potent analogues prepared were racemic mixtures of 29 and 32 , which had the 2-aminobutanoic acid tail and 
 the c - trityl scaffold with a m- or p - methyl substituent on one phenyl ring , respectively ( ec50 of 73  3 and 95  5 nm ) . the ki and ec50 of the resolved enantiomers 
 were also determined ( table 2 ) . the more active 
 ( r)-enantiomers ( r)-29 and ( r)-32 were low nanomolar inhibitors 
 of eg5 and have estimates of ki values of 6.4  3.9 and 5.4  1.7 nm , respectively , which 
 are close to the reported ki of 2 nm for ispinesib . the corresponding 
 butan-1-amine analogues ( 30 and 33 ) possess 
 similarly low ki estimates 
 ( 8.8  1.8 and 16.4  1.9 nm , respectively ) , indicating 
 that the presence of the carboxylic acid is not essential for inhibition . 
 on the other hand the carboxylic acid containing ( r)-29 and ( r)-32 analogues 
 are approximately 2-fold more active in cell - based growth inhibition 
 assays . this reverses the trend observed with the sulfur analogues , 
 where removal of the carboxylic acid from the tail of compounds with 
 substituted trityl head groups in general improved cellular activity . the ligand efficiencies , which depend on protein targets , were 
 calculated for all the compounds based on their ki values ( tables 13 ) to enable useful comparison 
 between compounds with a range of molecular weights and activities . the compounds without the carboxylic acid groups generally showed 
 better ligand efficiencies than their counterparts with the carboxylic 
 acid groups : compounds 29 and 32 had ligand 
 efficiencies of 0.4 and 0.41 , whereas 30 and 33 showed higher ligand efficiency of 0.46 and 0.44 , respectively . ispinesib has a calculated ligand efficiency of 0.32 based on the 
 reported ki ( 1.7  
 0.1 nm ) , indicating that our smaller triphenylbutanamine 
 scaffold possesses greater enhancement potential . next , we examined 
 the most potent analogues together with selected control compounds , 
 for the inhibition of the mt - stimulated eg5 atpase activity ( table 5 ) . this assay , with a salt concentration of 150 
 mm , performed in the presence of mts , is closer to physiological conditions 
 and allows ki to be determined 
 in the presence of 5 nm eg5 instead of 80 nm employed 
 for the basal assay , resulting in more reliable values , in particular 
 for the more potent analogues . the estimates for ki for both the inhibition of the basal and 
 mt - stimulated atpase activities are in good agreement . the most potent 
 compounds are ispinesib with ki of 2.5  1.2 nm followed by ( r)-32 with 3.4  0.9 nm , which is further confirmed in cell - based 
 assays . finally , we investigated the cross - reactivity of rac-29 against a kinesin panel of the five 
 human kinesins kif5a , kif7 , mpp1 , mklp-2 , and cenp - e by investigating 
 their inhibition of the basal and mt - stimulated atpase activities . we did not observe any significant inhibition for any of the kinesins 
 tested ( data not shown ) , indicating that the selectivity previously 
 observed for stlc is maintained in this more potent analogue . the sar reported 
 here confirms that a lipophilic substituent on one phenyl ring generally 
 improves potency , and we have demonstrated that the sulfur in the 
 tail group could be replaced by methylene to yield compounds with 
 systematically improved activity both against eg5 and in the k562 
 cell line . here , we report the crystal structures of eg5 complexed 
 with s - trityl analogue 25 and triphenylbutanamine 
 analogue 29 to a resolution of 2.75  . eg525 and eg529 complexes crystallized in space group p212121 and i213 with 
 7 and 1 molecule per asymmetric unit , respectively . in the eg525 complex , 6 out of 7 of the eg5 molecules were in the final 
 inhibitor - bound state , whereby loop l5 has swung downward to close 
 the inhibitor - binding pocket , the switch ii cluster has moved upward , 
 and the neck - linker has adopted a docked conformation . the seventh 
 molecule ( chain g ) was trapped in an intermediate state similar to 
 that in other eg5inhibitor structures , whereby local changes 
 at the inhibitor - binding pocket have not propagated to complete structural 
 changes at the switch ii cluster and the neck - linker remains undocked . in the eg529 complex , eg5 was in the final 
 inhibitor - bound state . examination of both complexes revealed that the three 
 phenyl rings of 25 ( figure 2c ) 
 and 29 ( figure 2d ) were buried 
 in the same pockets as stlc , with the same hydrophobic and aromatic 
 interactions described previously . as 
 we used the racemic mixture of 29 to crystallize the 
 eg5inhibitor complex , we observed electron density for both 
 enantiomers in the structure . several hydrogen bond interactions between 
 eg5 and the cysteine moiety of ( s)-29 were apparent : the amino group with the main chain carbonyl oxygen 
 of gly117 and the side chain oxygen ( oe1 ) of glu116 , and an oxygen 
 ( oxt ) of the carboxylic group of the cysteine moiety with one of the 
 side chain amino groups ( nh1 ) of arg221 ( figure 2d ) . instead , the amino group of the cysteine moiety interacts 
 with the protein via hydrogen bonding with a structural water molecule , 
 which does not produce any significant change in affinity . although 
 the sulfur in stlc has been substituted by a carbon in 29 , the interactions between eg5 and both inhibitors are virtually 
 identical , with the exception of the shorter c  c  c bonds 
 ( 3.9  ) compared to the c  s where the carboxylic acid is 
 absent , only hydrogen bond interactions were observed between the 
 amino group and the main chain carbonyl oxygen of gly117 and the side 
 chain oxygen ( oe1 ) of glu116 ( figure 2c ) . the loss of the 
 carboxylate group not only removed a hydrogen bond interaction but 
 also appeared to reduce restraints on the amine tail and increased 
 its conformational freedom , as evident from one molecule ( chain d ) 
 where the amino group pointed away from the side chain oxygen ( oe1 ) 
 of glu116 and toward the amino group of arg221 instead . this compound 
 has equivalent potency with stlc , which suggests that the loss of 
 the carboxylate group and the greater flexibility of tail group may 
 be compensated by an improved interaction with the trityl headgroup 
 bearing a meta - substituent . paradoxically , 24 ( the direct 
 analogue of 25 with a carboxylate in the tail ) has an 
 approximately 3-fold reduction in activity against eg5 in the basal 
 in vitro assay , which suggests that the substituent in the trityl 
 group may influence the relative position of the tail so that the 
 carboxylate has a detrimental effect on binding . craig plot for aromatic 
 substituents of  vs .  is the substituent hydrophobicity 
 constant and  the hammett substitution constant , a measure 
 of the electronic influence of the substituent . values were obtained 
 from model systems from refs ( 30 ) , ( 56 ) , and ( 57 ) . with well - defined electron density maps , we were 
 able to determine unequivocally the location of the meta - substituted 
 acetate and methyl groups of 25 and 29 , 
 respectively . both substituents sit in the hydrophobic p3 pocket formed 
 by ile136 , pro137 , leu160 , leu214 , phe239 , and the salt bridge between 
 glu116 and arg221 . the carbonyl oxygen of the acetate group of 25 appeared to from a hydrogen bond with one of the side chain amino 
 groups ( nh1 ) of arg221 and the main chain nitrogen of ala218 . the 
 methyl of the acetate group was buried in a largely hydrophobic pocket 
 formed by phe239 , leu160 , ile136 , and leu214 . overall , the crystal 
 structures of eg525 and eg529 provide insight into the arrangement of the substituents within 
 the binding site and help explain some of the structure for example , replacement of the m - acetyl with a m - ethyl ( 17 ) or m - isopropyl ( 18 ) group significantly 
 improved activity by approximately 20-fold , which could be attributed 
 to the replacement of a polar carbonyl group in an essentially hydrophobic 
 pocket with the more lipophilic alkyl group and facilitating a favorable 
 interaction with the side chain of leu214 . furthermore , the electronic 
 properties of the phenyl ring will be altered by an alkyl ( + i ) compared with an acetyl ( i ) , 
 which would impact on the strength of the ch interaction 
 with leu214 . the generally lower activities of the m - hydrophilic group series ( table 3 ) compared 
 with 25 provide further evidence that this hydrophobic 
 pocket has a low tolerance for polar groups , despite the proximity 
 of the glu116arg221 salt bridge . shifting the m - acetyl to the p - position ( compound 27 ) produced a 4-fold increase in activity , which could be due to an 
 improved alignment of the carbonyl with the side chain of arg221 . of the 47 compounds tested in proliferation assays using human k562 
 leukemia cells ( tables 13 ) , the most potent were subsequently investigated in four 
 additional human cell lines ( table 5 ) . these were derived from colon , lung , and pancreatic 
 cancer , as well as immortalized cells derived from normal breast tissue . 
 from our previous study , we included three potent s - trityl analogues ( 35 ) and stlc 1 as controls and to monitor progress and the clinical phase 
 ii candidate ispinesib to benchmark the new analogues . ( r)-32 improves at least 2-fold on our previously reported most potent 
 stlc analogues ( 35 ) in the selected cell lines and has comparable activity with ispinesib across all cell 
 lines . ( r)-29 , which differs from ( r)-32 only by the methyl group being in the 
 para - position , shows only slightly lower activity in the lung cell 
 line . although significant differences in activity were not 
 observed between s - trityl - l - cysteine and s - trityl - d - cysteine in previous studies , detectable 
 differences were evident among the enantiomers of the 2-aminobutanoic 
 acid series . when the sulfur of stlc was replaced 
 with carbon to produce ( s)-9 , there 
 was lower activity across all cell lines , while the corresponding 
 carbon analogue of stdc [ ( r)-9 ] had 
 comparable activity . it is only when substituents were introduced 
 into the headgroup of the tritylbutanamines that significant improvements 
 in activity became apparent . our two most potent compounds with and without the 
 carboxylic acid ( 29 and 30 , respectively ) 
 together with ispinesib to provide a benchmark were assessed in a 
 series of in vitro and in vivo adme assays ( table 6 ; for 29 , the separated enantiomers were used 
 for profiling ) . ispinesib had the lowest turbidimetric solubility 
 ( 65 m , partially soluble ) , whereas both 30 and rac-29 were soluble ( > 100 m ) . despite 
 the nonoptimal log  p of 30 and 
 ispinesib , the log  d at ph 7.4 was <3 and 
 favorable for all compounds . during the time course 
 of the experiments , all inhibitors either proved stable or exhibited 
 low clearance in human microsomal and hepatocyte stability assays . 
 in mouse microsomal assays , ispinesib and both enantiomers of 29 were stable whereas 30 showed high microsomal 
 clearance , indicating species - dependent differences between mouse 
 and human for this compound . consequently , the high clearance of 30 excluded it from in vivo mouse xenograft studies . a further 
 example of the need to balance efficacy with structural modifications 
 to improve potency is demonstrated by herg inhibition . both ispinesib 
 and 30 were moderately potent herg inhibitors with ic50 of 4.7  1.8 and 6.5  1.6 m , respectively . 
 among the most successful reported approaches for diminishing binding 
 to the herg channel are modulation of lipophilicity and structural 
 modifications that disrupt the -stacking and hydrophobic interactions 
 between the drug candidate and the channel cavity . it is therefore not unexpected that installation of a polar , 
 carboxylic acid functionality into 30 to produce 29 results in a dramatic reduction in herg binding . plasma 
 protein binding influences the distribution and elimination of compounds : 
 the fraction unbound of all compounds tested was > 90% with ( s)-29 being slightly less bound than the other 
 compounds and is acceptable for this program . ( s)-29 and ( r)-29 did not show any 
 inhibition up to 25 m , but ispinesib moderately to significantly 
 inhibited isoform cyp3a4 in vitro with an ic50 of 4.05 
  0.44 m , in agreement with two recent studies . the four isoforms cyp2c9 , cyp2c19 , cyp2d6 , and cyp3a4 were inhibited 
 by 30 with moderate to significant ic50 between 
 3.17  0.45 and 8.46  0.84 m . the racemic mixture was used . we subsequently determined the bioavailability for 30 , rac-29 , and ispinesib ( table 6 ) . compound 30 had particularly low bioavailability of 
 4% and low po levels , which confirms the in vitro high microsomal 
 clearance values seen in mice . ispinesib shows moderate clearance 
 with good po levels and has a moderate bioavailability of 45% . finally , rac-29 showed low clearance , good po levels , 
 and a bioavailability of 51% . on the basis of rac-29 having the better adme profile compared with 30 , particularly with respect to bioavailability , we tested 
 it in vivo using lung cancer patient explants ( lxfs 538 ) passaged 
 as subcutaneous xenografts in nude mice . in this model , by transplantation 
 of a tumor from a patient to a mouse , many of the characteristics 
 of the parental patient tumors including histology and sensitivity 
 to anticancer drugs are retained . moreover , earlier studies have shown 
 that such explants correctly replicate the response of the donor patient 
 to standard anticancer drugs in > 90% of the cases . rac-29 displayed good 
 antitumor activity in lxfs 538 : a minimum t / c of 26.3% was recorded , corresponding to transient partial 
 tumor remission ( i.e. , individual relative tumor volumes of < 100% ) 
 in four out of five tumors around day 10 and an obvious reduction 
 of growth rates compared to the vehicle control group in the latter 
 part of the experiment ( figure 5 ) . this resulted 
 in an increase of tumor volume doubling times from 8.8 days in the 
 control group to 28.7 days in the rac-29-treated group . the rac-29 treatment group (  ) received 20 mg / kg on days 0 , 
 2 , and 4 and 15 mg / kg rac-29 on days 
 17 , 19 , 24 ( indicated by arrows ) , whereas the control group (  ) 
 received only vehicle on the same days . the data are plotted as the 
 mean of the relative tumor volume  standard deviation . the difference 
 between treated group and vehicle is statistically significant ( p = 0.016 ) . triphenylbutan-1-amines represent a potent 
 class of eg5 inhibitors , which demonstrate good in vivo antitumor 
 activity against lung cancer xenografts in mouse models . the sar modifications 
 of meta or para lipophilic trityl substituents , isosteric replacement 
 of the sulfur with methylene , and inversion of the amino acid stereocenter 
 with respect to stlc have produced analogues that systematically improve 
 on the comparable s - trityl thioethanamines . with 
 very potent nanomolar inhibitory activity exhibited against the target 
 kinesin eg5 , favorable ligand efficiencies , and similar gi50 values over five tumor cell lines compared to the phase ii candidate 
 of equivalent potency the major effect of the -carboxylic 
 acid is in determining the cellular permeability , and its presence 
 in the amine tail reduces log  p , enhances 
 bioavailability , and attenuates interactions with herg and several 
 cyp isoforms . the continued development and further studies on this 
 scaffold will be reported in due course .
the human mitotic kinesin eg5 represents a novel mitotic 
 spindle target for cancer chemotherapy . 
 we previously identified s - trityl - l - cysteine ( stlc ) and related analogues 
 as selective potent inhibitors of eg5 . 
 we herein report on the development 
 of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from 
 the stlc scaffold . 
 this new generation systematically improves on 
 potency : the most potent c - trityl analogues exhibit kiapp  10 nm and gi50  50 nm , comparable to results from the phase ii clinical 
 benchmark ispinesib . 
 crystallographic studies reveal that they adopt 
 the same overall binding configuration as s - trityl 
 analogues at an allosteric site formed by loop l5 of eg5 . 
 evaluation 
 of their druglike properties reveals favorable profiles for future 
 development and , in the clinical candidate ispinesib , moderate herg 
 and cyp inhibition . 
 one triphenylbutanamine analogue and ispinesib 
 possess very good bioavailability ( 51% and 45% , respectively ) , with 
 the former showing in vivo antitumor growth activity in nude mice 
 xenograft studies .