mitotic 
 kinesins represent an exciting class of oncology targets . the members of this structurally divergent superfamily 
 of microtubule ( mt)-based molecular motor proteins have extensive 
 involvement throughout mitosis , with functions ranging from the organization 
 of the mitotic architecture to the transport of regulatory proteins . one of the most studied is the kinesin spindle protein ( ksp , also 
 known as eg5 , kif11 , knsl1 ; kinesin-5 family ) , whose primary role 
 is to establish the bipolar spindle in early prometaphase . inhibition of eg5 by small molecule inhibitors results in monopolar 
 spindles and mitotic arrest which can lead to cell death . this has proved highly effective across a variety 
 of in vitro and in vivo models of cancer , and total regression has 
 been demonstrated in a number of xenograft models . a great variety 
 of inhibitor scaffolds have been described in the literature , the majority of which are allosteric 
 inhibitors that bind to an induced fit pocket formed by loop l5 , approximately 
 10  from the nucleotide binding site . these allosteric inhibitors exhibit excellent selectivity for eg5 
 among kinesins due to the unique length of loop l5 among the kinesin 
 superfamily . a number of compounds have 
 progressed to clinical trials , with the most advanced in phase ii 
 ( e.g. , 13 , figure 1 ) ; however , to date the best outcome in the majority of studies 
 against advanced solid tumors has been limited to disease stabilization . better responses were achieved targeting hematological malignancies 
 with eg5 inhibitors . in a phase ii study with the 
 1,3,4,-thiadiazole - based candidate arry-520 ( 3 ) , a 19% objective response rate was recorded in 
 patients with relapsed / refractory multiple myelomas . additive responses were reported on treatment with 3 and the proteasome inhibitor bortezomib in xenograft models 
 of multiple myeloma , and combination clinical trials are now planned . targeting hematological malignancies with eg5 inhibitors as single 
 agents or in combination with existing therapies therefore represents 
 an exciting area of investigation which may offer clinical advantages 
 over current regimens . selected eg5 inhibitors ispinesib ( 1 ) , sb-743921 
 ( 2 ) , and arry-520 ( 3 ) in clinical development . our group previously identified s - trityl - l - cysteine ( stlc , 4 ) as a 
 tight - binding and selective 
 allosteric inhibitor of eg5 ( figure 2 ) . in common with other loop l5 eg5 inhibitors , 4 blocks 
 bipolar spindle formation causing mitotic arrest , which ultimately 
 leads to apoptotic cell death across a variety of tumor cell lines . this primarily occurs through activation of 
 the spindle checkpoint and the intrinsic apoptotic pathway . antitumor activity has been reported for 4 and the p - methoxyphenyl analogue 7 across leukemia , bladder , and prostate xenograft models . in initial sar investigations , analogues with improved cellular 
 potency were identified which incorporated a phenyl containing lipophilic 
 para substituents ( e.g. , 57 , figure 2 ) . the subsequent elucidation of the crystal structure 
 of 4 in complex with eg5 enabled us 
 to pursue the rational sar - based optimization of this scaffold , which 
 led to the development of butanamine analogues with improved activity 
 against eg5 ( 1114 , figure 2 ) . the lead triphenylbutanamine 
 from our previous study ( 11 ) had improved potency , favorable 
 drug - like properties , and in vivo antitumor activity in a lung cancer 
 xenograft model . we report here on the next phase of development , 
 which has produced analogues that significantly improve on both the 
 potency and in vivo efficacy of the previous leads . across multiple 
 cancer cell lines , these new leads demonstrate comparable in vitro 
 activity to the phase ii candidate ispinesib ( 1 ) , and 
 its more potent second generation analogue sb-743921 ( 3 ) which is also undergoing clinical studies ( figure 1 ) . favorable 
 drug - like properties are evident across a selection of the most potent 
 analogues , in addition to compelling in vivo pharmacokinetics . finally , 
 the new lead analogue induces complete tumor regression in a lung 
 cancer xenograft model . in addition to surpassing the activity of 
 all previously described s - trityl - l - cysteine 
 analogues , this in vivo efficacy is comparable with that of the drug 
 candidates 13 and strongly warrants 
 further development . the structure of s - trityl - l - cysteine 
 ( 4 ) and selected previously reported analogues . the crystal 
 structures of eg5 in complex with 4 , 5 , 
 and 11 have previously been determined and used for structure - based 
 drug design . s - tritylthioethanamine and 
 cysteine analogues were prepared by dehydration of trityl alcohols 
 in trifluoroacetic acid and thioetherification with l - cysteine 
 or cysteamine hydrochloride ( scheme 1 ) . intermediate trityl alcohols were synthesized 
 by the reduction of benzophenone analogues with lithiated aryl bromides ; 
 key thioethanamines were therefore accessible in two steps . the trityl 
 alcohol 59 containing a disubstituted m - ethyl , p - methylphenyl was prepared from the 5-bromobenzoic 
 acid derivative 55 by converting to the weinreb amide 56 to enable controlled access to ketone 57 , which was subsequently reduced with hydrazine 
 hydrate to afford the dialkyl aryl bromide 58 ( scheme 2 ) . subsequently , lithium bromine exchange and reduction 
 of benzophenone and thioetherification of 59 afforded 
 the target thioether 36 . reagents and conditions : 
 ( i ) n - buli , 78 c , 1 h ; ( ii ) benzophenone 
 or 3-hydroxybenzophenone , 
 thf , 78 c , 46 h , then rt overnight ; ( iii ) cysteamine 
 hydrochloride or l - cysteine , tfa , rt , 3 
 h. triphenylbutanamine ( ch2-trityl ) analogues 
 with the 
 carboxylic acid were prepared by the route reported previously from 
 common trityl alcohol intermediates , as illustrated for rac-42 ( scheme 3 ) . oxygen bond cleavage afforded 
 the carbon framework , prior to successive 
 oxidations by hydroboration oxidation to the primary alcohol 62 and then dess  martin periodinane ( dmp ) to aldehyde 63 . this was converted to -aminonitrile rac-64 by a modified version of the strecker 
 synthesis employing montmorillonite ksf clay . hydrolysis of rac-64 and subsequent 
 hydrogenation of benzylamine rac-65 using 
 ammonium formate gave the racemic amino acid 45 . attempts to resolve rac-42 by chiral hplc using a chiralpak ic column as described 
 previously were not successful . triphenylbutanamines 
 without the carboxylic acid were prepared by functional group interconversion 
 from primary alcohol intermediates ( e.g. , 62 ) to the 
 primary amine via the mesylate and azide ( supporting 
 information , schemes s6s8 ) . the -fluorinated triphenylbutanamine rac-52 was prepared by the enamine - based organocatalyzed 
 -fluorination of aldehyde 66 with l - proline 
 and n - fluorobenzenesulfonimide , followed by immediate 
 reduction to afford -fluorinated 
 alcohol rac-67 ( scheme 4 ) . expedient access to rac-28 was then achieved by stepwise conversion 
 via the tosylate and azide . the ,-difluorinated amine 53 was synthesized by a comparable methodology with selectfluor 
 as the electrophilic source of fluorine . direct access to azide 69 was possible from ,-difluorinated 
 alcohol 68 under microwave conditions , and subsequent 
 reduction of 69 yielded the ,-difluorinated - amine 53 . the -trifluoromethyl amine rac-54 was prepared from aldehyde 66 by trifluoromethylation 
 with tms - cf3 ; subsequent functional 
 group interconversion of trifluoromethyl alcohol rac-70 via the triflate and azide gave the desired product rac-54 . the most potent analogues identified previously 
 contained a lipophilic substituent in the meta or para position of 
 one phenyl ring . crystallographic studies with p - chlorophenyl- and m - tolyl - containing 
 analogues 5 and 11 revealed these substituents 
 to be preferentially positioned in the predominantly hydrophobic environment 
 of the p3 pocket ( figure 3b ) . in the meta position , the optimal substituents were typically small 
 alkyl groups that were electron - donating and hydrophobic , which we rationalized as being due to strengthening 
 the c h interaction between the isopropyl 
 side chain of leu 214 and the phenyl ring ( figure 3a ) . a small number of thioethanamines 
 was prepared to determine whether this was also applicable for substituents 
 in the para position ( 1521 , table 1 ) . the p - methyl and p - methoxy analogues 17 and 19 demonstrated 
 similar basal inhibition activity , that was comparable to 16 and improved 4-fold over 4 , and while a drop 
 in potency was apparent upon increasing from methyl to ethyl ( 17 and 18 ) , this was not seen with the ether 
 analogues 1921 . we evaluated all 
 inhibitors of eg5 in the basal assay for growth inhibition against 
 k562 human leukemia cells . this cell line has previously been shown 
 to be susceptible to eg5 inhibition and to overexpress eg5 . improved activity compared with 4 was exhibited in 
 the cellular assay by 17 , 18 , and 19 containing p - alkyl and p - methoxy substituents , with 18 and 19 matching 
 that of the potent meta - substituted analogue 16 . ethoxy and triofluoromethoxy derivatives 20 and 21 proved less active , which may be due 
 to less appropriate physicochemical properties such as reduced aqueous 
 solubility ( e.g. , 20 : turbidimetric solubility at ph 
 7.4 = 3.75 m ) . to determine whether the optimal thioethanamine 
 trityl substituents were transferrable to the butanamine - based inhibitors , 
 several triphenylbutanamine analogues containing a single substituent 
 in the meta or para position were prepared ( 2227 , table 1 ) . triphenylbutanamines 22 and 23 incorporating m - ethyl 
 and m - isopropyl substituents , respectively , proved 
 very potent in both assays , with 22 3-fold more 
 active against cells than the comparable thioether 16 . the p - methoxyphenyl analogues rac-25 and 26 with and without the -carboxylate 
 both displayed gi50 < 100 nm , which improved significantly 
 over the equivalent s - trityl pair 7 and 19 . while these findings confirmed the systematically improved 
 activity of the butanamines versus analogous thioethanamines , in general 
 these new compounds exhibited equivalent in vitro activity to the 
 previously described leads ( e.g. , rac-11 and 13 ) . the activity of the p - methoxy 
 analogue 26 was however improved ( 2-fold ) versus 
 other analogues without the carboxylate and now equivalent in the 
 cell - based assay to the zwitterionic rac-11 . interestingly , while rac-25 and 26 exhibited comparably strong activity across both assays , 
 the zwitterionic thioether 7 was clearly superior in 
 the cellular assay to 19 . ( a ) the eg54 allosteric binding site , illustrating 
 interactions with proximal ( 4  ) protein residues . ( b ) the eg54 inhibitor - binding pocket with a 
 solid surface illustrating the nomenclature for subpockets p1 , p2 , 
 and p3 . ( c ) stereoplot of ( r)-46 in 
 the allosteric binding site . hydrogen bonds between inhibitor ( blue ) 
 and eg5 residues ( beige ) are depicted by black broken lines . coordinate 
 and structure factor files for the eg546 complex 
 ( pdb i d : 4bbg ) were deposited at the pdb . having identified the optimal 
 substituents to improve affinity , we next investigated disubstituted 
 phenyl rings . by broadening the scope of our sar data , we hoped to 
 utilize this knowledge to allow for more permutations to modulate 
 the metabolic and pharmacokinetic properties of the ultimate lead 
 candidates by unearthing multiple modifications capable of imbuing 
 eg5 affinity . a 2,5-difluorophenyl ring in the p3 pocket produced 
 impressive increases in eg5 inhibitory activity during optimization 
 of the clinical candidate mk-0731 . in 
 our own series , although a m - fluoro substituent had 
 proved detrimental to eg5 affinity , we 
 had not examined the influence of fluoro substituents in alternative 
 positions in combination with the optimal alkyl and methoxy substituents . an o - fluoro substituent diminished inhibitory activity 
 against eg5 substantially when combined with a methyl substituent 
 in either meta or para ( 28 and 29 , table 2 ) . however , when combined with a p - methoxy substituent ( 31 ) , the o - fluorine 
 produced a 2-fold improvement in potency against eg5 compared 
 with the p - methoxyphenyl 19 . retaining 
 the p - methoxy substituent and repositioning the fluoro 
 substituent in the meta position was not tolerated ( 32 ) . in the cellular assay , 28 , 29 , 32 , and 33 proved weak inhibitors , but the improvements 
 evident in the basal assay for 31 translated to a modest 
 improvement in growth inhibition compared with the p - methoxyphenyl containing 19 . on introduction of the 
 carboxylic acid ( 30 ) , a further 3-fold improvement in 
 growth inhibitory activity was realized compared with 7 , equivalent to the butanamine lead rac-11 . interestingly the butanamine analogue of 30 , compound 40 proved less potent in both the enzymatic and cellular assays . next , we investigated combinations of dialkyl substitutuents following 
 from the observation that a -naphthyl analogue of 4 demonstrated good potency against both eg5 , in vitro and in hela 
 cells , with docking studies suggesting that the bicyclic ring occupied 
 the p3 pocket . compounds 3439 were prepared to investigate whether more 
 metabolically amenable and physiochemically favorable combinations 
 of dialkyl substituents could be implemented in place of the naphthyl 
 moiety ( table 2 ) . the dialkylphenyl and tetralene analogues ( 35 , 37 , and 39 ) all proved comparably effective inhibitors 
 of eg5 basal activity to singly substituted analogues ( e.g. , 1627 ) . when the terminal carboxylic acid 
 was introduced , incremental improvements in activity were apparent : 
 the dimethylphenyl compound 34 displayed the highest 
 affinity for eg5 of any s - trityl - l - cysteine 
 analogue , with an estimated ki  1.2 nm . however , in the cell - based assay , 35 , 37 , and 39 lacking the carboxylate were 
 disappointingly weak inhibitors , with poor solubility probably the 
 principal cause of their reduced cellular efficacy ( e.g. , turbidimetric 
 solubility at ph 7.4 : 35 = 3.75 m , supporting information , table s3 ) . the presence 
 of the terminal -carboxylic acid in the tail ameliorated this : 
 in addition to ki < 5 
 nm against eg5 in the basal assay , the l - cysteine analogues 34 , 36 , and 38 all exhibited improved 
 activity over the butanamine lead rac-11 in the cell - based assay . the most potent growth inhibition activity 
 was exhibited by 36 ( gi50  34 nm ) , 
 which was 30-fold better than 37 without the 
 -carboxylate , highlighting the importance of achieving the 
 correct physicochemical balance . s - alkylated derivatives 
 of cysteine were first designed as anticancer agents following the 
 observation that radiolabeled cysteine was incorporated by leukemic 
 white blood cells , so active uptake of the amino 
 acid zwitterion also can not be discounted as contributing to the improved 
 activity of 36 compared with 37 . next , we 
 transferred the optimal dialkyl substituent pattern to the butanamine 
 scaffold . the poor aqueous solubility of tetralene 38 even when the carboxylic acid was present precluded continuing with 
 this modification ( turbidimetric solubility at ph 7.4 = 20 m ) . although 36 was more potent in the cellular assay , the 
 superior eg5 affinity and physicochemical properties of the dimethylphenyl 
 analogue 34 marked this as the optimal modification from 
 this selection . the prepared compound rac-42 was less potent in the basal assay but importantly proved 3-fold 
 more active in the cellular growth inhibition assay ( gi50  23 nm ) , thereby improving on the activity of both the previously 
 reported butanamine leads 11 and 12 ( figure 2 ) and the l - cysteine analogues 34 , 36 , and 38 in this assay . all 
 the modifications to the trityl group described so far increased its 
 hydrophobicity , which in certain cases compromised cellular efficacy 
 due to poor aqueous solubility ( e.g. , 38 ) . in addition 
 to this immediately apparent penalty , excessively lipophilic ( i.e. , 
 log p / log d7.4 > 3 ) 
 compounds 
 are more likely to possess unfavorable adme properties and bind more 
 promiscuously , which is likely to result in greater side effects and 
 general toxicity . we were interested 
 in developing hydrophilic modifications to the trityl group to produce 
 a more physicochemically balanced scaffold to serve as a backup to 
 the lead amino acid - based compounds . one way to achieve this was by 
 replacing a phenyl ring with a heterocycle ; however , none of the investigated 
 analogues displayed noteworthy activity ( s1s5 , supporting information , table s1 ) , in 
 agreement with other reports on thiophene and pyridyl analogues . previously we identified certain hydrophilic phenyl substituents 
 that reduced the lipophilicity of the trityl headgroup and improved 
 cellular efficacy ( e.g. , 8 and 9 , figure 2 ) . the most effective 
 of these modifications was the m - hydroxy 8 , designed to form hydrogen - bonding interactions with the peptide 
 backbone of the p2 pocket ( figure 3a ) . modest improvements in cellular efficacy were also noted in analogues 
 containing primary and secondary amide phenyl substituents ( e.g. , 9 , figure 2 ) . these amides were proposed 
 to occupy the solvent - exposed p1 region because of the steric restrictions 
 in the p2 pocket observed in other sar studies ( figure 3b ) . we combined each of these modifications in turn , with a lipophilic 
 substituent on a different phenyl in the trityl group , to investigate 
 if synergistic increments in activity could be achieved ( 4451 , table 3 ) . while no 
 improvement was apparent for thioethanamine rac-45 containing a m - phenol and m - chlorophenyl , the related m - phenol analogues rac-46 and rac-47 with m - ethyl- and p - methylphenyl 
 substituents were 5-fold and 4-fold more potent inhibitors of eg5 
 basal activity , respectively , than comparable analogues 16 and 17 . these analogues also exhibited improved inhibition 
 over their respective benchmarks in the k562 assay : e.g. , rac-46 improved 2-fold over the benchmarks 8 and 16 , and rac-47 improved 5-fold over 8 and 17 . these data implied a synergistic binding motif was being realized , 
 with hydrogen bonding interactions from the m - hydroxyl 
 in the p2 pocket augmented by hydrophobic interactions with the alkyl - substituted 
 phenyl rings situated in the p3 pocket . while no increase in potency 
 was evident in the basal assay when a p - tolyl was 
 combined with a m-(primary amide)-containing phenyl , 
 this combination afforded a 2-fold improvement in the cellular 
 assay ( rac-49 compared with 9 and 17 ) which also supported the proposed binding conformation . 
 with gi50  100 nm , rac-47 is the most active thioethanamine without the terminal -carboxylic 
 acid in the cellular assay reported to date and comparable to the 
 lead butanamine 11 . these modifications did not prove 
 advantageous when applied in the comparable butanamine rac-51 , which was less active in both assays than rac-47 or 11 . better activity 
 was evident on addition of the terminal carboxylate in the basal assay 
 but not in the cellular assay ( dia-50 ) . we previously noted that passive cellular diffusion was adversely 
 affected by the presence of both the -carboxylate in the tail 
 and amide or hydroxyl phenyl substituents , which may offer a partial 
 explanation for the comparatively weak cellular activity of the zwitterionic 
 butanamine dia-50 . values provided 
 for reference from 
 previous paper . the binding mode proposed for analogues with two orthogonal phenyl 
 substituents was confirmed when the crystal structure of 46 in complex with eg5 was solved to a resolution of 2.65  . this 
 showed the m - phenol in p2 and the m - ethyl in p3 ( figure 3c ) . data collection and 
 refinement statistics are presented in table 4 . the eg546 complex crystallized in space group i213 with one molecule per asymmetric unit . although 
 the racemic mixture of 46 was used for crystallization , 
 only the r - enantiomer was observed in the inhibitor - binding 
 pocket . the three phenyl rings of ( r)-46 are positioned in the same overall conformation as 4 , and consequently the hydrophobic and aromatic interactions between 
 the three phenyl rings and the residues in the inhibitor - binding pocket 
 are conserved . in the thioethanamine tail , the 
 primary amine maintains the crucial hydrogen bonds exhibited by all 
 analogues of 4 with the carboxylate of glu116 and the 
 main chain amide carbonyl of gly117 . the well - defined electron density affords accurate 
 positioning of the two phenyl substituents and provides a clear structural 
 rationale for the improvements exhibited in binding affinity . the m - ethylphenyl is situated in the predominantly hydrophobic 
 p3 pocket bounded by leu160 , gly217 , ala218 , arg221 , mimicking the m - tolyl and p - chlorophenyl rings in previous 
 crystal structures . however , the terminal ch3 of the ethyl extends into previously unutilized space between 
 the methyl of ala218 and the aliphatic chain of arg221 . the m - phenol of 46 occupies the p2 pocket formed 
 by glu118 , arg119 , trp127 , asp130 , leu132 , ala133 , and pro137 , with 
 the hydroxyl pointing toward the core of the protein instead of outward 
 toward the solvent . hydrogen - bonding interactions are evident in this 
 position from the hydroxyl to the main chain carbonyl of glu118 , the 
 main chain nitrogen of ala133 and additional weak electrostatic interactions 
 with the main chain nitrogen of arg119 , one of the side chain oxygens 
 of asp130 , and the carbonyl oxygen of leu132 ( distance cutoff = 4.0 
  ) , similar to those observed in the eg5monastrol complex 
 and structurally related dihydropyrimidines , thereby stabilizing the 
 inhibitor in the allosteric site and increasing the affinity for the 
 protein . the racemic mixture was used for 
 crystallization . dpi : diffraction - component precision 
 index ; r.m.s.d . is the root - mean - square 
 deviation from ideal geometry . ` the inhibitory activity of the most potent analogues from the initial 
 in vitro assays was next examined in a mt - stimulated eg5 atpase assay , 
 together with selected control compounds ( 4 , 7 , rac-11 , and 13 ) and 
 external benchmarks ( 1 and 2 , table 5 ) . in cells , eg5 uses mt filaments as tracks to 
 push antiparallel mts apart , which makes this particular assay closer 
 to physiological conditions . three new analogues ( 36 , rac-42 , and rac-47 ) possessed ki estimates of < 10 nm . recently , the inhibitory activity of monastrol 
 against eg5 was shown to be dependent on the ionic strength of the 
 buffer . to investigate whether this was 
 the case for our scaffold , we assessed mt - stimulated atpase activity 
 of compounds 1 and rac-42 at different salt concentrations . for both compounds , the ic50 values systematically decrease with increasing salt concentrations , 
 indicating that activity is optimal under the more physiologically 
 relevant conditions represented by 150 mm nacl ( supporting information , figure s2 ) . estimates of ki values for the inhibition of 
 the mt - stimulated 
 atpase activity are also included . analogues 4 , 7 , and 11 were 
 all previously tested against multiple kinesins and found to specifically 
 inhibit eg5 . against a panel of seven human kinesins containing kif3b 
 ( kinesin-2 ; organelle transport ) , neuron - specific kif5a and kif5b / conventional 
 kinesin ( both kinesin-1 ; cargo transport ) , kif7 ( kinesin-4 , involved 
 in hedgehog signaling ) , kif9 ( kinesin-9 ; regulation of matrix degradation ) , 
 and kif20a / mklp-2 and kif20b / mpp1 ( both kinesin-6 ; required for cytokinesis),rac-42 displayed no activity at a 
 maximum inhibitor concentration of 200 m ( supporting information , table s4 and figure s1 ) , which confirms 
 the proposed high selectivity of this class of inhibitors within the 
 kinesin superfamily . our analogues have not yet been tested on 
 atpases beyond the kinesins , although other s - trityl - l - cysteine analogues were recently shown to be weak inhibitors 
 of hepatitis c virus ns5b polymerase . a selection 
 of the new lead compounds was evaluated in five additional 
 cell lines derived from colon ( hct116 ) , pancreas ( bxpc-3 ) , prostate 
 ( lncap and pc3 ) , and lung ( nci - h1299 ) tumors , to determine in vitro 
 efficacy against a histologically broader panel of cancer cell lines 
 ( table 5 ) . the lead compounds from our previous 
 study with and without the carboxylic acid ( 11 and 13 , respectively ) were included to assess progress , along with the original hit 4 and 
 the phase ii drug candidates 1 and 2 . in 
 general , hct116 cells were the most responsive to the eg5 inhibitors 
 tested , whereas nci - h1299 cells were the least affected . three of 
 the new analogues ( 34 , 36 , and rac-42 ) displayed activity higher than that of the original 
 lead ( rac-11 ) ; additionally both 36 and rac-42 were more potent than the clinical candidate 1 and equal to its more potent second generation analogue 2 . for those compounds without the carboxylic acid group , three ( 26 , rac-47 , rac-51 ) were more active than the previous lead 13 but less active than rac-11 . we have 
 shown previously that for 4 , 11 , and 12 , the absolute configuration of the stereocenter in the 
 amino acid tail has very little effect on efficacy . the phenol compounds rac-47 and rac-51 were prepared racemically and contain 
 a stereocenter at the trityl carbon more likely to affect activity , 
 especially given that only the ( r)-enantiomer was 
 observed in the crystal structure of the eg546 complex . compounds prepared via the tfa - mediated dehydration of 
 chiral tertiary alcohols form a 1:1 ratio of enantiomers ( scheme 1 ) . however , we did 
 not investigate the resolution of rac-47 and rac-51 further , as rac-47 displayed very high clearance in human hepatocytes 
 ( clint = 50.1  4.2 l min ; t1/2 = 27.7 min ) and thus was not suitable 
 for progression as a drug candidate . we subsequently subjected the new lead 
 compounds from this study 
 ( 34 , 36 , and rac-42 ) to a series of in vitro and in vivo physicochemical and 
 admet assays to evaluate if in addition to pharmacodynamics improvements , 
 they exhibited favorable drug - like properties ( table 6 ) . the phase ii candidate 2 was included as an 
 external benchmark ; the profile for the first generation candidate 1 has been reported previously . the p - methoxy analogue 7 was also 
 profiled , as this compound was reported to inhibit the rate of tumor 
 growth in subcutaneous prostate ( pc3 cells ) and bladder 
 xenograft tumor models . analogue 7 has a balanced profile , with favorable physiochemical characteristics 
 and reasonable metabolic stability in both human microsomes and hepatocytes . moderate affinity is displayed for the 2c19 cyp isoform . in comparison 
 to the other lead compounds , 7 is systematically less 
 potent in vitro ( table 5 ) , and so was not investigated 
 further . the most potent analogue lacking the carboxylic acid 26 , which also contains a p - methoxyphenyl , 
 was profiled and interestingly proved metabolically more resilient 
 in human microsomal and hepatocyte assays than 7 . several 
 liabilities were however evident , including low turbidimetric solubility 
 at physiological ph ( > 30 m ) , herg inhibition , and moderate 
 to significant inhibition of cyp isoforms from four of the main families . this agreed with the trend previously observed 
 that compounds lacking the carboxylic acid group have generally more 
 liabilities than those possessing it . at this stage , we investigated whether modulating the basicity of 
 the primary amine would be an effective strategy to ameliorate these . although alkylation of primary amines can overcome 
 herg inhibition , the hydrogen bonds formed 
 by the primary amine in 4 are crucial to maintaining 
 activity ( figure 3a ) , with secondary amines 
 only weakly active . we therefore applied the subtler 
 strategy of modulating the pka by -fluorination 
 to produce 5254 ( table 7 ) . in the basal assay , 
 while inhibitory activity comparable to that of the unmodified 26 was displayed by -fluorinated rac-52 , the binding affinity was substantially reduced 
 for ,-difluorinated 53 and lowered to micromolar 
 levels for -trifluoromethyl - containing rac-54 . despite good potency against eg5 , in cells rac-52 proved 8-fold less active than 
 the unmodified 26 , and 53 only exhibited 
 micromolar levels of growth inhibition . while these results reinforce 
 the importance of the protonated state of the primary amine at physiological 
 ph , the reasonable basal activity suggests that multiple factors contributed 
 to the reduced cellular efficacy . as this strategy did not appear 
 useful in this series consequently , we profiled 
 our two most potent cysteine - based analogues , 34 and 36 , which differ only by an extra methylene 
 in the meta position that imparts 3- to 4-fold higher potency across 
 cellular assays ( table 5 ) . this gain in potency 
 comes at a cost : 36 exhibits higher clearance in human 
 hepatocytes ( 24.4  4.7 versus 7.9  3.4 l / min / million 
 cells ) and is less available in human plasma ( 1.55% versus 4.51% , 
 table 6 ) . a distinct profile is also evident 
 toward cyp enzymes : like the p - methoxy analogue 7 , 34 inhibits cyp 2c19 ( ic50 = 7.9 
  1.3 m ) , whereas 36 does not but instead 
 weakly inhibits cyp 2c9 ( ic50 = 14.0  4.9 m ) . compound 36 displays high bioavailability ( 63% ) when 
 compared to that determined previously for 1 ( 45% ) , 13 ( 4% ) , and rac-11 ( 51% ) ( table 8) . the pharmacokinetic 
 data is in good agreement with that from the in vitro mouse metabolic 
 profiling : 36 has a shorter half - life and higher clearance 
 than the clinical candidate 1 , with rac-11 displaying low clearance in comparison . the butanamine 
 analogue of 34 and most active from 
 the in vitro profiling , rac-42 , was 
 investigated next . direct comparison with 34 illustrates 
 that rac-42 displays higher turbidimetric 
 solubility ( > 100 m versus 65 m ) , increased metabolic 
 resilience in human hepatocytes ( 5.9  1.9 versus 7.9  
 3.4 l / min / million cells ) and does not inhibit any of the common 
 cyp isoforms examined . in common with the profiled zwitterionic s - trityl analogues , no affinity is evident toward the herg 
 channel . this attractive profile is analogous to that reported for 
 our previous lead , rac-11 . examining clinical candidate 2 in the same assays , 
 we observed improved solubility at the three phs tested compared to 1 ( > 100 m versus 65 m ) . the reduced log p of 2 also 
 translates 
 to a higher proportion of unbound compound in human plasma ( 22% versus 
 8% ) . however , 2 retains the potentially physiologically 
 significant herg channel interactions and affinity for the cyp isoform 
 3a4 evident for 1 . therefore , 
 the butanamines rac-11 and rac-42 possess a more balanced admet profile , which may 
 offer distinct clinical advantages . we advanced the three most 
 active and balanced of the new analogues ( 34 , 36 , and rac-42 ) into in vivo xenograft 
 experiments with lung cancer patient explants ( lxfs 538 ) passaged 
 as subcutaneous xenografts in nude mice . as controls , our previous 
 lead analogue rac-11 and the clinical 
 candidate 1 were selected . we adopted lung cancer xenograft 
 models , as this is a key area for improving therapy due to the poor 
 prognosis for most patients ; the indications 
 that eg5 inhibitors may achieve better clinical outcomes in hematological 
 malignancies rather than in solid tumors came after this work had 
 commenced . we used explanted tumors , as these are a more realistic 
 model than solid tumors derived from in vitro cell lines . the transplanted 
 tumors retain the histological complexity from the patient , reflect 
 prior treatment and have been shown to be better predictors of clinical 
 response . administering our previous lead , rac-11 , by an improved dosing schedule resulted 
 in complete tumor regression at day 44 ( figure 4a ) , in comparison to partial remission recorded previously . when treatment stopped , tumor growth relapsed 
 around day 76 ( data not shown ) . xenograft experiments for 1 have previously been reported at a dose of 810 mg / kg in 
 breast tumor models . in mice implanted 
 with lxfs 538 tumors , treatment at this dose proved to be lethal ( data 
 not shown ) , and similar toxicity issues have been observed previously . when we repeated at a lower dose of 6 mg / kg , 
 total tumor regression was evident around day 13 and the mice remained 
 tumor free over the entire observation time ( figure 4a ) . for the cysteine containing 36 , although 
 significantly more potent than rac-11 in cell - based assays , only a reduced tumor growth rate was observed 
 ( figure 4b ) , although the difference was still 
 statistically significant ( p = 0.0124 ) . metabolic profiling of 34 and 36 explained this poor in vivo activity compared to the strong 
 in vitro efficacy : although stable in mouse microsomes , 36 showed high clearance in mouse hepatocytes ( 58.7  5.64 l / min / million 
 cells ; t1/2 = 23.6 min ) . a similar explanation 
 applied for 34 : while stable in human microsomes with 
 only low clearance in human hepatocytes ( 7.9  3.4 l / min / million 
 cells ) , medium clearance was evident in mouse microsomes ( 17.8  
 3.7 l / min / mg protein ) and extremely high clearance by mouse 
 hepatocytes ( 170  9.4 l / min / million cells ; t1/2 = 8.2 min ) . therefore , nude mice xenografts are not 
 an appropriate model for assessing the therapeutic potential of 34 and 36 : in the future , alternative models 
 such as nude rats will need to be employed , if these compounds prove 
 metabolically more stable in rat hepatocytes . in contrast to the thioethanamines 34 and 36 , the activity of the most potent butanamine rac-42 was translated effectively into the 
 xenograft model , with total tumor regression by day 17 , improving 
 on the activity recorded for rac-11 and 
 comparable to the response achieved on treatment with 1 ( figure 4c ) . antitumor efficacy of rac-11 , 34 , 36 , and rac-42 compared to the clinical candidate 1 in a subcutaneous 
 tumor xenograft model with lxfs 538 . the control groups ( black  ) 
 received only vehicle on the same days as treatment with inhibitors . ( a ) tumor xenografts for 1 ( purple  ) and rac-11 ( orange  ) showing total regression 
 after 13 days of treatment for 1 and at day 44 for rac-11 . the difference between the treated 
 groups and vehicle are statistically significant ( p < 0.0001 for both 1 and rac-11 ) . ( b ) reduced tumor growth rate by 36 . the 
 difference between the treated group ( 36 , green  ) 
 and vehicle is statistically significant ( p = 0.0124 ) . ( c ) differing responses of xenografts to 34 ( blue  ) 
 and rac-42 ( red  ) . treatment 
 with rac-42 results in complete tumor 
 regression within 17 days . the difference between the treated group ( rac-42 , red  ) and vehicle is statistically significant 
 ( p = 0.0003 ) . optimization of 4 has led 
 to structurally relatively 
 simple yet extremely effective eg5 inhibitors which display in vitro 
 and in vivo antitumor activity comparable to benchmark inhibitors 
 further advanced in clinical trials . the only other eg5 inhibitors 
 reported to induce complete tumor regression in mice xenograft models , 
 to the best of our knowledge , are the three clinical candidates 13 in phase ii . we propose that 
 the favorable drug - like properties , as highlighted by in vitro profiling , 
 strengthens the case for the progression of triphenylbutanamine analogues 
 based on rac-42 for further evaluation . in general , the butanamines proved systematically more potent than 
 the equivalent thioethanamines , but most notably their increased metabolic 
 stability imparted improved efficacy in xenograft studies with nude 
 mice . both the profiled butanamine and thioethanamine leads rac-11 and 36 show 
 good oral bioavailability and pharmacokinetics ; however , for an accurate 
 assessment of the potential of these s - trityl analogues , 
 alternative in vivo models will need to be examined . in this and our 
 preceding study , we have developed a range of modifications to the 
 core scaffold which impart excellent affinity for the target protein 
 eg5 . the potential of clinical candidates based on rac-42 will be determined by selecting those which provide 
 optimal bioavailability and robust pharmacokinetics , a process we 
 have initiated through the profiling described here . the wider challenge 
 facing eg5 inhibitors is translating the efficacy evident in preclinical 
 studies into the clinic . the responses to eg5 inhibitors in clinical 
 trials have been limited to date , and one explanation for this is 
 that tumor doubling times in xenograft models are significantly faster 
 than those in human patients and thus not sufficiently realistic models 
 for developing chemotherapeutics . this 
 hypothesis is corroborated by the improved responses observed against 
 faster growing hematological cancers and the principal dose - limiting 
 toxicities for most clinical candidates of neutropenia and myelosuppression . early clinical indications suggest a possible 
 application for eg5 inhibitors in advanced multiple myeloma , which 
 will most likely be in combination with existing therapies . in combined chemotherapies , the potential for 
 inducing drug  drug interactions and toxicities becomes increasingly 
 important . for example , the hepatic metabolism of the synthetic glucocorticoid 
 dexamethasone currently used in the treatment of multiple myeloma 
 is mediated primarily by cyp 3a4 . both 
 of the trial eg5 drugs we profiled , 1 and 2 , are cyp 3a4 inhibitors , so careful consideration of the physiological 
 relevance of these interactions would be required prior to undertaking 
 combination - based therapy . we have demonstrated that in comparison to the 
 clinical candidates 1 and 2 , the lead s - trityl - l - cysteine - derived analogues rac-11 and rac-42 exhibit 
 comparable in vivo efficacy by inducing complete tumor regression 
 in an advanced tumor explant xenograft model but display fewer liabilities 
 toward cyp enzymes and the herg channel . furthermore , unlike other 
 highly advanced eg5 inhibitors , the described compounds are not patented 
 and therefore are freely available for investigation in single or 
 combined cancer chemotherapies by the scientific and medical community , 
 which we propose , based on the recent results for arry-520 , should 
 focus on targeting hematological malignancies . compounds 4 and 2 were purchased from nova biochem , sigma aldrich , 
 and selleck chemicals , 
 respectively , and used without further purification . compound 7 ( nsc123528 ) was obtained from the 
 nci / dtp open chemical repository ( http://dtp.cancer.gov ) of the national cancer institute . anhydrous 
 reactions were carried out in oven - dried glassware under a nitrogen 
 atmosphere unless otherwise noted . microwave reactions were performed 
 using a biotage initiator-8 microwave synthesizer ( operating at 2.45 
 ghz ) . thin - layer chromatography ( tlc ) was carried out on aluminum - backed 
 sio2 plates ( silica gel 60 , f254 ) , and spots 
 were visualized using ultraviolet light ( 254 nm ) or by staining with 
 phosphomolybdic acid ( alcohols ) or ninhydrin ( amines ) . flash column 
 chromatography was performed on silica gel ( snap kp - sil , 60  , 
 4063 m cartridges ) using a biotage sp4 automated chromatography 
 system ( detection wavelength , 254 nm ; monitoring , 280 nm ) . melting 
 points were determined using a stuart scientific smp1 melting point 
 apparatus and are uncorrected . h and c nmr 
 spectra were recorded on a jeol ecx-400 ( 400 mhz ) , avance dpx400 ( 400 
 mhz ) , or avance dpx500 ( 500 mhz ) spectrometer . f nmr 
 spectra were recorded on an avance av400 ( 400 mhz ) instrument equipped 
 with a multinuclear probe . h chemical shifts (  ) 
 are reported in ppm relative to the residual signal of the deuterated 
 solvent ( 7.26 in cdcl3 , 3.31 in cd3od , and 2.50 
 in dmso - d6 ) . multiplicities are indicated 
 by s ( singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , m ( unresolved 
 multiplet ) , and br ( broad signal ) . c chemical shifts 
 (  ) are reported in ppm relative to the carbon resonance of 
 the deuterated solvent ( 77.16 in cdcl3 , 49.00 in cd3od , and 39.52 in dmso - d6 ) . high - resolution 
 mass spectra were recorded on a thermo electron ltq orbitrap mass 
 spectrometer using electrospray ionization . gas chromatography mass 
 spectra ( gc - ms ) using electron ionization ( ei ) were recorded on a 
 thermo scientific focus gc with dsq2 single quadrupole mass spectrometer . gc - ms using chemical ionization ( ci ) were recorded on an agilent technologies 
 7890a gc system and an agilent 5975c inert xl ei / ci msd with a dsq2 
 single quadrupole mass spectrometer , equipped with an agilent technologies 
 db5-ms column ( 30 m  0.25 mm  0.25 m ) . helium was 
 the carrier gas ( flow rate = 1 ml / min ) . elemental 
 analysis data were recorded on a perkin - elmer 2400 series 2 chn analyzer . lc - ms analyzes were performed with an agilent quaternary 1200 series 
 pump and an agilent 6130 dual source mass spectrometer with uv detection 
 at 254 nm . retention times ( tr ) were in 
 minutes , and purity was calculated as percentage of total area . the 
 method for determining purity consisted of the following : zorbax eclipse 
 xdb - c18 reverse phase column ( 15 cm  4.3 m , particle 
 size 5 m ) ; column temperature 40 c ; solvent a : h2o ( 5 mm ammonium acetate ) ; solvent b : mecn ( 5 mm ammonium 
 acetate ) ; gradient of a : b , 95:5 ( 03 min ) , a : b , 95:5  
 b , 100% ( 317 min ) , b , 100% ( 1727 min ) , b , 100%  
 a : b , 95:5 ( 2733 min ) , a : b , 95:5 ( 3336 min ) ; flow rate 
 1 ml min . new compounds were named according 
 to iupac nomenclature by acd chemsketch 12.01 ( windows , advanced chemistry 
 development , toronto , canada ) . oxalyl chloride ( 3.43 ml , 40 mmol ) was 
 added to a solution of 5-bromo-2-methylbenzoic acid 55 ( 4.30 g , 20 mmol ) in anhydrous ch2cl2 ( 20 
 ml ) , with a catalytic amount of dmf ( 1 drop ) , and stirred at room 
 temperature for 2 h. the reaction mixture was concentrated in vacuo , 
 and the residual oil was added by slow dropwise addition over 5 min 
 to a cooled ( 0 c ) solution of n , o - dimethylhydroxylamine hydrochloride ( 2.24 g , 23 mmol ) and triethylamine 
 ( 3.67 ml , 50 mmol ) in anhydrous ch2cl2 ( 20 ml ) . after being stirred for 1 h , the reaction mixture was warmed to room 
 temperature , stirred for 20.5 h , quenched with water followed by aqueous 
 hcl ( 0.5 m , 25 ml ) , and extracted with ch2cl2 ( 3  25 ml ) . the combined organic extracts were dried ( mgso4 ) , concentrated in vacuo , and purified by flash chromatography 
 ( sio2 ; 050% etoac in hexane ) to afford 56 as a colorless oil ( 3.63 g , 70% ) . h nmr ( cdcl3 , 500 mhz )  2.26 ( s , 3h , ch3 ) , 3.30 ( br s , 3h , 
 ch3 ) , 3.48 ( br s , 3h , ch3 ) , 7.07 ( d , j = 8.6 hz , 2h ) , 7.387.40 ( m , 2h ) . c nmr ( cdcl3 , 125 mhz )  18.77 , 61.33 , 118.95 , 129.13 , 
 131.95 , 132.23 , 134.06 , 137.20 . hrms ( esi+ ) calcd for c10h13brno2 ( m + h ) : 258.01242 ; found : 
 258.01244 . calcd for c10h12brno2 : c , 46.53 ; h , 4.69 ; n , 5.43 . found : c , 45.03 ; h , 4.78 ; n , 5.60 . the 
 title compound was prepared using an adaptation of the procedure 
 reported by hirashima et al . memgcl ( 3.0 
 m in thf , 8.27 ml , 24.8 mmol ) was added by slow dropwise addition 
 over 10 min to a cooled ( 0 c ) solution of 5-bromo - n - methoxy - n,2-dimethylbenzamide 56 ( 3.27 
 g , 12.4 mmol ) in thf ( 12.4 ml ) and stirred for 2 h. the reaction mixture 
 was warmed to room temperature and stirred for 2 h , before quenching 
 with saturated aqueous nh4cl solution ( 20 ml ) and extracting 
 with etoac ( 3  25 ml ) . the combined organic extracts were washed 
 successively with water and brine ( 75 ml each ) , dried ( mgso4 ) , and concentrated in vacuo to give 57 as a clear , 
 pale brown oil ( 2.40 g , 91% ) , which was used without further purification . h nmr ( cdcl3 , 500 mhz )  2.45 ( s , 3h , ch3 ) , 2.56 ( s , 3h , ch3 ) , 7.11 ( d , j = 8.2 hz , 1h ) , 7.48 ( dd , j = 2.1 , 8.2 hz , 1h ) , 
 7.77 ( d , j = 2.1 hz , 1h ) . c nmr ( cdcl3 , 125 mhz )  22.10 , 29.62 , 119.24 , 132.11 , 133.77 , 134.39 , 
 137.29 , 139.46 . gc - ms ( ei , 70 ev ) tr = 
 4.68 min ( m / z = 211.9 , m ) calcd for c9h9bro c , 50.73 ; h , 4.26 . 
 found : c , 50.44 ; h , 4.30 . the 
 title compound was prepared using an adaptation of the procedure reported 
 by chackal - catoen et al . hydrazine hydrate 
 monohydrate ( 1.46 ml , 30 mmol ) was added to a solution of 1-(5-bromo-2-methylphenyl)ethanone 57 ( 2.13 g , 10 mmol ) and powdered koh ( 1.68 g , 30 mmol ) in 
 anhydrous ethylene glycol ( 10 ml ) and refluxed for 4 h. after being 
 cooled to room temperature , the reaction was quenched was aqueous 
 hcl ( 1.0 m , 30 ml ) and extracted with etoac ( 3  30 ml ) . the 
 combined organic extracts were washed successively with water and 
 brine ( 75 ml each ) , dried ( mgso4 ) , and concentrated in 
 vacuo , and the residue was purified by flash chromatography ( sio2 ; hexane ) to afford alkane 58 as a colorless 
 oil ( 1.10 g , 55% ) . h nmr ( cdcl3 , 400 mhz )  
 1.20 ( t , j = 7.5 hz , 3h , ch3 ) , 2.24 ( s , 
 3h , ch3 ) , 2.59 ( q , j = 7.5 hz , 2h , ch2 ) , 7.00 ( d , j = 8.0 hz , 1h ) , 7.22 ( dd , j = 2.0 , 8.0 hz , 1h ) , 7.28 ( d , j = 1.9 
 hz , 1h ) . c nmr ( cdcl3 , 100 mhz )  14.21 , 
 18.84 , 26.18 , 119.63 , 128.72 , 130.80 , 131.71 , 134.83 , 144.68 . gc - ms 
 ( ei , 70 ev ) tr = 4.90 min ( m / z = 199.8 , m ) . n - butyllithium ( 2.5 m 
 in hexane , 2.21 ml , 5.53 
 mmol ) was added by slow dropwise addition over 2 min to a cooled ( 78 
 c ) solution of 4-bromo-2-ethyl-1-methylbenzene 58 ( 918 mg , 4.61 mmol ) in anhydrous thf ( 4.61 ml ) and stirred for 1 
 h at 70 c . a solution of benzophenone ( 966 mg , 
 5.30 mmol ) in anhydrous thf ( 5.30 ml ) was added by slow dropwise addition 
 over 5 min , and the reaction mixture was stirred with the temperature 
 maintained at 70 c for 5 h , before allowing 
 the reaction to warm to room temperature and stirring for a further 
 20 h. the reaction was quenched with saturated aqueous nh4cl solution ( 10 ml ) and extracted with etoac ( 3  10 ml ) . the 
 combined organic extracts were then washed successively with h2o and brine ( 30 ml each ) , dried ( mgso4 ) , and concentrated 
 in vacuo . purification by flash chromatography ( sio2 ; 010% 
 etoac in hexane ) afforded trityl alcohol 59 as a yellow 
 oil ( 1.263 g , 91% ) . h nmr ( cdcl3 , 500 mhz ) 
 1.14 ( t , j = 7.6 hz , 3h , ch3 ) , 2.31 ( s , 
 3h , ch3 ) , 2.59 ( q , j = 7.6 hz , 2h , ch2 ) , 2.77 ( s , 1h , oh ) , 6.93 ( dd , j = 2.0 , 7.9 
 hz , 1h ) , 7.07 ( d , j = 7.9 hz , 1h ) , 7.12 ( d , j = 1.9 hz , 1h ) , 7.267.32 ( m , 10h ) . c nmr ( cdcl3 , 125 mhz )  14.60 , 18.92 , 82.11 , 125.60 , 
 127.24 , 127.66 , 127.98 , 128.06 , 129.67 , 134.96 , 142.11 , 144.77 , 147.26 . hrms ( esi ) calcd for c21h21 ( m  
 oh ) : 285.16378 ; found : 285.16348 . a solution of the tertiary alcohol 59 ( 254 mg , 0.84 mmol ) with l - cysteine ( 112 mg , 0.93 
 mmol ) in tfa ( 1.0 ml ) was stirred for 3 h at room temperature . the aqueous mixture was extracted with ch2cl2 ( 3  10 ml ) and the organic layer dried ( mgso4 ) 
 and concentrated in vacuo . purification by flash chromatography ( sio2 ; 025% meoh in ch2cl2 ) afforded 
 the thioether 36 as a white solid ( 242 mg , 71% ) : mp 149152 
 c . h nmr ( 500 mhz , meod )  = 1.10 ( t , 3h , j = 7.6 hz ) , 2.27 ( s , 3h ) , 2.56 ( q , 2h , j = 7.6 hz ) , 2.69 ( dd , 1h , j = 9.2 , 13.4 hz ) , 2.82 
 ( dd , 1h , j = 4.2 , 13.4 hz ) , 3.05 ( dd , 1h , j = 4.2 , 9.2 hz ) , 7.06 ( d , 1h , j = 8.0 
 hz ) , 7.14 ( dd , 1h , j = 2.1 , 8.0 hz ) , 7.187.24 
 ( m , 3h ) , 7.277.32 ( m , 4h ) , 7.427.46 ( m , 4h ) . c nmr ( 125 mhz , meod )  = 14.94 , 18.73 , 27.28 , 34.36 , 
 55.13 , 68.05 , 127.92 , 128.07 , 129.04 , 130.47 , 130.70 , 130.74 , 135.60 , 
 143.16 , 143.29 , 145.91 , 145.95 , 172.53 . hrms ( esi+ ) calcd for c25h28no2s ( m + h ) : 406.1835 ; 
 found 406.1843 . calcd for c25h27no2s/2h2o : c , 72.43 ; 
 h , 6.81 ; n , 3.38 . found : c , 72.18 ; h , 6.37 ; n , 3.14 . the title compound was prepared using an 
 adaptation of 
 the method reported by kabalka et al.n - butyllithium ( 2.5 m in hexane , 6.12 ml , 15.3 
 mmol ) was added cautiously by dropwise addition to a cooled ( 0 c ) 
 solution of ( 3,4-dimethylphenyl)(diphenyl)methanol 61 ( 4.00 g , 13.9 mmol ) in anhydrous ch2cl2 ( 85 
 ml ) , and the reaction mixture was allowed to warm to room temperature 
 and stirred for 30 min . allyltrimethylsilane ( 6.02 ml , 36.9 mmol ) 
 and iron trichloride ( 5.19 g , 32.0 mmol ) were then added , and the 
 reaction was stirred at room temperature for 6 h. the reaction was 
 quenched with h2o ( 100 ml ) and extracted with ch2cl2 ( 3  100 ml ) . the combined organic extracts were 
 washed successively with saturated aqueous nahco3 solution 
 ( 150 ml ) and brine ( 200 ml ) , dried ( mgso4 ) , and concentrated 
 in vacuo . purification by flash chromatography ( sio2 ; 010% 
 etoac in hexane ) afforded alkene 61 as a brown oil [ 3.119 
 g , 91% ( based on 79% conversion ) ] and recovered starting material 60 ( 834 mg ) . h nmr ( 400 mhz , cdcl3 ) 
  = 2.22 ( s , 3h , ch3 ) , 2.26 ( s , 3h , ch3 ) , 3.433.46 ( m , 2h ) , 4.97 ( ddd , j = 1.4 , 
 3.4 , 10.3 hz , 1h ) , 5.06 ( ddd , j = 1.5 , 3.5 , 17.0 
 hz , 1h ) , 5.70 ( ddd , j = 6.6 , 10.4 , 17.1 hz , 1h ) , 
 6.946.98 ( m , 1h ) , 7.037.08 ( m , 2h ) , 7.197.34 
 ( m , 10h ) . c nmr ( 100 mhz , cdcl3 )  = 
 19.40 , 20.24 , 45.67 , 56.01 , 117.18 , 125.97 , 126.97 , 127.79 , 129.14 , 
 129.58 , 130.62 , 134.23 , 135.87 , 136.35 , 144.92 , 147.65 . gc - ms ( ci , 
 methane ) tr = 16.44 min ( m / z = 311.2 , [ m  h ] ) . calcd 
 for c24h24 : c , 92.26 ; h , 7.74 . found : c , 91.51 ; 
 h , 7.28 . a solution of bh3thf ( 1.0 m in thf , 20.0 
 ml , 20.00 mmol ) was added to a cooled ( 0 c ) solution of alkene 61 ( 3.11 g , 9.95 mmol ) in thf ( 20 ml ) , and stirred at room 
 temperature for 19 h. the reaction was cooled to 0 c and quenched 
 cautiously with h2o ( 5 ml ) and aqueous naoh ( 3.0 m , 6.8 
 ml , 20.40 mmol ) , followed by slow dropwise addition of hydrogen peroxide 
 ( 30% in h2o , 5.11 ml , 50.01 mmol ) over 5 min . the mixture 
 was maintained at 0 c for 30 min and then allowed to warm to 
 room temperature and stirred for a further 3.5 h , diluted with h2o ( 50 ml ) , and extracted with et2o ( 3  50 
 ml ) . the combined organic extracts were washed successively with saturated 
 aqueous nahco3 solution ( 200 ml ) and brine ( 200 ml ) , dried 
 ( mgso4 ) , and concentrated in vacuo . purification by flash 
 chromatography ( sio2 ; 022% etoac in hexane ) afforded 
 the primary alcohol 62 as a colorless oil ( 1.90 g , 58% ) . h nmr ( 400 mhz , cdcl3 )  = 1.311.40 
 ( m , 2h , ch2 ) , 2.19 ( s , 3h , ch3 ) , 2.21 ( s , 3h , 
 ch3 ) , 2.592.66 ( m , 2h , ch2 ) , 3.62 ( t , j = 6.4 hz , 2h , ch2 ) , 6.977.03 ( m , 2h ) , 
 7.047.09 ( m , 1h ) , 7.147.19 ( m , 2h ) , 7.227.31 
 ( m , 8h ) . c nmr ( 100 mhz , cdcl3 )  = 
 19.36 , 20.25 , 29.36 , 36.61 , 56.06 , 63.54 , 125.85 , 126.75 , 127.89 , 
 129.19 , 129.36 , 130.44 , 134.11 , 135.90 , 144.94 , 147.67 . gc - ms ( ci , 
 methane ) tr = 17.91 min ( m / z = 371.1 , [ m + c3h5 ] ) . calcd for c24h26o1/4etoac : 
 c , 85.19 ; h , 8.01 . found : c , 85.11 ; h , 7.81 . the title compound was prepared using an 
 adaptation of the method 
 developed by dess et al . and the procedure reported by wang et al . martin periodinane ( 3.069 g , 7.24 mmol ) was added to 
 a solution 4-(3,4-dimethylphenyl)-4,4-diphenylbutan-1-ol 62 ( 1.993 g , 6.03 mmol ) in anhydrous ch2cl2 ( 24 
 ml ) . the reaction was stirred at room temperature for 4 h and then 
 quenched cautiously with sodium thiosulfate solution ( 0.26 m in saturated 
 aqueous nahco3 solution , 100 ml ) and extracted with ch2cl2 ( 3  50 ml ) . the combined organic extracts 
 were washed successively with saturated aqueous nahco3 solution , 
 h2o , and brine ( 150 ml each ) , dried ( mgso4 ) , 
 and concentrated in vacuo . purification by flash chromatography ( sio2 ; 016% etoac in hexane ) afforded aldehyde 63 as a colorless oil ( 830 mg , 42% ) . h ( 400 mhz , cdcl3 )  = 2.20 ( s , 3h , ch3 ) , 2.22 ( s , 3h , ch3 ) , 2.292.35 ( m , 2h , ch2 ) , 2.882.94 
 ( m , 2h , ch2 ) , 6.977.06 ( m , 3h ) , 7.157.22 
 ( m , 2h ) , 7.247.30 ( m , 8h ) . c nmr ( 100 mhz , cdcl3 )  = 19.36 , 20.24 , 31.99 , 41.30 , 55.63 , 126.17 , 126.60 , 
 128.13 , 129.18 , 129.40 , 130.26 , 134.48 , 136.21 , 144.11 , 146.96 , 202.01 . gc - ms ( ci , methane ) tr = 17.57 min ( m / z = 329.2 [ m + h ] ) . the title compound 
 was prepared by an adaptation of the method reported by yadav et al . montmorillonite ksf clay ( 4.5 g ) and 1-phenylmethanamine 
 ( 549 l , 5.02 mmol ) were added to a solution of 4-(3,4-dimethylphenyl)-4,4-diphenylbutanal 63 ( 300 mg , 1.0 mmol ) in anhydrous ch2cl2 ( 45 ml ) at room temperature . trimethylsilyl cyanide ( 686 l , 
 5.48 mmol ) was then added , the reaction mixture was stirred at room 
 temperature for 2.5 h and filtered , and the clay was rinsed with ch2cl2 . the filtrate was concentrated in vacuo and 
 purification of the residue by flash chromatography ( sio2 ; 016% etoac in hexane ) afforded the racemic -aminonitrile 64 as a pale yellow oil [ 931 mg , 68% ( based on 79% conversion ) ] 
 and unreacted 4-(3,4-dimethylphenyl)-4,4-diphenylbutanal 63 ( 485 mg ) . h ( 500 mhz , cdcl3 )  = 1.561.62 ( m , 
 2h , ch2 ) , 2.20 ( s , 3h , ch3 ) , 2.23 ( s , 3h , ch3 ) , 2.712.81 ( m , 2h , ch2 ) , 3.363.40 
 ( m , 1h , ch ) , 3.78 ( d , j = 12.8 hz , 1h , chahbbn ) , 4.02 ( d , j = 12.8 
 hz , 1h , chahbbn ) , 6.987.06 
 ( m , 3h ) , 7.177.21 ( m , 2h ) , 7.257.31 ( m , 9h ) , 7.337.36 
 ( m , 4h ) . c nmr ( 100 mhz , cdcl3 )  = 
 19.34 , 20.22 , 30.12 , 36.31 , 50.17 , 51.78 , 55.95 , 120.33 , 126.14 , 126.64 , 
 127.72 , 128.10 , 128.49 , 128.74 , 129.18 , 129.39 , 130.22 , 134.43 , 136.17 , 
 138.42 , 144.13 , 147.03 , 147.06 . hrms ( esi+ ) calcd for c32h33n2 ( m + h ) : 445.2638 ; found 
 445.2640 . the title compound 
 was prepared using an adaptation of the procedure reported by wang 
 et al . concentrated hcl ( 12 m , 25 ml ) 
 was added to a solution of the nitrile rac-64 ( 900 mg , 2.02 mmol ) in dioxane ( 25 ml ) and the mixture 
 heated at reflux for 48 h. after cooling , the mixture was concentrated 
 in vacuo . ph 9.5 ) with saturated 
 aqueous sodium carbonate solution and extracted with ch2cl2 ( 3  100 ml ) . purification by 
 flash chromatography ( sio2 ; 020% meoh in ch2cl2 with 1% nh4oh ) afforded the protected 
 amino acid rac-65 as a white solid ( 538 
 mg , 57% ) : mp 176178 c . h ( 400 mhz , meod ) 
  = 1.511.70 ( m , 2h , ch2 ) , 2.16 ( s , 3h , ch3 ) , 2.19 ( s , 3h , ch3 ) , 2.642.86 ( m , 2h , 
 ch2 ) , 2.642.85 ( m , 2h , ch2 ) , 3.403.44 
 ( m , 1h , ch ) , 3.92 ( d , j = 12.8 hz , 1h , chahbph ) , 4.02 ( d , j = 12.8 
 hz , 1h , chahbph ) , 6.957.05 
 ( m , 2h ) , 7.117.17 ( m , 2h ) , 7.207.31 ( m , 8h ) , 7.347.40 
 ( m , 5h ) . c nmr ( 100 mhz , meod )  = 19.23 , 20.09 , 
 27.89 , 36.66 , 51.22 , 57.06 , 63.35 , 126.93 , 127.81 , 128.87 , 130.09 , 
 130.30 , 130.41 , 131.08 , 131.50 , 132.78 , 135.20 , 136.91 , 145.69 , 148.55 , 
 173.40 . hrms ( esi+ ) calcd for c32h34no2 ( m + h ) : 464.2584 ; found 464.2585 . the title compound 
 was prepared 
 using an adaptation of the method reported by ram et al . a solution of benzylamine rac-65 ( 520 mg , 1.12 mmol ) , 10% pd / c ( 250 mg ) , and hcoonh4 ( 354 mg , 5.61 mmol ) in anhydrous meoh ( 7.5 ml ) was heated 
 at reflux for 2 h. after being cooled to room temperature , the mixture 
 was filtered through a thick pad of celite , the residue washed with 
 meoh ( 25 ml ) , and the filtrate concentrated in vacuo . purification 
 by flash chromatography ( sio2 ; 025% meoh in ch2cl2 ) afforded the racemic amino acid 42 as a white solid ( 346 mg , 83% ) : mp 258259 c . h ( 500 mhz , meod )  = 1.571.65 ( m , 2h , ch2 ) , 2.17 ( s , 3h , ch3 ) , 2.20 ( s , 3h , ch3 ) , 2.652.86 ( m , 2h , ch2 ) , 3.53 ( t , 1h , j = 6.0 hz , ch ) , 6.977.05 ( m , 2h ) , 7.137.17 
 ( m , 2h ) , 7.227.31 ( m , 8h ) . c nmr ( 125 mhz , meod ) 
  = 19.22 , 20.08 , 28.93 , 36.75 , 56.33 , 57.04 , 126.92 , 127.80 , 
 128.85 , 130.06 , 130.33 , 131.52 , 135.18 , 136.89 , 145.72 , 148.55 , 148.59 , 
 166.57 . hrms ( esi+ ) calcd for 
 c25h27no2h2o : c , 
 76.70 ; h , 7.47 ; n , 3.58 . found : c , 76.74 ; h , 7.27 ; n , 3.45 . the inhibition of the basal and mt - stimulated atpase activities 
 was measured as previously described using a tecan sunrise photometer . the inhibition of the basal atpase activity was measured 
 at a concentration of 80100 nm eg5 in the presence of 150 
 mm nacl . the inhibition of the mt - stimulated atpase activity was determined 
 at an eg5 concentration of 5 nm in the presence of 150 mm 
 nacl . for this eg5 construct , it has been noted that the ki estimates are dependent on the salt concentration 
 of the buffer.s - trityl - l - cysteine and related inhibitors are tight - binding inhibitors ; estimates for ki values were therefore calculated using the morrison 
 equation . ligand efficiencies 
 ( le ) were calculated from the equation : le = g / hac  rt ln(ki)/hac , where g is 
 the change in gibbs free energy , t is the absolute 
 temperature , r represents the gas constant , and hac 
 is the heavy atom count for non - hydrogen atoms . ligand efficiencies were calculated in pipeline pilot 7.4 
 for windows ( accelerys software , san diego , ca ) . hct116 ( atcc ccl-247 ) cells were cultured 
 in dmem ( invitrogen , paisley , u.k . ) , supplemented with 10% fetal bovine 
 serum ( paa , pasching , austria ) . k562 ( atcc ccl-243 ) , lncap ( atcc crl-1740 ) , 
 pc-3 ( atcc crl-1435 ) , and nci - h1299 ( crl-5803 ) cells were cultured 
 in rpmi ( invitrogen , paisley , u.k . ) , supplemented with 10% fetal bovine 
 serum ( paa , pasching , austria ) . bxpc-3 ( atcc crl-1687 ) cells were 
 cultured in rpmi ( invitrogen , paisley , u.k . ) , supplemented with 1% 
 nonessential amino acids ( invitrogen , paisley , u.k . ) , 1% sodium pyruvate 
 ( invitrogen , paisley , u.k . ) , 1% glutamine ( invitrogen , paisley , u.k . ) , 
 and 10% fetal bovine serum ( paa , pasching , austria ) . cells were maintained 
 at 37 c , 95% humidity , and 5% carbon dioxide in a humidified 
 incubator and used for experiments for 68 weeks before being 
 replaced with fresh stocks that had been stored in liquid nitrogen . cells were seeded in triplicate 
 in 96-well assay plates at 1.250 cells ( bxpc-3 , hct116 ) , 1.500 cells 
 ( pc-3 ) , 2.500 cells ( nci - h1299 ) , 3.000 cells ( lncap ) , or 5.000 cells 
 ( k562 ) per well in 100 l of the respective growth medium . medium 
 blanks and cell blanks were also prepared for every cell line . on 
 the next day , inhibitors were added with a starting concentration 
 of 100 m in a 3-fold serial dilution series . at 72 h after 
 addition of inhibitors , 10% alamar blue ( invitrogen , paisley , u.k . ) 
 was added , and depending on the cell line , between 2 and 12 h later 
 the absorbance was measured at 570 and 600 nm . all values were corrected 
 for the absorbance of the medium blank , and the corrected cell blanks 
 were set to 100% . calculations for determining the relative proliferation 
 were performed using the equations described in the manufacturer s 
 manual . the gi50 values were subsequently determined using 
 a sigmoidal dose  response fitting ( variable slope ) with graphpad 
 prism 5.03 for windows ( graphpad software , san diego , ca ) . profiling of drug - like 
 properties of interesting compounds including physicochemical properties 
 ( turbidimetric solubility , permeability ) , metabolism and safety ( human 
 and mouse microsomal and hepatocyte stability , plasma protein binding , 
 cytochrome p450 inhibition , herg ) , and pharmacokinetics were determined 
 at cyprotex , as previously described . mice were randomized to the various groups , 
 and dosing was started when the required number of mice carried a 
 tumor of 50250 mm volume , preferably 80200 
 mm . vehicle for 1 : 10% ethanol , 10% cremophor , 
 80% d5w ( dextrose 5% ) ; vehicle for all other compounds : 8% dmso , 2% 
 tween 80 , distilled water ( ph 5 ) . all treatments were given intraperitoneally . vehicle control mice ( group 1 ) were treated with 10 ml / kg vehicle 
 on days 0 , 3 , 6 , 8 , 10 , 13 , 20 , 22 , 24 , 29 , 31 , 34 , 36 , 38 , 48 , 51 , 
 55 , 58 , 62 , 65 , and 69 . the rac-11 treatment group ( group 2 ) received 20 mg / kg rac-11 on day 0 ; 14 mg / kg rac-11 on day 3 ; 
 18 mg / kg rac-11 on days 6 and 8 ; 14 
 mg / kg rac-11 on days 10 and 13 ; 16 mg / kg rac-11 on day 20 ; 18 mg / kg rac-11 on days 22 , 24 , 29 , and 31 ; 20 mg / kg rac-11 on day 34 ; 16 mg / kg rac-11 on days 36 and 38 ; 20 mg / kg rac-11 on days 48 , 51 , 55 , and 62 ; and 22.5 mg / kg rac-11 on days 65 , 69 , and 80 . the treatment group for 1 ( group 3 ) received 10 mg / kg of 1 on days 0 and 4 , and 
 6 mg / kg of 1 on days 7 , 20 , and 24 . vehicle control 
 mice ( group 1 ) were treated with 10 ml / kg vehicle 
 on days 2 , 4 , 7 , 11 , 14 , 16 , 18 , 21 , 23 , 25 , 28 , 30 , and 32 . the 36 treatment group ( group 2 ) received 15 mg / kg 36 on days 0 , 2 , 4 ; 17 mg / kg 36 on day 7 ; 14 mg / kg 36 on days 11 , 14 , and 16 ; 17 mg / kg 36 on days 
 18 , 21 , and 23 ; 20 mg / kg 36 on days 25 and 28 ; and 22.5 
 mg / kg 36 on days 30 and 32 . vehicle control mice 
 ( group 1 ) were treated with 10 ml / kg vehicle 
 on days 0 , 3 , 7 , 10 , and 14 . the rac-42 treatment group ( group 2 ) received 15 mg / kg rac-42 on days 0 , 3 , 7 , 10 , and 14 . the 34 treatment group ( group 3 ) received 15 mg / kg 34 on days 
 0 , 3 , 7 , 10 , and 14 and up to 30 mg / kg during later treatment , without 
 indications of toxicity at this dose . the tumor volume was determined by two - dimensional 
 measurement with a caliper on the day of randomization ( day 0 ) and 
 then twice weekly ( i.e. , on the same days on which mice were weighed ) . tumor volumes were calculated according to the formula ( ab )  0.5 where a represents the 
 largest and b the perpendicular tumor diameter . tumor 
 inhibition for a particular day ( t / c in % ) was calculated from the ratio of the median rtv ( relative 
 tumor volumes ) values of test versus control groups multiplied by 
 100% . for the evaluation of the statistical significance of tumor 
 inhibition , the mann individual 
 rtvs were compared on days on which the minimum t / c was achieved , as long as sufficient animals were 
 left for statistical analysis or otherwise on days as indicated . by 
 convention , p  0.05 indicates significance 
 of tumor inhibition . statistical calculations were performed using 
 graphpad prism 5.03 ( graphpad software , san diego , ca ) . purified eg5 ( 20 
 mg / ml ) in complex with 1 mm mg2atp 
 was incubated with 2 mm rac-46 ( in dmso ) 
 for 2 h on ice . crystals of eg5 with 46 appeared after 
 1 week in hanging drops by mixing 1 l of protein inhibitor 
 complex with 1 l of reservoir solution containing 22% peg-3350 , 
 0.3 m ammonium sulfate , 0.1 m mes ph 5.5 , and 0.01 m trimethylamine 
 hydrochloride in vdx plates ( hampton research ) at 4 c . dehydrating 
 solution ( 32% peg-3350 , 0.36 m of ammonium sulfate , 0.12 m mes ph 
 5.5 , 0.012 m trimethylamine hydrochloride , and 10% glycerol ) was added 
 slowly to the crystal droplet until the total volume of the drop was 
 eight times the original . the drop was then equilibrated against air 
 in 4 c for 30 min . a cubic crystal with dimensions of approximately 
 0.1 mm on each side diffraction data for 
 the eg5inhibitor complexes eg546 were 
 recorded at the esrf id23 - 2 . data were processed using imosflm and scaled using scala from the ccp4 suite of programs . the eg546 structure was solved by molecular replacement 
 ( phaser ) using one molecule of eg5 motor 
 domain from 3ken . electron - density and 
 difference density maps , all a - weighted , were inspected , 
 and the model was improved using coot . for the eg546 complex , 97.0% ( 322 ) 
 of the residues are in the preferred regions , 2.7% ( 9 ) are in the 
 allowed regions , and 0.3% ( 1 ) are outliers .
the mitotic kinesin eg5 is critical 
 for the assembly of the mitotic 
 spindle and is a promising chemotherapy target . 
 previously , we identified s - trityl - l - cysteine as a selective inhibitor of 
 eg5 and developed triphenylbutanamine analogues with improved potency , 
 favorable drug - like properties , but moderate in vivo activity . 
 we 
 report here their further optimization to produce extremely potent 
 inhibitors of eg5 ( kiapp < 
 10 nm ) with broad - spectrum activity against cancer cell lines comparable 
 to the phase ii drug candidates ispinesib and sb-743921 . 
 they have 
 good oral bioavailability and pharmacokinetics and induced complete 
 tumor regression in nude mice explanted with lung cancer patient xenografts . 
 furthermore , they display fewer liabilities with cyp - metabolizing 
 enzymes and herg compared with ispinesib and sb-743921 , which is important 
 given the likely application of eg5 inhibitors in combination therapies . 
 we present the case for this preclinical series to be investigated 
 in single and combination chemotherapies , especially targeting hematological 
 malignancies .