smyd2 is a catalytic set domain containing 
 protein methyltransferase reported to monomethylate lysine residues 
 on histone and nonhistone proteins .
its 
 overexpression has been reported in cancer cell lines as well as in 
 esophageal squamous carcinoma , bladder carcinoma , gastric cancer , 
 and pediatric acute lymphoblastic leukemia patients . in these studies ,
smyd2 overexpression often correlated with lower 
 survival rate and was suggested to be a clinically relevant prognostic 
 marker .
knockdown of smyd2 in overexpressing escc , bladder , and gastric 
 cancer cell line models significantly reduced cell proliferation .
initially , smyd2 was characterized as methylating 
 h3 lysine 36 and lysine 4 when interacting 
 with hsp90a .
methylation of histones by 
 smyd2 has been connected to increased transcription of genes involved 
 in cell - cycle regulation , chromatin remodeling , and transcriptional 
 regulation .
in addition , several studies 
 have uncovered an important role of smyd2 methylation activity toward 
 nonhistone proteins closely connected to cancer .
this is in line with 
 the emerging concept that posttranslational methylation of nonhistone 
 proteins ( e.g. , of transcription factors ) by protein methyltransferases 
 can also substantially alter protein function .
thereby , a regulatory 
 role of lysine methylation can probably be extended to multiple cellular 
 pathways besides transcriptional regulation and histones .
so far , the best - characterized example of smyd2 methylation of a 
 nonhistone protein is the tumor suppressor transcription factor p53 .
transcriptional activity of p53 is inhibited by smyd2-mediated posttranslational 
 methylation at lysine 370 ( k370 ) .
the structural 
 basis of p53 methylation by smyd2 has been characterized by solving 
 the crystal structure of a ternary complex with the cofactor product s - adenosylhomocysteine and a p53-derived substrate peptide .
it has been proposed that methylation at k370 
 reduces the dna - binding efficiency of p53 and subsequently prevents 
 the transcriptional activation of p53 target genes . in the same study ,
a knockdown of smyd2 and treatment with 
 doxorubicin led to an increase in p53-mediated cell - cycle arrest and 
 apoptosis . in line with these observations ,
low smyd2 gene expression 
 was suggested as a predictive marker for an improved response to neoadjuvant 
 chemotherapy in breast cancer patients . besides p53 ,
several other proteins have been identified as smyd2 
 substrates , including the estrogen receptor ( er ) , parp1 , retinoblastoma protein ( rb ) , and hsp90 .
mechanistically , methylation 
 of hsp90 has been connected to the normal physiological role of smyd2 
 in muscle biology , as well as in cancer .
these studies indicate that smyd2 has many substrates 
 and various potentially tissue - specific physiological and pathogenic 
 functions .
smyd2 therefore represents a very attractive target for 
 further exploration in different disease - relevant models .
nevertheless , 
 the biology of smyd2 is still poorly understood and the molecular 
 contribution of individual substrates to specific knockdown phenotypes 
 remains largely unknown . for a more unbiased interpretation of biological 
 experiments ,
although first 
 cellular - active probe inhibitors of smyd2 have been described ( figure 1 ) , there is a need 
 for structurally orthogonal chemical probes to enable cross - validation 
 studies and thereby rule out unspecific effects .
the publication of 1 ( az505 ) was the first disclosure of a co - crystal structure of an 
 inhibitor bound to smyd2 and paved the way for further studies , leading 
 to the discovery of 2 ( lly-507 ) , and 3 ( a-893 ) , which have 
 reported significantly improved potency .
analysis of the respective 
 cocrystal structures reveals that all three inhibitors bind in a similar 
 fashion , occupying the same binding pockets .
furthermore , the cellular 
 activity of the known inhibitors is limited , and no data about in 
 vivo applicability have been published . here
we report the discovery 
 of a potent and selective aminopyrazoline - based small - molecule inhibitor 
 ( s)-4 ( bay-598 ) .
we show that ( s)-4 has a distinctly 
 different binding mode compared to previous inhibitors , utilizing 
 a dichlorophenyl moiety as so far unprecedented chemical motif for 
 addressing the methyl - lysine binding pocket of smyd2 . for the first 
 time
, we are presenting in vivo xenograft and dmpk data for a smyd2 
 inhibitor .
in addition to previously described inhibitors , ( s)-4 shows very potent cellular activity combined 
 with reasonable dmpk properties ( figure 1 ) . furthermore , we are indicating the potential 
 that this inhibitor might offer to the field of protein methyltransferases 
 in the quest to fully explore the underlying complex biology and therapeutic 
 potential of smyd2 by validating ahnak protein as a new cellular substrate .
comparison of so far 
 described smyd2 inhibitors with ( s)-4 discovered in this study .
data from respective references as well 
 as assay results from this study are presented .
the potential link between smyd2 
 and cancer motivated us to screen the bayer compound collection , with 
 the aim of identifying small - molecule inhibitors of the enzyme . to 
 this end , a scintillation proximity assay ( spa ) was set up using recombinant 
 his - tagged smyd2 , a biotinylated p53-derived peptide substrate , and 
 tritiated s - adenosyl - l - methionine ( h - sam ) ( figure 2a ) .
of the three million compounds tested in a primary hts , we identified 
 more than 2300 confirmed hits which inhibited smyd2 with ic50 values below 15 m . among the multiple structural clusters 
 and singletons in the hit list ,
several offered starting points with 
 low micromolar potency and tractable chemical matter , and our attention 
 was drawn to pyrazolines such as compound 5 .
initial 
 hits and later derivatives of the pyrazoline series showed stabilizing 
 effects in an smyd2 thermal shift assay ( tsa ) ( figure 2b ) .
in addition , binding of compound 6 to smyd2 was validated by isothermal titration calorimetry 
 ( itc ) , which indicated a submicromolar binding constant ( kd = 540 nm , figure 2c ) and a high enthalpic contribution to the binding energy . 
 as the latter reduces the likelihood of nonspecific off - target activities
it transpired that compounds 
 of this structural series had been prepared during the course of an 
 in - house program as antagonists for protease - activated receptor 1 
 ( par1 ) , a g - protein - coupled receptor which is highly expressed in 
 platelets and plays an important role in thrombin signaling and platelet 
 aggregation .
identification and biophysical validation 
 of the pyrazoline smyd2 inhibitor series leading to ( s)-4 .
( a ) scintillation proximity assay ( spa ) screening 
 approach leading to the pyrazoline hit cluster , with representative 
 hit compound 5 with ic50 = 1.7 m .
( b ) 
 thermal shift assay ( tsa ) for smyd2 protein stabilization for inhibitors 
 of the aminopyrazoline series [  ] , with aminopyrazoline hit 5 ( tm = 3.3 k ) and ( s)-4 ( tm = 7.1 k ) highlighted .
( c ) isothermal titration calorimetry ( itc ) of compound 6 indicating a submicromolar binding constant ( kd = 540 nm ) and a high enthalpic contribution to the binding 
 energy .
synthesis of the required intermediates 10a  f started from the commercially available 
 2-amino-1-phenylethanones 7 ( scheme 1 ) .
protection of the amines as the allyl 
 carbamates 8 was followed by mannich reaction with formaldehyde 
 and piperidine , and the resulting product mixture was treated with 
 hydrazine monohydrate to install the pyrazoline moiety ( compounds 9 ) .
reagents and conditions : ( a ) allyl 
 chloroformate , k2co3 ( 1.5 m in water ) , dcm , 
 rt , overnight ; ( b ) formaldehyde ( 37 wt % in water ) , piperidine , etoh , 
 rt , overnight ; ( c ) hydrazine monohydrate , etoh , reflux ; ( d ) diphenyl n - cyanocarbonimidate , i - proh , reflux , 1 
 h , then rt , overnight . the synthesis of compounds 5 , 6 , and 1224 began with 10a ( scheme 2 ) .
treatment of 10a with the respective 
 aniline or amine provided intermediates 11a  i .
deprotection to the 4-aminopyrazolines and further elaboration 
 led to the desired amides 5 , 6 , and 1223 by a sequence of optional reductive 
 n - alkylation and amide formation .
for the hydroxyacetyl derivatives 5 , 6 , and 1221 , the amide was formed by using acetoxyacetyl chloride , followed 
 by treatment of the crude amide with methanolic potassium carbonate 
 to remove the acetyl moiety .
the amino acid amide 22 was 
 prepared by a coupling with 9-fluorenylmethoxycarbonyl ( fmoc ) protected 
 glycine ( hatu , nmm , dmf ) , followed by removal of the fmoc group with 
 piperidine .
furthermore , oxoimidazolidine 24 was prepared 
 by reaction of the n - unsubstituted 4-aminopyrazoline derivative with 
 chloroacetyl chloride and treatment of the resulting chloroacetamide 
 with urotropine .
reagents 
 and conditions : ( a ) aniline derivative , n - buli , thf , 
 78 c , or alkylamine , 1,4-dioxane , reflux ; ( b ) 1,3-dimethylbarbituric 
 acid , pd(pph3)4 , thf ; ( c ) acetaldehyde , nabh4 , dcm , 0 c ; ( d ) ( 1 ) acetoxyacetyl chloride , aqueous 
 nahco3 solution , dcm , ( 2 ) k2co3 , 
 meoh ; ( e ) skip ( for compound 19 ) , or aldehyde derivative , 
 nabh4 , meoh , 0 c ; ( f ) fmoc - glycine , hatu , nmm , dmf ; 
 ( g ) piperidine , dmf ; ( h ) methoxyacetyl chloride , aqueous nahco3 solution , dcm ; ( i ) chloroacetyl chloride , n , n - diisopropylethylamine , dcm ; ( j ) urotropine , etoh . compounds 4 and 2529 were prepared from intermediates 10b  f by the addition of 3-(difluoromethoxy)aniline , followed 
 by installation of the n - ethylated hydroxyacetamide as described above 
 ( scheme 3 ) . reagents and conditions : ( a ) 3-(difluoromethoxy)aniline , n - buli , thf , 78 c ; ( b ) 1,3-dimethylbarbituric 
 acid , pd(pph3)4 , thf ; ( c ) acetaldehyde , nabh4 , meoh , 0 c ; ( d ) ( 1 ) acetoxyacetyl chloride , aqueous 
 nahco3 solution , dcm , ( 2 ) k2co3 , 
 meoh .
the synthesis of compounds 3034 started from intermediate 10d by addition of the respective aniline derivatives , followed by introduction 
 of the n - ethylated hydroxyacetamide ( scheme 4 ) .
reagents and conditions : 
 ( a ) aniline derivative , n - buli , thf , 78 c ; 
 ( b ) 1,3-dimethylbarbituric acid , pd(pph3)4 , 
 thf ; ( c ) acetaldehyde , nabh4 , meoh , 0 c ; ( d ) ( 1 ) 
 acetoxyacetyl chloride , aqueous nahco3 solution , dcm , ( 2 ) 
 k2co3 , meoh .
compounds 4 , 28 , and 3034 were separated into their enantiomers by preparative chiral hplc 
 or chiral supercritical fluid chromatography . for compounds 6 and 4 ,
the compounds were stable to racemization in aqueous solution 
 at ph 7 , as well as in mouse and human plasma at 37 c , for at 
 least 48 h. however , clean racemization was obtained under basic conditions 
 and microwave irradiation ( dbu , thf , 90 c ) .
our initial structure  activity relationship 
 ( sar ) studies of 3-(4-chlorophenyl)pyrazoline derivatives with respect 
 to smyd2 inhibition are summarized in table 1 . for the carboximidamide n - substituent ( r ) , a meta - substituted phenyl seemed optimal , with the 3-(difluoromethoxy)phenyl 
 derivative 6 displaying highest potency and binding efficiency ( ic50 = 0.8 m , bei = 12.5 ) . 

small para substituents are tolerated , such as with the 3-chloro-4-fluorophenyl , 
 3,4-dichlorophenyl , and 2,2-difluoro-1,3-benzodioxol-5-yl derivatives 5 , 12 , and 13 ( ic50 = 
 1.7 , 3.3 , and 2.2 m , respectively ) .
in contrast , the 4-(difluoromethoxy)phenyl 
 derivative 14 is more than 15-fold less active than the 
 corresponding meta - derivative 6 .
the extended 3-chlorophenethyl 
 derivative 15 showed no measurable activity ( ic50 > 20 m ) , implying that the space within the relevant binding 
 pocket is limited .
nonaromatic derivatives , such as 4,4,4-trifluorobutyl - substituted 16 or the cyclopropylethyl derivative 18 , are 
 less potent than 6 ( ic50 = 6.9 and 8.3 m 
 for 16 and 18 , respectively ) . surprisingly , 
 the corresponding n - butyl derivative 17 is inactive ( ic50 > 20 m ) . the sar for par1 
 antagonism at the carboximidamide n - substituent appears to be rather 
 flat : all compounds 5 , 6 , and 1218 are potent par1 antagonists , with ic50 values below 100 nm .
with respect to the amide 
 moiety , it became clear that the presence and orientation of a hydrogen - bond 
 donor has a large impact on potency toward smyd2 ( table 2 ) .
variation of the amide n - alkyl substituents ( r ) revealed that the n - ethyl derivative is preferred : secondary amide 19 ( ic50 = 10.9 m ) is about 10-fold less active and 
 the n - propyl derivative 20 is also less 
 potent than the corresponding n - ethyl derivative 6 ( ic50 = 0.8 m ) , whereas the larger cyclopropylmethyl 
 substituent in 21 resulted in a loss of potency ( ic50 > 20 m ) .
replacement of the hydroxyacetyl in compound 12 ( ic50 = 3.3 m ) by an aminoacetyl group 
 gave the equipotent derivative 22 ( ic50 = 
 2.8 m ) .
in contrast , the methoxyacetyl derivative 23 , lacking the hydrogen - bond donor , is inactive ( ic50 > 
 20 m ) . in compound 24 , where the hydrogen - bond 
 donor is fixed in an oxoimidazolidine ring , there is significantly 
 reduced potency ( ic50 = 6 m ) relative to the n - ethyl derivative 6 .
on the basis of the available 
 amide derivatives , with bei values in the same range ( 1011 ) , 
 an improvement in the binding efficiency was not envisaged .
furthermore , 
 alterations of the amide moiety did not offer a path forward to selectivity 
 against par1 .
compounds 22 , 23 , and 24 are in a similar potency range for par1 antagonism and 
 greater than 10-fold more potent against par1 ( ic50 = 130 , 
 30 , and 100 nm , respectively ) than smyd2 .
binding efficiency 
 index : pic50/mw  1000 . at this stage , we selected 
 compound 6 for co - crystal structure determination with 
 smyd2 based on its potency and promising biophysical properties .
compound 6 was soaked into crystals of smyd2 grown in the presence 
 of sam .
the crystal structure revealed that compound 6 binds into the substrate peptide binding pocket of smyd2 ; the observed 
 binding mode is consistent with the previously established sar .
there 
 is a very good steric and electrostatic fit of 6 to the 
 substrate binding site of smyd2 ( figure 3a , b ) .
the 4-chlorophenyl substituent inserts 
 into the lysine binding channel and is engaged in -stacking 
 interactions with phe184 and tyr240 .
there is a good fit of the 3-(difluoromethoxy)phenyl 
 substituent into the adjacent hydrophobic pocket-1 , with the difluoromethoxy 
 group pointing into a hydrophobic subpocket .
although racemic 6 was used for soaking , the density maps reveal that only 
 the s - enantiomer is bound .
the s configuration at the pyrazoline provides an optimal exit vector 
 for the hydroxyacetamide substituent , which occupies pocket-2 and 
 forms two hydrogen bonds with thr185 ( figure 3a ) .
the n-nitrile 
 of the carboximidamide contributes to a water - bridged hydrogen bond 
 with ser196 . on the basis of the crystal structure of compound 6
, we envisioned that further exploration of the aniline and 
 amide moieties would not lead to a significant improvement of potency 
 and binding efficiency .
therefore , we elected to focus on derivatives 
 of the 4-chlorophenyl substituent , which were underrepresented in 
 our compound library .
( a , b ) two different views of the co - crystal structure 
 of 6 in complex with smyd2 .
there is a good steric and 
 electrostatic fit of 6 into the substrate binding site 
 of smyd2 , and multiple hydrogen - bond interactions are involved .
the 
 lysine channel is occupied by the 4-chlorophenyl residue of the ligand , 
 representing a novel lysine channel binding motif . in addition , pockets-1 
 and -2 are addressed .
( c ) visualization of the watermap calculations 
 suggesting an optimal displacement of water molecules by a 3,4-dichlorophenyl 
 residue .
calculated water sites are shown as spheres and colored based 
 on their free energy . only sites within the lysine channel
watermap results are based on the crystal structure of the monochloro 
 derivative 6 ( pdb code 5arf ) .
the modeled ligand structure is shown as colored sticks ( chlorine , 
 green ; carbon , yellow ; hydrogen , white ; nitrogen , blue ; oxygen , red ; 
 fluorine , light green ) .
( d ) overlay of the crystal structures of compound 6 ( magenta ) and ( s)-4 ( yellow , 
 pdb code 5arg ) , showing nearly identical binding modes . with the co - crystal structure in hand , we employed molecular 
 modeling for the prioritization of derivatives .
in particular , watermap 
 calculations , which estimate the position and 
 the thermodynamic properties of water molecules in the ligand - free 
 structure , suggested the introduction of a second substituent at the 
 3-position of the 4-chlorophenyl group . to test this hypothesis ,
the unsubstituted phenyl derivative 25 is inactive , while the 4-bromo derivative 26 ( ic50 = 1.1 m ) is as potent as the 4-chloro derivative 6 , indicating the importance of a hydrophobic substituent 
 at the 4-position . as predicted by watermap calculations , introduction 
 of a second substituent at the meta position , as exemplified by 3,4-dichloro 
 derivative 4 ( ic50 = 0.08 m , bei = 
 13.5 ) and 4-chloro-3-methyl derivative 28 ( ic50 = 0.08 m , bei = 14 ) , resulted in significantly improved potency 
 ( ca
as highlighted in figure 3c , the two chloro substituents are colocated with two calculated 
 water sites that have high free energy , suggesting an optimal water 
 displacement by 3,4-dichlorophenyl and thus a lower binding free energy 
 than for the 4-chlorophenyl or the unsubstituted phenyl derivative , 
 consistent with the observed potency difference for these derivatives
. 
 however , introduction of a third substituent leads to a decrease in 
 potency compared to the 3,4-disubstituted derivatives , as exemplified 
 by 4-chloro-3,5-dimethyl derivative 27 ( ic50 = 0.57 m ) .
compound 29 , with the bulkier 1,3-benzodioxole 
 moiety , is also less potent ( ic50 = 4.2 m ) , suggesting 
 that the sar at this position is rather steep .
the co - crystal was obtained by soaking 
 ( s)-4 into crystals of sam - bound smyd2 . 
 structure determination revealed that ( s)-4 features an almost identical binding mode as compound 6 , as evidenced in the overlay of both structures ( figure 3d ) . in line with the observed s configuration of compound 6 and ( s)-4
, we observed a greater than 50-fold difference 
 in the ic50 values of the two enantiomers of 4 [ 27 nm for ( s)-4 vs 1.7 m for 
 ( r)-4 , see table 4 ] .
introduction of the additional 3-chloro 
 substituent also resulted in a dramatic decrease in the antagonistic 
 effect on par1 [ ic50 = 1.7 m for ( s)-4 and > 30 m for ( r)-4 ] .
although ( s)-4 proved to 
 be the active isomer for both smyd2 inhibition and par1 antagonism , 
 there is a greater than 50-fold selectivity for smyd2 relative to 
 par1 .
the co - crystal structures of compound 6 and ( s)-4 revealed that the pyrazoline - based smyd2 
 inhibitors feature a different binding mode to other recently reported 
 inhibitors . thus , 1(12 ) and 2(28 ) ( figure 4a ) have published ic50 values 
 of 0.12 m and < 15 nm , respectively , and like ( s)-4 they bind to the substrate binding site of smyd2 . 

all ligands occupy 
 the lysine binding channel and the adjacent hydrophobic pocket-1 ; 
 however , ( s)-4 employs pocket-2 via 
 hydrogen - bond interactions with its hydroxyacetyl moiety ( cf .
figure 2a ) , which are not 
 present in 1 or 2 . 1 and 2
, on the other hand , occupy a remote hydrophobic pocket-3 
 which is not addressed by ( s)-4 .
figure 4c provides a view 
 into the lysine binding channel ( the respective motifs are highlighted 
 in color on the structures in figure 4a ) .
2 binds with its n - alkylpyrrolidine moiety at this position , which is structurally 
 closely related to the lysine side chain of a methylated substrate 
 peptide . 1 and ( s)-4 address 
 the lysine channel with aromatic substituents .
it is noteworthy that 
 the 3,4-dichlorophenyl group of 1 and of ( s)-4 bind to different positions .
comparison of ( s)-4 ( this work ) with the recently reported 
 smyd2 inhibitors 1(11 ) and 2 .
( b , c ) two different views of 
 an overlay of ( s)-4 ( yellow ) with 1 ( green ) and 2 ( blue ) .
( s)-4 has a distinct binding mode , addressing pocket-2 which is 
 not occupied by 1 or 2 , while only 1 and 2 occupy the distant hydrophobic pocket-3 . on the basis of
3,4-dichlorophenyl 
 as a novel lysine channel binding motif , we identified several pyrazoline 
 derivatives as potent smyd2 inhibitors ( see table 4 ) . aiming to identify a candidate for in 
 vivo experiments , we focused on the potent s - enantiomers 
 of these derivatives , profiling them in pharmacokinetic assays in 
 vitro .
( s)-4 showed moderate stability 
 upon incubation with rat hepatocytes ( clblood = 2.5 l / h / kg ) 
 as well as moderate apparent permeability ( 34 nm / s ) and a hint of 
 active transport in the caco2 assay ( efflux ratio = 5 ) . in comparison , 
 the 4-chloro-3-methylphenyl derivative ( s)-28 had similar permeability and efflux ( 33 nm / s , efflux ratio 
 = 7 ) and slightly lower metabolic stability in rat hepatocytes ( clblood = 2.8 l / h / kg ) .
compounds ( s)-30 and ( s)-3234 exhibited 
 high metabolic stability ( clblood = 1.80.3 l / h / kg ) ; 
 however , these derivatives have very low aqueous solubility ( < 5 

on the other hand , the methoxyphenyl derivative ( s)-31 has high aqueous solubility ( 163 mg / l ) and moderate 
 permeability with a hint of active transport ( 62 nm / s , efflux ratio 
 = 3 ) in the caco2 assay ; nevertheless , ( s)-31 displayed low stability upon incubation with rat hepatocytes 
 ( clblood = 3.2 l / h / kg ) , and thus bioavailability is expected 
 to be low .
on the basis of these data , ( s)-4 was selected for further in vitro and in vivo studies .
the 
 in vivo pharmacokinetic properties of ( s)-4 were first evaluated by a single - dose administration of ( s)-4 at 0.4 mg / kg by iv bolus or 0.8 mg / kg 
 po , respectively , to rats ; there was moderate blood clearance ( 1.6 
 l / h / kg ) and a low bioavailability of 24% ( see supporting information figure s1a ) .
these data prompted us to assess 
 the exposure of ( s)-4 following oral 
 administration .
hence , we treated mice with 10100 mg / kg po 
 qd which are well tolerated doses of ( s)-4 . as a result , unbound ic50 [ cellular methylation in - cell 
 western ( icw ) assay ; see figure 7a  c ] is covered for 9 to 12 
 h at steady state when 10 and 100 mg / kg , respectively , are administered 
 ( see supporting information figure s1b ) .
( s)-4 showed potent in vitro inhibition of smyd2 with 
 an ic50 of 27  7 nm in the biochemical spa assay 
 ( figure 5a ) . to analyze the mode of inhibition and affinity 
 of ( s)-4 , our smyd2 protein preparation 
 was characterized with respect to its apparent michaelis 
. 
 the km(app ) values of 60 nm and 1 m , 
 respectively , were in excellent agreement with the constants previously 
 reported using a similar assay .
then , 
 we performed ic50 determinations at increasing concentrations 
 of one substrate and a fixed amount of the other ( [ s ] = km(app ) ) and applied the cheng 
increasing the concentration of peptide substrate resulted in a 
 linear increase in ic50 ( figure 4b , upper panel ) , as would be expected for 
 a competitive mode of inhibition .
fitting these data to the corresponding 
 cheng  prusoff competitive inhibition model revealed a ki(app ) of 8  1 nm ( sd ) . on the other hand , 

when sam concentrations were titrated to saturation , we observed a 
 decrease in ic50 which converged to a constant value ( figure 4b , lower panel ) . 

consequently , the ic50 vs [ s]/km(app ) plot fits best to the cheng  prusoff model 
 for uncompetitive inhibition , yielding an inhibitor constant ki(app ) of 28  3 nm ( sd ) .
our 
 data suggest that ( s)-4 is a peptide - competitive , 
 sam - uncompetitive inhibitor of smyd2 methyltransferase activity , which 
 preferably binds to the smyd2sam substrate complex . interestingly , 
 smyd2 has been reported to follow a random sequential bi bi mechanism 
 of substrate binding , but the uncompetitive mode of 
 inhibition of ( s)-4 regarding the sam 
 cofactor suggests an ordered sequential bi bi mode of substrate binding , 
 where sam would be required to bind before the peptide substrate . 

similar results have recently been described for inhibitors of the 
 sam - dependent arginine methyltransferase prmt5 .
( a ) activity in the scintillation proximity assay ( spa ) . 
 ic50 ( n > 10 ) for smyd2 inhibition 
 = 26  7 nm ( representative inhibition curve shown ) . ( b )
ic50 values obtained from spa were plotted against the indicated 
 substrate concentrations ( represented as [ substrate]/km(app ) ) .
data were fitted to competitive and uncompetitive 
 models of the cheng  prusoff equation .
data points are the mean of eight replicates , error bars indicate 
 1  sd .
the data indicate that ( s)-4 is a peptide - competitive , sam - uncompetitive inhibitor of smyd2 methyltransferase 
 activity .
( c ) selectivity profile on a panel of 32 additional methyltransferases 
 showing high selectivity of ( s)-4 for 
 smyd2 .
for further evaluation of selectivity , 
 ( s)-4 was tested on a panel of 32 additional 
 methyltransferases , including closely related family members smyd3 , 
 suv420h1 , and suv420h2 . as a result , ( s)-4 displayed > 100-fold selectivity for smyd2 , with very weak residual 
 activity toward the closest related methyltransferase smyd3 ( ic50  3 m ) ( figure 5c ) .
in addition to the methyltransferases , ( s)-4 was also profiled in the commercially 
 available kinomescan ( discoverx ) and 
 leadprofilingscreen ( eurofins panlabs ) assay panels to fully determine 
 relative selectivity and specificity for kinases and other primary 
 molecular targets , including several cns targets .
overall , we were 
 able to confirm the high selectivity and specificity of ( s)-4 for smyd2 inhibition ( see supporting information tables s1 and s2 ) .
the ability of ( s)-4 to inhibit smyd2 was tested by monitoring its effects on cellular 
 p53 methylation using different cellular mechanistic assays .
first , 
 we generated a polyclonal antibody ( sy46 ) for the specific detection 
 of p53 monomethylation at lysine 370 , as described elsewhere .
this antibody was then tested on recombinant 
 p53 protein which had been in vitro methylated by smyd2 in a western 
 blot .
specificity for methylated p53 was confirmed by the exclusive 
 detection of the in vitro methylated p53 protein isoforms , whereas 
 the nonmethylated p53 was not detected ( figure 6a ) .
endogenous methylation 
 of p53 protein was characterized by treatment of kyse-150 esophageal 
 cancer cells with increasing concentrations of ( s)-4 for 5 days .
the kyse-150 cell line model was selected 
 based on a described smyd2 gene amplification and a heterozygous r248q mutation in p53 ( cosmic ) , leading to p53 
 protein accumulation without a stress stimulus .
after treatment with 
 ( s)-4 , a significant reduction of methylation 
 was detected confirming that p53 is a cellular target of smyd2-dependent 
 methylation ( figure 6b ) .
nevertheless overall endogenous detection of p53 protein methylation 
 led to weak signals , hence this method was not useful for the determination 
 of a cellular ic50 .
therefore , we additionally employed 
 an established system with a transient flag - tagged smyd2 and flag - tagged 
 p53 overexpression in hek293 t cells as benchmark assay .
this assay 
 has been used previously to characterize the structurally unrelated 
 smyd2 inhibitor 2 . as shown 
 in figure 6c , ( s)-4 showed a concentration - dependent decrease 
 in p53 methylation without affecting p53 total protein levels . a cellular 
 ic50 of 58 nm was determined ( figure 6d ) , which confirms that ( s)-4 is the most potent cellular - active smyd2 inhibitor 
 known to date ( figure 1 ) .
( a ) a specific antibody 
 directed against p53k370me1 ( sy46 ) was generated and tested for specificity 
 on recombinant p53 ( rec .
( b ) endogenous methylation of p53 protein 
 was characterized by treatment of kyse-150 esophageal cancer cells 
 with increasing concentrations of ( s)-4 for 5 days ( co = control ) .
( c ) cellular p53 methylation assay using 
 transient flag - tagged smyd2 and flag - tagged p53 overexpression in 
 hek293 t cells .
increasing concentrations of ( s)-4 reduce the methylation of overexpressed p53 ( for this assay 
 p53k730me1-specific antibody kindly provided by dr .
( d ) ic50 determination in the cellular p53 methylation 
 assay . to further characterize 
 the effects of our aminopyrazoline - based inhibitors on the cellular 
 methylation activity of smyd2
, we generated a polyclonal cell line 
 derived from kyse-150 with stable n - terminal 2xc - myc - tagged smyd2 
 overexpression to maximize cellular methylation activity .
in an immunofluorescence 
 analysis , smyd2 was localized mainly in the cytosol ( supporting information figure s2a ) , as reported by others .
surprisingly , the antibody sy46 directed against 
 methylated p53 showed a very strong signal specifically in the clones 
 with smyd2 overexpression in the immunochemistry staining not derived 
 from p53 protein ( supporting information figure s2b , c ) .
we validated the novel smyd2 substrate giving rise 
 to the strong immunochemistry staining signal by knockdown and overexpression 
 experiments in additional cell lines to be derived from ahnak protein ( figure 7b and supporting information figure 2d  f ) .
ahnak methylation has been 
 very recently reported in a proteomics study by other as an additional 
 methylation substrate of smyd2 , further confirming our results .
importantly , we used the strong ahnak methylation 
 signal to set up an in cell western ( icw ) assay for cellular optimization 
 of our lead series ( figure 7a ) .
the methylation signal was reduced by treatment with increasing 
 concentrations of ( s)-4 , derivatives 
 of the aminopyrazoline series or 1 ( figure 7a ) .
in addition , ( s)-4 specifically reduced methylation of ahnak without 
 altering ahnak protein expression in a western blot ( figure 7b ) .
importantly , ic50 values in the cellular icw assay for aminopyrazolines correlated 
 with potency in the scintillation proximity assay ( figure 7c ) and were also comparable 
 to the p53 methylation assay ( see figure 6d ) .
our results of the methylation of ahnak 
 and the recent identification of many additional methylation targets clearly point toward additional roles beyond 
 p53 regulation of smyd2 , and further studies supported by potent and 
 selective inhibitors as ( s)-4 are needed 
 to fully elucidate the underlying biology .
ahnak is a novel substrate 
 of smyd2 and methylation can be inhibited by aminopyrazoline inhibitors . 

each row represents a different inhibitor tested 
 in increasing concentrations ranging from 39 nm up to 5 m . 
 ( s)-4 and 1 are highlighted . 

( b ) inhibition of ahnak methylation in mda - mb231 cells overexpressing 
 smyd2 .
( c ) good correlation of cellular activity ( icw ) to biochemical 
 activity ( spa ) of ( s)-4 and pyrazoline 
 cluster derivatives .
( s)-4 has a cellular 
 ic50  60 nm . to explore the potential effects of ( s)-4 on proliferation , we tested a panel of 240 different 
 cancer cell lines ( oncopanel 240/eurofins panlabs )
. cell lines were 
 long - term cultured with ( s)-4 for 10 
 days to allow for a sufficient translation of demethylation of smyd2 
 substrates to potential antiproliferative effects , which takes 4872 
 h ( supporting information figure s2c ) . 

( s)-4 exposure resulted in only limited 
 responses in a subset of cell lines ; an antiproliferative response 
 with ic50 < 10 m was seen in 21 cell lines ( 9% ) , 
 however , most cell lines ( 83% ) did not reach 50% proliferation inhibition 
 with 20 m of ( s)-4 ( figure 8a ) .
in addition , there was no clear preference for a specific 
 tissue origin of the responding cancer cell line .
thus , smyd2 inhibition 
 by ( s)-4 has only limited proliferation 
 effects in a small subset of cancer cell lines under the employed 
 conditions . on the basis of the observation that p53 protein is methylated 
 by smyd2 , which should lead to suppression of apoptosis , we were additionally 
 interested in the effects of ( s)-4 in 
 combination with an apoptotic stimulus .
kyse-150 , u2os , and a2780 
 cell lines were pretreated with ( s)-4 or inactive derivative 25 ( see table 3 ) for 2 days ( demethylation phase ) , followed 
 by treatment with doxorubicin ( apoptotic trigger ) .
( s)-4 , but not 25 , significantly improved 
 caspase 3/7 activation in all three tested cell lines without inducing 
 apoptosis alone ( figure 7b ) .
( a ) 
 a panel of 240 different cancer cell lines ( oncopanel 240/eurofins 
 panlabs ) was long - term cultured with ( s)-4 for 10 days and proliferation effects were determined .
( s)-4 showed proliferation effects with an ic50 < 10 m in a subset of cell lines .
( b ) effects 
 on apoptosis induction were determined by caspase 3/7 activation . 

kyse-150 , u2os , and a2780 cell lines were pretreated with ( s)-4 or inactive derivative 25 ( see table 3 ) for 
 2 days , then apoptosis was induced by treatment with doxorubicin . 

functional validation of novel potential 
 cancer targets such as the protein methyltransferase smyd2 relies 
 on appropriate model systems in vitro as well as in vivo .
additionally , 
 chemical probe inhibitors also suitable for in vivo applications are 
 highly desirable .
most reported activities of smyd2 are not directly 
 involved in survival signaling of cancer cells .
hence , in vitro proliferation 
 assays may not adequately cover the full phenotype of smyd2 inhibition , 
 and more complex ( in vivo ) assays are thus required .
the research 
 work characterizing smyd2 in heart and skeletal muscle cells by knockdown 
 experiments in vivo clearly illustrates the complex underlying biology of smyd2 and 
 the necessity for chemical probes suitable for in vivo applications . 

therefore , we were interested in establishing if smyd2 methylation 
 activity in tumor cells can be inhibited by ( s)-4 in vivo . to this end , mice bearing subcutaneous tumor xenografts 
 ( tumor tissue derived from the smyd2-overexpressing kyse-150 cell 
 line ) were treated orally with 10 , 30 , 70 , or 100 mg / kg ( s)-4 , or vehicle ( peg 400/water 8:2 ) , once daily for 
 3 days .
after the treatment period , tumors were harvested and analyzed 
 ex vivo for methylation of ahnak by dot - blotting . for detection of 
 the methylation signals ,
sy46 methylation antibody was used ( see figure 7a and supporting 
 information figure s2a  f ) .
( s)-4 significantly reduced the methylation 
 with doses starting from 30 mg / kg , with most significant effects in 
 the 100 mg / kg treated group ( p < 0.001 , student s t test ) ( figure 9a ) .
treatment with 10 mg / kg ( s)-4 resulted in no significant effect on the methylation 
 level .
exposure at 10 mg / kg is close to the level of the cellular 
 ic50 for 9 h , which may indicate a need for an 
 even higher exposure as the ic50 to achieve in vivo effects 
 on demethylation . in vivo characterization of ( s)-4 .
mice ( n = 12 per group ) bearing subcutaneous tumor 
 xenografts ( tumor tissue derived from the smyd2-overexpressing kyse-150 
 cell line ) were treated as indicated , then the tumors were analyzed 
 for methylation signals by dot - blotting .
( s)-4 significantly reduced the methylation with doses starting 
 from 30 mg / kg , with most significant effects in the 100 mg / kg group 
 ( p < 0.001 , student s t test ) .
( b ) tumor area graph summarizing the in vivo tumor efficacy 
 study with the kyse-150 xenograft model .
average tumor area  
 standard error of mean per group is plotted as the mean .
treatment 
 was started at day 4 after tumor inoculation ( black arrow ) , and groups 
 were treated as indicated .
( c ) tumor weight graphs corresponding to 
 the same experiment shown in ( b ) .
significant ( p value < 0.05 ) difference 
 between vehicle control and treatment group ( dunn s method ) . 

group 2 : one animal was excluded on the 14th treatment day due to 
 animal welfare reasons ( ulcerated tumor ) , tumor size 82 mm .
then the kyse-150 esophageal xenograft model was used to 
 evaluate if the observed improved apoptosis induction in the in vitro 
 setting after treatment with doxorubicin ( figure 8b ) could translate to antitumor efficacy 
 in vivo .
four groups of tumor - bearing mice were treated as follows : 
 group 1 ( control group ) was only treated with vehicle ( solutol / ethanol / water 
 1:1:8 ) iv qd and once at day 4 with the vehicle used for doxorubicin 
 ( saline ) iv ; group 2 was treated with ( s)-4 at 500 mg / kg po qd ; group 3 was treated with 10 mg / kg doxorubicin 
 iv once at day 4 ; group 4 was treated with a combination of ( s)-4 and doxorubicin .
there was a slight reduction 
 in area ( figure 9b ) 
 and weight ( figure 9c ) of tumors from mice treated with the combination of ( s)-4 and doxorubicin relative to tumors from the control 
 group .
combination treatment only resulted in a minor increase in 
 treatment - related body weight loss ( figure 9d ) .
the combination treatment reached a t / c 
 level ( based on tumor weight ) of 0.46 , which is significant ( figure 9c ) .
therefore , combination 
 of a smyd2 inhibitor with a chemotherapeutic agent resulted in reduced 
 cancer cell growth in vivo . in comparison ,
the monotherapy groups 
 treated with only doxorubicin or ( s)-4 showed no significant antitumor efficacy relative to the vehicle 
 control group .
these data are consistent with the observed limited 
 cellular proliferation effects of ( s)-4 in the cell line panel ( figure 8a ) and indicate that , in contrast to smyd2 knockdown , a catalytic inhibition may be insufficient to 
 induce cell death in the kyse-150 esophageal model .
furthermore , the 
 monotherapeutic approach with doxorubicin did not result in any antitumor 
 efficacy in the kyse-150 xenograft model .
in 
 summary , we have identified ( s)-4 as 
 a potent , selective , and cell - active , substrate - competitive inhibitor 
 of smyd2 .
our data show that smyd2 inhibition can enhance efficacy 
 of doxorubicin in vivo , which confirms our in vitro observation of 
 higher caspase 3/7 activation ( figure 8b ) .
our results ( s)-4 are also in agreement with an earlier study , where an increased apoptosis induction in cells with an 
 smyd2 knockdown was observed . nonetheless , in our initial explorative 
 in vivo study with ( s)-4 , effects on 
 xenografted tumors were only moderate .
in addition , high doses of 
 ( s)-4 were needed in vitro as well as 
 in vivo relative to the concentration needed to achieve effects on 
 methylation .
therefore , we can not exclude the possibility that additional , 
 so - far unexplored activities of smyd2 might be responsible for the 
 observed effects .
this again underlines the necessity to identify 
 suitable chemical probes for more extensive target validation campaigns 
 to fully explore the complex biology of smyd2 and other targets . in 
 this regard ,
( s)-4 will be a highly 
 valuable tool for the further exploration of smyd2 biology , not only 
 for in vitro but also for in vivo studies .
all 
 final products were at least 95% pure , as determined by analytical 
 hplc .
intermediate 10a and compounds 5 , 6 , and 1224 were synthesized according to the methods described previously.h nmr were determined to be > 95% .
h nmr spectra were recorded on bruker avance iii hd spectrometers 
 operating at 300 , 400 , or 500 mhz .
the chemical shifts (  ) reported 
 are given in parts per million ( ppm ) , and the coupling constants ( j ) are in hertz ( hz ) .
the spin multiplicities are reported 
 as s = singlet , br s = broad singlet , d = doublet , t = triplet , q 
 = quartet , m = multiplet , and br = broad .
the lc / ms analysis was performed 
 on waters acquity uplcms singlequad with a acquity uplc beh c18 column 
 ( 1.7 m , 50 mm  2.1 mm ) at 60 c , using water + 0.1 
 vol % formic acid ( 99% ) and acetonitrile as mobile phase at a flow 
 rate of 0.8 ml / min and a dad detector ( 210400 nm ) .
lc / ms / ms 
 was performed on a ctc pal autosampler , an agilent 1200 hplc , and 
 a absciex 4000 mass spectrometer .
for all separated enentiomers , it was assumed based on the co - crystal 
 structure of 4 , that the active enantiomer ( smyd2 inhibition ) 
 has s - configuration .
compound 10b was prepared 
 as described for 10d . starting from 7b ( 37.7 
 g , 0.18 mol )
, 10b was obtained as white solid ( 40.1 g , 
 49% over 4 steps ) .
h nmr ( 400 mhz , cdcl3 ) : 
  4.264.42 ( m , 3h ) , 4.66 ( dd , j = 13.3 , 
 6.0 hz , 1h ) , 4.74 ( dd , j = 13.1 , 5.8 hz , 1h ) , 5.30 
 ( d , j = 10.3 hz , 1h ) , 5.40 ( d , j = 16.8 hz , 1h ) , 5.67 ( td , j = 8.8 , 4.5 hz , 1h ) , 
 6.00 ( ddt , j = 16.8 , 11.0 , 5.6 hz , 1h ) , 7.027.11 
 ( m , 2h ) , 7.197.34 ( m , 5h ) , 7.58 ( d , j = 8.5 
 hz , 2h ) .
lc - ms ( esi ) : m / z = 468.1/470.1 
 [ m + h ] .
compound 10c was prepared 
 as described for 10d , starting from 7c ( 35 
 g , 0.15 mol ) to obtain 10c ( 29.3 g , 43% over 4 steps ) 
 as white solid .
h nmr ( 400 mhz , [ d]6dmso ) : 
  2.32 ( s , 6h ) , 4.034.18 ( m , 1h ) , 4.394.61 ( m , 
 2h ) , 5.065.27 ( m , 2h ) , 5.495.66 ( m , 1h ) , 5.795.94 
 ( m , 1h ) , 5.755.95 ( m , 1h ) , 7.26 ( d , j = 7.8 
 hz , 2h ) , 7.307.37 ( m , 1h ) , 7.417.55 ( m , 2h ) , 7.567.73 
 ( m , 2h ) , 8.17 ( d , j = 8.7 hz , 1h ) .
lc - ms ( esi ) : m / z = 452.2 [ m + h ] .
to 
 a stirred solution of 2-amino-1-(3,4-dichlorophenyl)ethanone hydrochloride 
 ( 1:1 ) ( 7d ; 116 g , 0.480 mol ) in water ( 500 ml ) was added 
 allyl chloroformate ( 56.5 ml , 0.530 mol ) in dcm ( 800 ml ) .
the reaction 
 mixture was cooled to 0 c , and k2co3 ( 207 
 g , 1.49 mol ) in water ( 1 l ) was added dropwise over 1 h. the mixture 
 was allowed to warm to rt , stirred overnight , then diluted with dcm 
 ( 500 ml ) . the organic phase was washed with saturated aqueous nh4cl solution ( 400 ml ) followed by brine ( 500 ml ) , then dried 
 over mgso4 , filtered , and concentrated in vacuo .
the crude 
 mixture was purified by dry flash column chromatography ( eluent : dcm / heptane 
 2:1 , 3:1 , 4:1 ; dcm ; etoac ) to yield allyl [ 2-(3,4-dichlorophenyl)-2-oxoethyl]carbamate 
 ( 8d ; 120 g , 87% ) as a white crystalline solid .
h nmr ( 400 mhz , cdcl3 ) :  4.62 ( d , j = 5.5 hz , 2h ) , 4.68 ( d , j = 4.6 hz , 2h ) , 5.25 ( dd , j = 1.4 , 10.5 hz , 1h ) , 5.35 ( dd , j = 1.4 , 
 16.9 hz , 1h ) , 5.72 ( br s , 1h ) , 5.94 ( m , 1h ) , 7.60 ( d , j = 8.2 hz , 1h ) , 7.79 ( dd , j = 2.1 , 8.5 hz , 1h ) , 
 8.06 ( d , j = 1.8 hz , 1h ) .
lc - ms ( esi ) : m / z = 288.1 [ m + h ] . to a stirred suspension 
 of 8d ( 50.0 g , 0.174 mol ) in etoh ( 390 ml )
was added 
 formaldehyde solution ( 20 ml , 0.261 mol ; 37 wt % in water ) followed 
 by the dropwise addition of piperidine ( 26 ml , 0.261 mol ) in etoh 
 ( 130 ml ) over 30 min .
the volatiles were removed 
 by evaporation to yield an orange oil which was not further purified . 
 to a solution of this crude material in etoh ( 480 ml ) was added hydrazine 
 monohydrate ( 29.6 ml , 0.609 mol ) , and the reaction mixture was heated 
 to reflux for 2.5 h. then the mixture was allowed to cool to rt , concentrated , 
 and poured over ice - cooled saturated nh4cl solution ( 300 
 ml ) .
the crude product was extracted with etoac ( 1.5 l ) , and the combined 
 organic layers were washed with brine ( 300 ml ) then dried over mgso4 , filtered , and concentrated to yield allyl [ 3-(3,4-dichlorophenyl)-4,5-dihydro-1h - pyrazol-4-yl]carbamate ( 9d ; 50.0 g , 91% ) 
 as a pale - yellow solid .
h nmr ( 400 mhz , [ d]6dmso ) :  3.24 ( m , 1h ) , 3.59 ( m , 1h ) , 4.394.54 ( m , 2h ) , 
 5.085.25 ( m , 3h ) , 5.795.90 ( m , 1h ) , 7.52 ( dd , j = 1.8 , 8.3 hz , 1h ) , 7.57 ( br s , 1h ) , 7.59 ( m , 1h ) , 7.68 
 ( d , j = 1.8 hz , 1h ) , 7.84 ( d , j = 
 8.7 hz , 1h ) .
lc - ms ( esi ) : m / z = 
 314.1 [ m + h ] . to a stirred suspension of 9d ( 50.0 g , 0.159 mol ) in i - proh ( 860 ml )
was added 
 diphenyl n - cyanocarbonimidate ( 38.0 g , 0.159 mol ) . 

the reaction mixture was heated to reflux , at which point the suspension 
 dissolved into solution ; after a further 10 min at reflux , a white 
 precipitate formed .
the mixture was stirred at reflux for a further 
 1 h then allowed to slowly cool to rt overnight .
the precipitate was 
 collected by filtration and washed with et2o ( 2  
 250 ml ) .
the resulting white solid was allowed to dry to yield 10d ( 48.6 g , 67% ) as a white solid .
h nmr ( 400 
 mhz , [ d]6dmso ) :  4.13 ( apparent d , j = 8.2 hz , 1h ) , 4.47 ( m , 3h ) , 5.14 ( m , 2h ) , 5.515.63 ( m , 
 1h ) , 5.795.90 ( m , 1h ) , 7.23 ( d , j = 7.3 hz , 
 2h ) , 7.30 ( t , j = 7.8 hz , 1h ) , 7.45 ( m , 2h ) , 7.79 
 ( br m , 2h ) , 7.97 ( br s , 1h ) , 8.19 ( d , j = 8.7 hz , 
 1h ) .
lc - ms ( esi ) : m / z = 458.0 [ m 
 + h ] .
compound 10e was prepared 
 as described for 10d , starting from 7e ( 3.1 
 g , 13.9 mmol ) to obtain 10e ( 2.12 g , 34.4% over 4 steps ) 
 as white solid .
h nmr ( 400 mhz , cdcl3 ) :  
 2.18 ( s , 3h ) , 4.30 ( d , j = 6.4 hz , 2h ) , 4.64 ( d , j = 6.0 hz , 2h ) , 5.24 ( d , j = 10.5 hz , 
 1h ) , 5.34 ( d , j = 16.9 hz , 1h ) , 5.605.70 
 ( m , 1h ) , 5.94 ( ddt , j = 5.6 , 10.9 , 16.8 hz , 1h ) , 
 6.897.35 ( m , 7h ) , 7.48 ( m , 1h ) , 7.54 ( m , 1h ) .
lc - ms ( esi ) : m / z = 438.2 [ m + h ] .
compound 10f was prepared as described 
 for 10d , starting from 7f ( 12.0 g , 55.7 
 mmol ) to obtain 10f ( 9.1 g , 38% over 4 steps ) as white 
 solid .
h nmr ( 400 mhz , [ d]6dmso ) :  4.054.08 
 ( m , 1h ) , 4.434.47 ( m , 3h ) , 5.15.2 ( m , 2h ) , 5.505.55 
 ( m , 1h ) , 5.805.90 ( m , 1h ) , 6.08 ( s , 2h ) , 7.05 ( d , j = 7.8 hz , 1h ) , 7.217.27 ( m , 2h ) , 7.277.41 
 ( m , 3h ) , 7.437.50 ( m , 2h ) , 8.16 ( d , j = 8.7 
 hz , 1h ) .
lc - ms ( esi ) : m / z = 434.2 
 [ m + h ] . to a stirred solution of the respective aniline 
 derivative ( 3 equiv ) in anhydrous thf ( 2 ml / mmol ) at 78
c 
 was added n - buli ( 3 equiv , 2 m in hexane ) dropwise 
 while maintaining the reaction temperature below 65 c . 

the reaction mixture was stirred at 78 c for 1 h , then 
 the respective phenyl 4-{[(allyloxy)carbonyl]amino}-n - cyano-3-phenyl-4,5-dihydro-1h - pyrazole-1-carboximidate 10 ( 1 equiv ) in anhydrous thf ( 30 ml / mmol ) was added dropwise 
 while maintaining the reaction temperature below 65 c . 

the reaction mixture was stirred at 78 c for 2 h , then 
 slowly poured over saturated nh4cl solution ( 30 ml / mmol 
 based on 10 ) .
the crude product was extracted into etoac 
 ( 30 ml / mmol ) ; the combined organic layers were washed with brine ( 15 
 ml / mmol ) , dried over mgso4 , filtered , and concentrated . 

the crude solid was precipitated from a minimum volume of etoac , collected 
 by filtration , and washed with et2o to give the corresponding 
 intermediate 11 .
compound 11j was prepared from 10b ( 5.0 g , 10.7 mmol ) 
 and 3-(difluoromethoxy)aniline .
11j was not pure after 
 chromatography and was used as such in further steps ( 3.8 g , purity 
 77% ; uv , 51% ) .
lc - ms ( esi ) : m / z = 
 533.1 [ m + h ] .
compound 11k was prepared from 10c ( 10.0 g , 22.1 mmol ) 
 and 3-(difluoromethoxy)aniline .
h nmr ( 400 mhz , cdcl3 ) : 
  2.32 ( s , 6h ) , 4.34.6 ( m , 4h ) , 5.13 ( dd , j = 1.1 , 10.3 hz , 1h ) , 5.19 ( dd , j = 1.4 , 17.4 hz , 
 1h ) , 5.55.6 ( m , 1h ) , 5.75.9 ( m , 1h ) , 6.53 ( t , j = 73.8 hz , 1h ) , 6.87 ( br d , j = 8.7 hz , 
 1h ) , 7.17.3 ( m , 4h ) , 7.50 ( s , 2h ) , 8.35 ( s , 1h ) .
lc - ms ( esi ) : m / z = 517.2 [ m + h ] .
compound 11l was prepared from 10d ( 20.0 g , 43.6 mmol ) 
 and 3-(difluoromethoxy)aniline .
h nmr ( 400 mhz , [ d]6dmso ) : 
  4.08 ( dd , j = 4.6 , 11.9 hz , 1h ) , 4.364.53 
 ( m , 3h ) , 5.11 ( dd , j = 1.4 , 10.5 hz , 1h ) , 5.17 ( dd , j = 1.4 , 17.4 hz , 1h ) , 5.505.59 ( m , 1h ) , 5.775.90 
 ( m , 1h ) , 6.99 ( dd , j = 2.3 , 8.2 hz , 1h ) , 7.16 ( m , 
 1h ) , 7.21 ( t , j = 73.7 hz , 1h ) , 7.22 ( m , 1h ) , 7.39 
 ( t , j = 8.3 hz , 1h ) , 7.737.81 ( m , 2h ) , 8.15 
 ( d , j = 1.4 hz , 1h ) , 8.17 ( d , j = 
 8.7 hz , 1h ) , 9.79 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 523.2 [ m + h ] . compound 11 m was prepared from 10e ( 10.0 g , 22.8 mmol ) 
 and 3-(difluoromethoxy)aniline .
h nmr ( 400 mhz , [ d]6dmso ) : 
  2.332.40 ( m , 3h ) , 4.034.15 ( m , 1h ) , 4.414.56 
 ( m , 3h ) , 5.135.36 ( m , 2h ) , 5.535.63 ( m , 1h ) , 5.825.94 
 ( m , 1h ) , 7.007.08 ( m , 1h ) , 7.197.29 ( m , 2h ) , 7.25 
 ( t , j = 74 hz , 1h , partially masked by other peaks ) , 
 7.407.49 ( m , 1h ) , 7.55 ( d , j = 8.4 hz , 1h ) , 
 7.73 ( dd , j = 1.7 , 8.2 hz , 1h ) , 7.847.90 
 ( m , 1h ) , 8.2 ( br d , j = 8.6 hz , 1h ) , 9.75 ( br s , 
 1h ) .
lc - ms ( esi ) : m / z = 503.2 [ m 
 + h ] .
compound 11n was prepared from 10f ( 2.0 g , 4.6 mmol ) and 
 3-(difluoromethoxy)aniline .
( dd , j = 4.6 , 11.9 hz , 1h ) , 4.43 ( t , j = 11.2 hz , 1h ) , 4.50 ( d , j = 4.1 hz , 
 2h ) , 5.16 ( dd , j = 1.4 , 10.5 hz , 1h ) , 5.22 ( dd , j = 1.6 , 17.2 hz , 1h ) , 5.495.60 ( m , 1h ) , 5.825.93 
 ( m , 1h ) , 6.11 ( s , 2h ) , 6.957.09 ( m , 2h ) , 7.167.32 
 ( m , 4h ) , 7.39 ( m , 1h ) , 7.61 ( d , j = 1.4 hz , 1h ) , 
 8.21 ( d , j = 8.7 hz , 1h ) , 9.63 ( s , 1h ) .
lc - ms ( esi ) : m / z = 499.2 [ m + h ] .
compound 11o was prepared from 10d ( 10 g , 21.8 mmol ) and 
 3-(trifluoromethoxy)aniline .
h nmr ( 400 mhz , [ d]6dmso ) : 
  4.13 ( dd , j = 4.8 , 11.9 hz , 1h ) , 4.424.56 
 ( m , 3h ) , 5.125.25 ( m , 2h ) , 5.505.63 ( m , 1h ) , 5.835.94 
 ( m , 1h ) , 7.19 ( br d , j = 8.1 hz , 1h ) , 7.357.44 
 ( m , 2h ) , 7.467.56 ( m , 1h ) , 7.777.85 ( m , 2h ) , 8.138.24 
 ( m , 2h ) , 9.89 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 540.8 [ m + h ] .
compound 11p was prepared from 10d ( 2.0 g , 4.4 mmol ) and 
 3-methoxyaniline .
h nmr ( 400 mhz , [ d]6dmso ) :  
 3.3 ( s , 3h , masked by water signal ) , 3.974.07 ( m , 1h ) , 4.334.48 
 ( m , 1h ) , 5.40 ( s , 1h ) , 5.745.82 ( m , 1h ) , 6.95 ( br d , j = 7.1 hz , 1h ) , 7.147.26 ( m , 3h ) , 7.347.43 
 ( m , 1h ) , 7.73 ( d , j = 8.5 hz , 1h ) , 7.81 ( dd , j = 1.9 , 8.5 hz , 1h ) , 8.08 ( s , 1h ) , 8.76 ( br d , j = 8.8 hz , 1h ) , 9.51 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 487.1 [ m + h ] .
compound 11q was prepared from 10d ( 5.0 g , 10.9 mmol ) 
 and 6-(difluoromethoxy)pyridin-2-amine .
11q was obtained 
 as off - white solid ( 2.9 g , 43% ) .
h nmr ( 400 mhz , [ d]6dmso ) :  3.95 ( dd , j = 5.0 , 12.4 hz , 
 1h ) , 4.32 ( t , j = 11.7 hz , 1h ) , 4.44.5 ( m , 
 2h ) , 5.13 ( m , 2h ) , 5.51 ( m , 1h ) , 5.84 ( m , 1h ) , 6.82 ( d , j = 8.2 hz , 1h ) , 7.13 ( d , j = 7.8 hz , 1h ) , 7.58 ( t , j = 72.8 hz , 1h ) , 7.77.8 ( m , 2h ) , 7.91 ( t , j = 7.8 hz , 1h ) , 8.05 ( d , 1h ) , 8.18 ( d , j = 1.8 hz , 1h ) , 10.45 ( s , 1h ) .
lc - ms ( esi ) : m / z = 524.1 [ m + h ] .
compound 11r was prepared from 10d ( 2.0 g , 4.4 mmol ) and 
 4-chloro-3-(difluoromethoxy)aniline .
h nmr ( 400 mhz , [ d]6dmso ) :  4.014.17 ( m , 1h ) , 4.424.56 ( m , 3h ) , 
 5.125.25 ( m , 2h ) , 5.535.64 ( m , 1h ) , 5.825.94 
 ( m , 1h ) , 7.28 ( t , j = 73 hz , 1h , partially masked 
 by other peaks ) , 7.34 ( dd , j = 2.4 , 8.7 hz , 1h ) , 
 7.42 ( m , 1h ) , 7.61 ( d , j = 8.6 hz , 1h ) , 7.81 ( s , 
 2h ) , 8.138.25 ( m , 2h ) , 9.91 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 557.1 [ m + h ] .
compound 11s was prepared from 10d ( 1.6 g , 3.4 mmol ) and 
 3-(difluoromethoxy)-5-fluoroaniline .
h nmr ( 400 mhz , [ d]6dmso ) :  4.13 ( dd , j = 4.6 , 11.9 hz , 1h ) , 
 4.414.53 ( m , 3h ) , 5.105.25 ( m , 2h ) , 5.515.63 
 ( m , 1h ) , 5.785.94 ( m , 1h ) , 6.96 ( dd , j = 
 2.2 , 9.7 hz , 1h ) , 7.10 ( d , j = 5.1 hz , 1h ) , 7.18 
 ( br d , j = 10.9 hz , 1h ) , 7.29 ( t , j = 73 hz , 1h , partially masked by other peaks ) , 7.747.85 
 ( m , 2h ) , 8.108.30 ( m , 2h ) , 9.88 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 541.2 [ m + h ] . to a stirred solution of 11j ( 3.8 g
, 77%-uv , 5.5 mmol ) in degassed thf ( 150 ml ) was added 1,3-dimethylbarbituric 
 acid ( 1.8 g , 11.0 mmol ) , followed by tetrakis(triphenylphosphine)palladium(0 ) 
 [ pd(pph3)4 ; 0.25 g , 0.22 mmol ] .
the reaction 
 mixture was stirred under argon for 12 h , then cautiously quenched 
 with saturated nahco3 solution ( 150 ml ) and extracted into 
 etoac ( 150 ml ) .
the organic layer was washed with brine ( 100 ml ) , 
 then dried over na2so4 , filtered , and concentrated . 

the crude material was purified by dry flash column chromatography 
 ( eluent : etoac , meoh ) to yield 4-amino-3-(4-bromophenyl)-n-cyano - n-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1h - pyrazole-1-carboximidamide ( 2.4 g , 95% ; uv , 92% ) .
h nmr ( 400 mhz , [ d]6dmso ) :  2.38 ( br s , 
 2h ) , 4.02 ( dd , j = 4.4 , 11.5 hz , 1h ) , 4.37 ( dd , j = 9.9 , 11.4 hz , 1h ) , 4.81 ( dd , j = 4.5 , 
 9.9 hz , 1h ) , 6.98 ( dd , j = 2.0 , 8.1 hz , 1h ) , 7.22 
 ( t , j = 74.0 hz , 1h ) , 7.207.23 ( m , 1h ) , 7.27 
 ( ddd , j = 0.8 , 2.0 , 8.1 hz , 1h ) , 7.40 ( t , j = 8.1 hz , 1h ) , 7.647.70 ( m , 2h ) , 7.988.03 
 ( m , 2h ) , 9.62 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 449.3 [ m + h ] . to the amine product ( 2.30 
 g , 5.1 mmol ) in meoh ( 70 ml ) at rt was added acetaldehyde ( 0.27 g , 
 6.1 mmol ) .
the reaction mixture was stirred at rt for 1 h , and at 
 40 c for 1 h , then it was cooled to 0 c and nabh4 ( 0.22 g , 5.7 mmol ) was added in small portions .
the mixture was 
 stirred at rt for 1 h , then poured over saturated nahco3 solution ( 10 ml ) .
the volatiles were removed by evaporation , and 
 the resulting aqueous slurry was extracted with etoac ( 2  10 
 ml ) .
the combined organic layers were washed with brine ( 100 ml ) , 
 then dried over na2so4 , filtered , and concentrated 
 to yield a crude black oil .
the crude material was purified by dry 
 flash column chromatography ( eluent : dcm , meoh ) to yield 3-(4-bromophenyl)-n-cyano - n-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1h - pyrazole-1-carboximidamide ( 1.11 g , 45% ) as the main n - ethylation 
 product .
lc - ms ( esi ) : m / z = 477.1 
 [ m + h ] .
the corresponding debrominated derivative n-cyano - n-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-3-phenyl-4,5-dihydro-1h - pyrazole-1-carboximidamide ( 0.41 g , 19% ) was obtained 
 as a byproduct .
lc - ms ( esi ) : m / z = 398.3 [ m + h ] . to the debrominated byproduct ( 410 
 mg , 1 mmol ) in dcm ( 15 ml ) were added et3n ( 0.15 ml , 1.1 
 mmol ) , acetoxyacetyl chloride ( 0.12 ml , 1.1 mmol ) , and dmap ( 126 mg , 
 1 mmol ) , and the mixture was stirred at rt for 1 h. etoac ( 15 ml ) 
 was added , and the mixture was washed with saturated aqueous nh4cl solution ( 20 ml ) and brine ( 20 ml ) .
the crude 
 product was purified by dry flash column chromatography ( eluent : dcm , 
 meoh ) to yield 504 mg of an impure intermediate , which was dissolved 
 in meoh ( 12 ml ) , k2co3 ( 140 mg , 1.01 mmol ) was 
 added , and the mixture was heated to reflux for 30 min . after cooling , 
 to the reaction mixture was added saturated aqueous nh4cl , and the mixture was extracted with etoac .
the organic phase was 
 dried over na2so4 , concentrated , and the residue 
 purified by flash column chromatography ( eluent : dcm , meoh ) and subsequent 
 preparative hplc to yield n-(1-{n-cyano - n-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-phenyl-4,5-dihydro-1h - pyrazol-4-yl)-n - ethyl-2-hydroxyacetamide 
 ( 25 ; 50 mg , 10% ) .
h nmr ( 300 mhz , [ d]6dmso ) :  1.03 ( br s , 3h ) , 3.163.33 ( m , 2h ) , 
 3.924.25 ( m , 3h ) , 4.47 ( br t , j = 11.7 hz , 
 1h ) , 4.75 ( br t , j = 5.5 hz , 1h ) , 6.947.03 
 ( m , 1h ) , 7.197.30 ( m , 3h ) , 7.367.52 ( m , 4h ) , 7.757.84 
 ( m , 2h ) , 9.77 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 457.2 [ m + h ] . to the main product 
 from the n - ethylation step ( 1.1 g , 2.3 mmol ) in dcm ( 30 ml )
were added 
 et3n ( 0.34 ml , 2.4 mmol ) , acetoxyacetyl chloride ( 0.27 
 ml , 2.4 mmol ) , and dmap ( 282 mg , 2.3 mmol ) and the mixture was stirred 
 at rt for 1 h. etoac ( 30 ml ) was added , and the mixture was washed 
 with saturated aqueous nh4cl solution ( 40 ml ) and brine 
 ( 40 ml ) .
the crude product was purified by dry flash column 
 chromatography ( eluent : dcm , meoh ) to yield 1.2 g of an impure intermediate . 

then 200 mg of this intermediate were dissolved in meoh ( 5 ml ) , k2co3 ( 48 mg , 0.35 mmol ) was added , and the mixture 
 was heated to reflux for 30 min . after cooling , the reaction mixture 
 was concentrated and the residue was purified by hplc to yield ( n-[3-(4-bromophenyl)-1-{n-cyano - n-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1h - pyrazol-4-yl]-n - ethyl-2-hydroxyacetamide 
 ( 26 ; 50 mg , 24% ) .
h nmr ( 400 mhz , [ d]6dmso ) :  1.05 ( br s , 3h ) , 3.273.36 ( m , 2h ) , 
 3.984.18 ( m , 3h ) , 4.48 ( t , j = 11.7 hz , 1h ) , 
 4.72 ( t , j = 4.9 hz , 1h ) , 7.00 ( d , j = 7.8 hz , 1h ) , 7.21 ( s , 1h ) , 7.23 ( t , j = 74.0 
 hz , 1h ) , 7.26 ( d , j = 8.3 hz , 1h ) , 7.41 ( t , j = 8.2 hz , 1h ) , 7.67 ( d , j = 8.5 hz , 2h ) , 
 7.73 ( d , j = 8.3 hz , 2h ) , 9.78 ( br s , 1h ) .
lc - ms 
 ( esi ) : m / z = 535.1 [ m + h ] .
compound 27 was prepared as 
 described for 4 , starting from 11k ( 4.9 
 g , 9.6 mmol ) .
27 was obtained as gray solid ( 1.6 g , 32% 
 over 3 steps ) .
h nmr ( 300 mhz , [ d]6dmso ) :  
 1.05 ( br s , 3h ) , 2.32 ( s , 6h ) , 3.233.44 ( m , 2h ) , 3.944.15 
 ( m , 3h ) , 4.44 ( br t , j = 11.7 hz , 1h ) , 4.69 ( br s , 
 1h ) , 6.927.02 ( m , 1h ) , 7.147.27 ( m , 2h ) , 7.337.43 
 ( m , 1h ) , 7.56 ( s , 2h ) , 9.73 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 518 [ m + h ] . to a stirred solution of 11l ( 14.2 g , 27.0 mmol ) in degassed thf ( 370 ml )
was added 1,3-dimethylbarbituric 
 acid ( 17.0 g , 108 mmol ) , followed by pd(pph3)4 ( 2.40 g , 2.16 mmol ) .
the reaction mixture was stirred under argon 
 for 15 min , then cautiously quenched with saturated nahco3 solution ( 400 ml ) and extracted into etoac ( 400 ml ) .
the organic 
 layer was washed with brine ( 200 ml ) , then dried over mgso4 , filtered , and concentrated .
the crude material was purified by 
 dry flash column chromatography ( eluent : etoac / heptane 1:1 , 2:1 ; etoac ; 
 meoh / etoac 1:100 ) to yield 4-amino - n-cyano-3-(3,4-dichlorophenyl)-n-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1h - pyrazole-1-carboximidamide ( 9.3 g , 78% ) as an orange oil .
h nmr ( 400 mhz , [ d]6dmso ) :  3.954.02 ( m , 
 1h , under etoac signal ) , 4.35 ( m , 1h ) , 4.83 ( dd , j = 4.6 , 9.6 hz , 1h ) , 7.01 ( dd , j = 2.1 , 8.0 hz , 
 1h ) , 7.22 ( t , j = 2.1 hz , 1h ) , 7.24 ( t , j = 73.7 hz , 1h ) , 7.27 ( m , 1h ) , 7.38 ( t , j = 7.8 
 hz , 1h ) , 7.487.61 ( m , 1h ) , 7.72 ( d , j = 8.2 
 hz , 1h ) , 8.00 ( dd , j = 2.1 , 8.5 hz , 1h ) , 8.31 ( d , j = 1.8 hz , 1h ) , 9.67 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 439.1 [ m + h ] . to a stirred solution 
 of the amine product ( 9.30 g , 21.2 mmol ) in meoh ( 170 ml ) at 0 c 

was added acetaldehyde ( 1.12 g , 25.4 mmol ) , followed by the portionwise 
 addition of nabh4 ( 0.96 g , 25.4 mmol ) over 20 min .
the 
 mixture was stirred for 30 min , then poured over saturated nahco3 solution ( 100 ml ) .
the volatiles were removed by evaporation , 
 and the resulting aqueous slurry was extracted with etoac ( 2  
 100 ml ) .
the combined organic layers were washed with brine ( 100 ml ) , 
 then dried over mgso4 , filtered , and concentrated to yield 
 a crude black oil .
the crude material was purified by dry flash column 
 chromatography ( eluent : etoac / heptane 1:1 , etoac ) to give a black 
 oil , which was triturated with et2o to yield n-cyano-3-(3,4-dichlorophenyl)-n-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1h - pyrazole-1-carboximidamide ( 7.40 g , 75% ) as a gray solid .
h nmr ( 400 mhz , [ d]6dmso ) :  0.98 ( t , j = 6.6 hz , 3h ) , 2.402.64 ( m , 2h , partially under 
 dmso signal ) , 4.164.27 ( m , 2h ) , 4.83 ( m , 1h ) , 6.98 ( dd , j = 2.3 , 8.2 hz , 1h ) , 7.21 ( t , j = 2.1 
 hz , 1h ) , 7.25 ( t , j = 73.7 hz , 1h ) , 7.27 ( dt , j = 1.0 , 8.1 hz , 1h ) , 7.42 ( t , j = 8.2 
 hz , 1h ) , 7.76 ( d , j = 8.7 hz , 1h ) , 8.01 ( dd , j = 2.1 , 8.5 hz , 1h ) , 8.33 ( d , j = 1.8 
 hz , 1h ) , 9.71 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 467.2 [ m + h ] . to this product
( 7.24 g , 
 15.5 mmol ) in dcm ( 62 ml ) was added saturated aqueous nahco3 solution ( 72 ml ) .
the biphasic mixture was stirred vigorously and 
 cooled to 5 c ; acetoxyacetyl chloride ( 2.50 ml , 23.2 mmol ) in 
 dcm ( 10 ml ) was added dropwise over 15 min .
the mixture was stirred 
 at 5 c for 10 min , after which time lc analysis indicated total 
 consumption of the starting material and only one major peak .
the 
 volatiles were removed by evaporation to yield an oily aqueous suspension 
 to which k2co3 ( 4.28 g , 31.0 mmol ) was added , 
 followed by meoh ( 110 ml ) .
the mixture was brought to reflux for 5 
 min then allowed to cool to rt , whereupon the product precipitated 
 as a white solid .
the solid was collected by filtration , washed with 
 water ( 50 ml ) and et2o ( 50 ml ) , and dried in vacuo to yield 4 ( 7.58 g , 93% ) as a white powder .
h nmr ( 400 
 mhz , [ d]6dmso ) :  0.981.10 ( m , 3h ) , 3.173.42 
 ( m , 2h , partially under water signal ) , 3.944.17 ( m , 3h ) , 4.46 
 ( t , j = 11.7 hz , 1h ) , 4.75 ( t , j = 5.7 hz , 1h ) , 6.98 ( dd , j = 7.8 , 1.8 hz , 1h ) , 
 7.18 ( br t , 1h ) , 7.22 ( m , 1h ) , 7.23 ( t , j = 8.2 hz , 
 1h ) , 7.40 ( t , 1h ) , 7.62 ( dd , j = 8.2 , 1.8 hz , 1h ) , 
 7.7 ( d , j = 8.2 hz , 1h ) , 8.11 ( d , j = 1.4 hz , 1h ) , 9.85 ( br s , 1h ) . lc - ms ( esi ) : m / z = 525.0 [ m + h ] .
compound 4 was separated into its enantiomers by chiral sfc ( instrument , sepiatec 
 prep sfc 100 ; column , chiralpak i d 5 m , 250 mm  20 mm ; 
 eluent , co2/i - proh 7:3 ; flow , 80 ml / min ; 
 temperature , 40 c ) : tr ( min ) = 3.354.40 
 [ ( s)-4 ] , 7.319.00 [ ( r)-4 ] .
enantiomer ( s)-4 : sfc ( chiralpak i d 5 m , 100 mm  4.6 mm , co2/i - proh 7:3 ; flow , 4 ml / min ; temperature , 
 37.5 c ) tr ( min ) = 2.41 .
enantiomer ( r)-4 : sfc ( chiralpak i d 5 m , 100 mm 
  4.6 mm , co2/i - proh 7:3 ; flow , 4 
 ml / min ; temperature , 37.5 c ) tr ( min ) 
 = 5.66 .
compound 28 was prepared as 
 described for 4 , starting from 11 m ( 3.4 
 g , 6.6 mmol ) .
28 was obtained as yellow solid ( 537 mg , 
 16% over 3 steps ) h nmr ( 500 mhz , [ d]2dcm ) : 
  1.11 ( br t , j = 6.8 hz , 3h ) , 2.43 ( s , 3h ) , 
 3.113.34 ( m , 3h ) , 4.114.27 ( m , 2h ) , 4.434.54 
 ( m , 2h ) , 6.65 ( t , j = 74.1 hz , 1h ) , 7.007.08 
 ( m , 1h ) , 7.32 ( d , j = 7.3 hz , 2h ) , 7.407.46 
 ( m , 2h ) , 7.49 ( dd , j = 10.1 , 8.2 hz , 1h ) , 7.72 ( d , j = 1.3 hz , 1h ) , 8.27 ( s , 1h ) .
lc - ms ( esi ) : m / z = 505.0 [ m + h ] .
compound 28 was separated into its enantiomers by chiral hplc ( instrument , 
 agilent prep 1200 ; column , chiralpak i d 5 m , 250  30 
 mm ; eluent , hexane / etoh 7:3 ; flow , 50 ml / min ) : tr ( min ) = 7.29.0 [ ( r)-28 ] , 10.912.9 [ ( s)-28 ] .
enantiomer 
 ( s)-28 : hplc ( chiralpak i d 3 m , 
 100 mm  4.6 mm , hexane / etoh 7:3 , 1.0 ml / min ) tr ( min ) = 6.22 .
compound 29 was prepared as 
 described for 4 , starting from 11n ( 870 
 mg , 2.0 mmol ) .
29 was obtained as off - white solid ( 432 
 mg , 43% over 3 steps ) h nmr ( 400 mhz , [ d]6dmso ) : 
  0.99 ( br s , 3h ) , 3.083.31 ( m , 2h ) , 3.944.18 
 ( m , 3h ) , 4.41 ( br t , j = 11.2 hz , 1h ) , 4.75 ( br s , 
 1h ) , 6.07 ( br d , j = 2.3 hz , 2h ) , 6.96 ( br d , j = 8.2 hz , 2h ) , 7.117.27 ( m , 4h ) , 7.357.43 
 ( m , 1h ) , 7.487.63 ( m , 2h ) , 9.68 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 501.2 [ m + h ] .
compound 30 was prepared as 
 described for compound 4 , starting from 11o ( 385 mg , 0.71 mmol ) .
30 was obtained as white solid 
 ( 244 mg , 66% over 3 steps ) .
h nmr ( 400 mhz , [ d]6dmso ) :  1.03 ( t , j = 6.6 hz , 3h ) , 3.33.5 
 ( m , 2h ) , 3.94.1 ( m , 3h ) , 4.44.5 ( m , 1h ) , 4.74.8 
 ( m , 1h ) , 7.15 ( d , 1h ) , 7.37.8 ( m , 6h ) , 8.09 ( s , 1h ) , 9.88 
 ( s , 1h ) .
lc - ms ( esi ) : m / z = 542.9 
 [ m + h ] .
compound 30 was separated 
 into its enantiomers by chiral sfc ( instrument , sepiatec prep sfc100 ; 
 column , chiralpak ie 5 m 250  20 mm ; eluent , co2/2-propanol 67/33 ; flow , 80 ml / min ; temperature , 40 c ) : tr ( min ) = 4.85.9 [ ( r)-30 ] , 8.610.3 [ ( s)-30 ] .
enantiomer ( s)-30 : sfc ( chiralpak 
 ie 5 m 100 mm  4.6 mm , co2/2-propanol 67/33 ; 
 flow , 4.0 ml / min ; temperature , 37.5 c ) tr ( min ) = 5.24 .
compound 31 was prepared as 
 described for 4 , starting from 11p ( 1.5 
 g , 3.1 mmol ) .
31 was obtained as yellow solid ( 325 mg , 
 21% over 3 steps ) .
h nmr ( 400 mhz , [ d]6dmso ) : 
  1.04 ( br s , 3h ) , 3.143.27 ( m , 1h ) , 3.74 ( s , 3h ) , 3.994.14 
 ( m , 3h ) , 4.38 ( br t , j = 11.9 hz , 1h ) , 4.72 ( br t , j = 5.6 hz , 1h ) , 5.85 ( br s , 1h ) , 6.84 ( m , 3h ) , 7.20 ( br 
 t , j = 8.5 hz , 1h ) , 7.517.64 ( m , 2h ) , 7.697.73 
 ( m , 1h ) , 8.07 ( br s , 1h ) , 9.75 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 489.2 [ m + h ] .
compound 31 was separated into its enantiomers by chiral hplc ( instrument , 
 agilent prep 1200 ; column , chiralpak ic 5 m , 250 mm  
 20 mm ; eluent , meoh / etoh / et2nh 50:50:0.1 ; flow , 20 ml / min ; 
 temperature , 23 c ) : tr ( min ) = 5.06.0 
 [ ( s)-31 ] , 6.758.0 [ ( r)-31 ] . enantiomer ( s)-31 : hplc ( chiralpak ic 5 m , 150 mm  4.6 mm , meoh / etoh / et2nh 50:50:0.1 , 1.0 ml / min ) tr ( min ) 
 = 2.65 .
compound 32 was prepared as 
 described for 4 , starting from 11q ( 1.9 
 g , 3.6 mmol ) .
32 was obtained as pale - yellow solid ( 558 
 mg , 29% over 3 steps ) .
h nmr ( 400 mhz , [ d]6dmso ) :  1.08 ( br t , j = 7.1 hz , 3h ) , 3.323.50 
 ( m , 2h ) , 3.934.17 ( m ,
3h ) , 4.41 ( br t , j = 
 11.7 hz , 1h ) , 4.76 ( br s , 1h ) , 6.84 ( d , j = 8.2 hz , 
 1h ) , 7.16 ( br d , j = 8.2 hz , 1h ) , 7.26 ( t , j = 65.9 hz , 1h ) , 7.63 ( s , 1h ) , 7.75 ( br d , j = 8.2 hz , 1h ) , 7.94 ( t , j = 8.0 hz , 1h ) , 8.04 ( s , 
 1h ) , 10.52 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 526.1 [ m + h ] .
compound 32 was 
 separated into its enantiomers by chiral sfc ( instrument , sepiatec 
 prep sfc 100 ; column , chiralpak i d 5 m , 250 mm  20 mm ; 
 eluent , co2/i - proh 71:29 ; flow , 80 ml / min ; 
 temperature , 40 c ) : tr ( min ) = 3.55.0 
 [ ( r)-32 ] , 5.07.0 [ ( s)-32 ] .
enantiomer ( s)-32 : sfc ( chiralpak i d 5 m , 100 mm  4.6 mm , co2/i - proh 71:29 , 4.0 ml / min ) tr ( min ) = 3.82 .
compound 33 was prepared as 
 described for 4 , starting from 11r ( 1.5 
 g , 2.7 mmol ) .
33 was obtained as yellow solid ( 18 mg , 
 12% over 3 steps ) .
h nmr ( 400 mhz , [ d]6dmso ) : 
  0.81 ( br s , 1h ) , 0.99 ( br s , 3h ) , 2.92 ( br s , 0.4h ) , 3.013.29 
 ( m , 3h ) , 3.944.33 ( m , 6h ) , 4.40 ( br s , 0.4h ) , 4.72 ( t , j = 5.3 hz , 1h ) , 5.13 ( br s , 0.3h ) , 5.73 ( br s , 0.4h ) , 5.94 
 ( br s , 1h ) , 6.937.42 ( m , 6h ) , 7.527.72 ( m , 3h ) , 7.867.96 
 ( m , 1h ) .
lc - ms ( esi ) : m / z = 559.2 
 [ m + h ] .
compound 33 was separated 
 into its enantiomers by chiral sfc ( instrument , sepiatec prep sfc 
 100 ; column , chiralpak ic 5 m , 250 mm  20 mm ; eluent , 
 co2/etoh 7:3 ; flow , 80 ml / min ; temperature , 40 c ) : tr ( min ) = 4.755.6 [ ( s)-33 ] , 6.257.5 [ ( r)-33 ] .
enantiomer ( s)-33 : sfc ( chiralpak 
 ic 5 m , 100 mm  4.6 mm , co2/etoh 7:3 , 4.0 
 ml / min ) tr ( min ) = 2.99 .
compound 34 was prepared as 
 described for 4 , starting from 11s ( 415 
 mg , 0.8 mmol ) .
34 was obtained as white solid ( 65 mg , 
 15% over 3 steps ) .
h nmr ( 400 mhz , [ d]6dmso ) : 
  1.08 ( br t , 3h ) , 3.39 ( m , 1h ) , 4.004.20 ( m , 3h ) , 4.50 
 ( br t , j = 11.5 hz , 1h ) , 4.77 ( br t , j = 5.8 hz , 1h ) , 5.596.16 ( m , 1h ) , 6.95 ( br d , j = 8.1 hz , 1h ) , 7.10 ( br s , 1h ) , 7.19 ( br d , j = 
 10.4 hz , 1h ) , 7.31 ( t , j = 73.5 hz , 1h ) , 7.66 ( br 
 d , j = 8.4 hz , 1h ) , 7.77 ( d , j = 
 8.4 hz , 1h ) , 8.11 ( s , 1h ) , 9.94 ( br s , 1h ) .
lc - ms ( esi ) : m / z = 543.2 [ m + h ] .
compound 34 was separated into its enantiomers by chiral hplc ( instrument , 
 agilent prep 1200 ; column , chiralpak i d 5 m , 250 mm  
 30 mm ; eluent , hexane / etoh / et2nh 70:30:0.1 ; flow , 40 ml / min ; 
 temperature , 23 c ) : tr ( min ) = 6.57.7 
 [ ( s)-34 ] , 8.19.7 [ ( r)-34 ] .
enantiomer ( s)-34 : hplc ( chiralpak i d , 3 m , 100 mm  4.6 mm ( hexane / etoh / et2nh 70:30:0.1 , 1.0 ml / min ) tr ( min ) 
 = 3.81 .
cell 
 lines were obtained from the american type culture collection ( hek293 , 
 u2os ) , the german collection of microorganisms and cell cultures ( kyse-150 ) , 
 and the european collection of cell cultures ( a2780 ) .
they were maintained 
 in the recommended cell culture media at 37 c in 5% co2 .
smyd2 enzyme 
 kinetics and inhibitory activities of compounds were analyzed using 
 a scintillation proximity assay ( spa ) which measured methylation by 
 the enzyme of the synthetic , biotinylated peptide btn - ahx - gsrahsshlkskkgqstsrh - amide 
 ( biosyntan ) derived from the c - terminal domain of p53 .
the smyd2 full - length 
 enzyme with an n - terminal 6xhis tag was expressed in escherichia coli and purified by affinity chromatography 
 on a ni - nta sepharose column , followed by size - exclusion chromatography 
 on a superdex 200 16/60 column ( ge healthcare ) .
assays were conducted 
 in 384-well microtiter plates in a buffer containing 50 mm tris / hcl 
 ph 9.0 , 1 mm dtt , 0.01% ( w / v ) bsa , and 0.0022% ( v / v ) pluronic , and 
 a final volume of 5 l .
the smyd2 concentration in the assay 
 was 3 nm , while tritiated s - adenosyl - l - methionine 
 ( h - sam ) and the peptide substrate were present at 60 nm 
 and 1 m , respectively , to ensure  balanced  conditions .
apparent michaelis  menten constants for 
 sam and the p53 peptide were determined by titrating one substrate 
 to saturation at cosubstrate concentrations of 1  km(app ) .
enzyme kinetics were followed over 2 h by quenching 
 the reactions as described above at time points 0 , 5 , 10 , 15 , 30 , 
 60 , 90 , and 120 min .
compounds were tested in 11-point , 3.5-fold dilution 
 series ranging from 0.1 nm to 20 m .
reactions were run for 
 30 min and quenched by adding streptavidin ps spa imaging beads ( perkinelmer ) 
 to a concentration of 3.12 g/l and 25 m  cold  

the amount of product was evaluated using a viewlux ( perkinelmer ) 
 ccd plate imaging device [ emission filter 613/55 ] .
the data were normalized 
 using two sets of control wells for high (= enzyme reaction with dmso 
 instead of test compound = 0% = minimum inhibition ) and low (= all 
 assay components without enzyme = 100% = maximum inhibition ) smyd2 
 activity .
ic50 values were calculated by fitting the normalized 
 inhibition data to a four - parameter logistic equation using either 
 a bayer proprietary tool or genedata screener analysis software . for mechanism - of - inhibition studies ,
ic50 determinations 
 were basically performed as described above , but incubation times 
 were adjusted to guarantee kinetic linearity at each concentration 
 of substrate analyzed . for peptide competition studies ,
ic50 values were determined at six different concentrations of p53 peptide 
 ( 0.5 , 1 , 2 , 4 , 6 , and 12 m ) . for sam competition , 25 , 50 , 100 , 
 200 , 300 , and 600 nm h - sam were used . in both cases ,
prusoff equations : where [ s ] = substrate concentration , km(app ) = apparent michaelis  menten constant , ki(app ) = apparent inhibitor constant for binding to free enzyme , 
 and ki(app ) = apparent inhibitor 
 constant for binding to enzyme  substrate complex . the effect of ( s)-4 on 
 the methyltransferase activity of g9a , ehmt1 , suv39h1 , suv39h2 , setdb1 , 
 setd7 , setd8 , suv420h1 , suv420h2 , prmt1 , prmt3 , prmt6 , prmt8 , prdm9 , 
 setd2 , and smyd3 , and mll1 , mll3 , ezh2 , and prmt5 complexes , as well 
 as dnmt1 and bcdin3d , was assessed using the spa method . for dnmt3a/3l , dnmt3b/3l , ash1l , dot1l , nsd1 , nsd2 , nsd3 ,
mettl21a , and mettl21d , 
 a trichloroacetic acid ( tca ) precipitation method in filter plates 
 were employed .
the 
 identification of par1 antagonistic effects and the quantification 
 of those effects , as well as the resulting ic50 values , 
 were performed as described previously .
thermal shift assays 
 ( tsa ) were carried out with the thermofluor system ( johnson & 
 johnson pharmaceutical research and development ) .
melting curves were 
 obtained at a protein concentration of 1.5 m , 8xsypro orange 
 ( invitrogen ) , and a ligand concentration of 100 m , using buffer 
 containing 20 mm tris ph 8.0 , 100 mm nacl , 8.5% .
scans were measured 
 from 25 to 95 c at a scanning rate of 2 c / min .
isothermal titration calorimetry ( itc ) measurements were carried 
 out using a microcal itc200 system . in the experiments ,
compound ( 400 
 m ) was titrated into smyd2 protein ( 67 m ) dissolved 
 in 25 mm tris ph 8.5 , 50 mm nacl , 0.0022% pluronic f-127 , and 2% dmso 
 at 25 c . after an initial injection of 0.2 l ,
which was 
 not considered for data fitting , 10 injections of 4 l each 
 in 150 s intervals were performed .
recombinant human smyd2 ( uniprot 
 q9nrg4 , amino acids 2433 ) was expressed in insect cells ( sf9 ) 
 containing an n - terminal tev - cleavable 6xhis tag .
cell pellets were 
 resuspended in lysis buffer ( 40 mm tris ph 8 , 500 mm nacl , 0.1% igepal , 
 5 mm imidazole , 1 mm dtt ) supplemented with complete edta - free protease 
 inhibitor tablets and 50 u / ml benzonase .
the cell lysate was loaded 
 onto a ni - nta column , eluted with imidazole , and concentrated using 
 an ultracentrifugal filter unit .
then , smyd2 was gel - filtrated on 
 a superdex s200 column equilibrated in 20 mm tris ph 8 , 100 mm nacl , 
 5% glycerol , and 1 mm dtt .
the 6xhis tag was cleaved with tev protease 
 in solution at 6 c overnight .
uncleaved smyd2 and tev protease 
 were separated from the cleaved product by a second ni - nta affinity 
 step .
the cleaved smyd2 protein was further purified by a second gel - filtration 
 step using a superdex 200 column equilibrated in 20 mm tris ph 8 , 
 150 mm nacl , 5% glycerol , and 1 mm tris(2-carboxyethyl)phosphine ( tcep ) . 

the protein was concentrated to 15.5 mg / ml ( 313 m ) ( uv  vis ) 
 using an ultracentrifugal filter unit and shock - frozen in liquid nitrogen . 
 for crystallization
, the cofactor sam was added to a final concentration 
 of 3.8 mm as follows : sam stock solution ( 100 mm in dmso , 1.2 l ) 
 was added to concentrated smyd2 solution ( 30 l ) and incubated 
 at 4 c for 2 h. crystals grew at 20 c within 3 d using 
 the hanging drop method .
drops were made from the smyd2/sam solution 
 ( 1 l ) and reservoir solution [ 2024% ( w / v ) peg 3350 , 
 100 mm hepes ph 7.0 ; 0.8 l ] .
thirty minutes after drop setup , 
 a seed solution ( 0.2 l ) was added .
the seed solution was made 
 from smyd2:sam crystals ( obtained with the same reservoir conditions 
 in a previous experiment ) , which were crashed manually ( using seed 
 beads , hampton research ) , diluted in reservoir solution , shock - frozen , 
 and stored at 80 c . for complex formation , a crystal 
 was transferred into a new drop reservoir solution ( 1.5 l ) . 

a stock solution of the respective inhibitor ( 100 mm in dmso ) was 
 10-fold diluted with reservoir solution .
over the course of 2 h , this 
 diluted stock solution ( 1.5 l ) was added in three steps of 
 0.5 l to the drop containing the smyd2:sam crystal , resulting 
 in a final concentration of 5 mm inhibitor in the soaking drop .
the 
 crystal was soaked in this drop at 20 c for 4 days in the case 
 of ( s)-4 and for 1 h for compound 6 .
the soaked crystal was briefly 
 immersed in cryo buffer ( 0.1 m hepes ph 7.0 , 22% peg 3350 , 20% glycerol , 
 2 mm inhibitor ) and shock - frozen in liquid nitrogen .
diffraction data 
 sets were collected using beamline 14.1 at the helmholtz - zentrum berlin 
 at 100 k using a wavelength of 0.91841  and a pilatus detector . 

the crystal form described here was first solved 
 for an smyd2:sam crystal in the absence of an inhibitor , using the 
 molecular replacement method ( program phaser from the ccp4 program suite and pdb 
 entry 3tg5 as 
 search model ) .
the data sets described here were then solved by rigid 
 body refinement using the smyd2:sam structure as starting model and 
 the program refmac from the ccp4 program 
 suite .
3d models for compound 6 and ( s)-4 were generated using the program discovery studio , 
 and parameter files for crystallographic refinement and model building 
 were generated using the software prodrg .
( s)-4 was manually built into the 
 electron - density maps using coot , followed 
 by several cycles of refinement with refmac and rebuilding in coot . 

watermap calculations were based on the crystal 
 structure of the 4-chlorophenyl derivative 6 ( pdb code 5arf ) .
the smyd2compound 6 complex structure was prepared using the protein preparation 
 wizard functionality in maestro .
preparation involved assignment 
 of bond orders , addition of hydrogens , creation of zero - order bonds 
 to metals , deletion of water molecules beyond 5  from heteroatoms , 
 assignment of protonation states according to ph 7.0 , and optimization 
 of the hydrogen - bonding network and restrained minimization . here , 
 and for all further minimizations and simulations , the opls 2005 force 
 field was used .
the calculation involved the following ( default ) settings : water 
 molecules within 10  of the ligand were investigated , the ligand 
 as well as any crystallographic water molecules were removed prior 
 to simulation , the protein was truncated beyond 20  from the 
 ligand , and the simulation time was 2 ns . in brief ( see ref ( 40 ) for details ) , the different 
 stages of the calculation were solvation of the system in an orthorhombic 
 box of tip4p water with a minimum distance 
 between box edge and solute of 10  and a series of minimizations 
 and short simulations to equilibrate the system , followed by a 2 ns 
 production simulation .
all nonwater heavy atoms were harmonically 
 restrained during all minimizations and simulations using a force 
 constant of 5 kcal mol  .
coordinates were saved every 1.5 ps , yielding a total of 1334 snapshots 
 for further analysis .
water molecules were subsequently clustered 
 such that nonoverlapping spheres ( i.e. , hydration sites ) with radius 
 1  were obtained .
thermodynamic properties of these sites ( i.e. , 
 enthalpies , entropies , and thus free energies ) were approximated as 
 follows : enthalpies were estimated as the difference between the average 
 interaction energy of the water molecule with the rest of the system 
 and the average interaction energy in bulk water .
, 
 we used a lysine monomethylation specific rabbit polyclonal antibody 
 ( sy46 ) .
the antibody was generated ( eurogentec ) against a p53 peptide 
 containing the monomethylated k370 epitope , as described elsewhere .
antibody has been purified against unmethylated 
 p53 peptide . using this antibody in cellular systems revealed that 
 it also recognized methylated ahnak , which is also methylated by smyd2 
 ( see supporting information figure s2 ) .
556439 ) was incubated with full - length 
 smyd2 protein ( in - house production ) and sam in reaction buffer ( 50 
 mm tris ph 9 , 1 mm dtt , 0.01% bsa , 0.0022% pluronic ) at 30 c 
 overnight .
cells were lysed in 
 ripa buffer pierce ( thermo fisher scientific ) with benzonase ( merck ) 
 and protease inhibitors ( roche diagnostics ) .
proteins were separated 
 by sds - page and blotted onto nitrocellulose membranes ( invitrogen ) . 

membranes were blocked in 5% milk pbs - t ( phosphate buffered saline 
 with 0.1% tween 20 ) and immunoprobed with antibodies raised against 
 histone h3 ( cell signaling technology no .
ab8224 , 
 1:1000 ) , or with the methylation specific antibody sy46 ( eurogentec ) . 

the secondary antibodies used were goat antimouse / rabbit irdye 800 
 cw ( li - cor biosciences ) and alexa fluor 680 goat antimouse / rabbit 
 igg .
bands were detected and quantified with odyssey fc software ( li - cor 
 biosciences ) .
western blot assay for p53 methylation in hek293 t cells 
 was performed as described elsewhere with an antibody directed against 
 p53k370me1 kindly provided by dr .
effects on apoptosis induction 
 were measured with the caspase - glo 3/7 assay system ( promega ) . in 
 brief ,
10000 cells / well were treated for 2 d with compound 25 or ( s)-4 ( to allow for demethylation 
 of p53 .
then , caspase activation was induced by doxorubicin ( sigma ) 
 ( 1.25 m for u2os and a2780 cells , 2.5 m for kyse-150 
 cells ) treatment for 24 h. the apoptotic index was determined by normalization 
 of caspase 3/7 activation signals to proliferation effects , measured 
 by alamarblue ( thermo fisher scientific ) .
for the detection 
 of smyd2 cellular methylation activity , an in - cell western ( icw ) assay 
 was established .
for the icw , kyse-150 cells stably transfected with 
 a construct expressing wild - type n - terminal 2xc - myc - tagged smyd2 ( ncbi 
 reference sequence : np_064582.2 ) were used . for further detection 
 and validation experiments of ahnak methylation
, we additionally generated 
 stable hela and mda - mb-231 cell lines using the same construct .
for 
 conducting the icw assay , 5000 smyd2-engineered kyse-150 cells / well 
 were seeded in 96-well plates ( sigma ) and cultivated for 24 h at 37 c 
 in 5% co2 .
nontransfected kyse-150 cells were used as a 
 control for maximal inhibition of methylation activity .
cells were 
 grown in 49% rpmi 1640 and 49% ham s f12 media supplemented 
 with 2% heat - inactivated fcs . for the determination of smyd2
inhibitory 
 activity , cells were treated for 72 h in the presence of test compound 
 ( at a final concentration range of 3.9  10 to 5  10 m ) or with dmso .
media were removed , 
 and 3.7% ( w / v ) formaldehyde in pbs was added for 20 min .
after two 
 washes with pbs , 0.25% ( v / v ) triton x-100 in pbs was added for 15 
 min of permeabilization .
after one wash with pbs , cells were blocked 
 in 5% ( w / v ) nonfat dry milk in pbs for 1 h. fixed cells were exposed 
 to primary methylation antibody ( sy46 , 1:200 ) in 5% nonfat dry milk 
 in pbs for 24 h. one row of cells on each plate was not exposed to 
 methylation antibody ( sy46 ) and was reserved for background control 
 measurements .
the wells were washed three times with pbs , then secondary 
 ir800-conjugated antibody ( li - cor , no .
92632211 , 1:1000 ) and 
 dna - intercalating dye draq5 ( thermo fisher scientific , no . 62251 , 
 1:1000 ) were added for 3 h in blocking buffer .
after five washes with 
 pbs , the fluorescence in each well was measured on an odyssey scanner 
 ( li - cor ) at 800 nm ( sy46 methylation signal , 764 nm excitation ) and 
 700 nm ( draq5 signal , 683 nm excitation ) .
the normalized 
 data were further analyzed by four - parameter logistic regression analysis 
 using a bayer proprietary tool to determine the ic50 value 
 for each tested compound .
for ic50 determinations , c0 (= no inhibition ) was defined as the signal 
 measured for the dmso - treated controls .
ci ( maximal inhibition ) was defined as the signal measured for nontransfected 
 kyse-150 cells . for the characterization 
 of proliferation effects ,
animal experiments were 
 conducted in accordance with the german animal welfare laws , approved 
 by local authorities and in accordance with the ethical guidelines 
 of bayer ag .
eight - week - old female balb / c nude mice obtained from 
 charles river laboratories ( germany ) were acclimated for at least 
 for 24 h before tumor cell injection .
kyse-150 cells ( 4  10 ) were resuspended in 100% matrigel ( 100 l ) and injected 
 subcutaneously into the right flank region of the mice ; 4 days after 
 tumor cell inoculation , mice were randomized into four treatment groups . 

group 1 ( n = 12 ) was treated with vehicle only ( solutol / ethanol / water 
 1:1:8 ) iv qd and once at day 4 with saline iv ; group 2 ( n = 6 ) was treated with 500 mg / kg ( s)-4 po qd ; group 3 ( n = 12 ) was treated with 10 mg / kg 
 doxorubicin iv once at day 4 ; group 4 ( n = 6 ) was 
 treated with a combination of ( s)-4 and 
 doxorubicin .
tumor weight is assumed to be log normally distributed , thus all tumor 
 weights were logarithmically transformed ( base = 2 ) prior to statistical 
 inference .
one - sided dunnett s comparison of log 2 tumor weights 
 of all treatment groups with the vehicle group were carried out on 
 an overall significance level of 0.05 .
the results were transformed 
 from the log 2 scale to the original scale so that the difference 
 from the pairwise comparisons and the respective 95% confidence intervals 
 calculated on the log 2 scale correspond to t / c ratios of geometric 
 mean values plus respective 95% confidence intervals on the original 
 tumor weight scale ( mg ) . data was analyzed with r 3.0.1 .
eight - week - old female balb / c 
 nude mice obtained from charles river laboratories ( germany ) were 
 acclimated for at least for 24 h before tumor cell injection . smyd2-engineered 
 kyse-150 cells ( 4  10 ) were resuspended in 100% 
 matrigel ( 100 l ) and injected subcutaneously into the right 
 flank region of the mice .
treatment was started when tumors reached 
 a tumor area of 6070 mm ( day 7 after inoculation ) . 

mice ( n = 12 per group ) were treated with 10 , 30 , 
 70 , or 100 mg / kg ( s)-4 po qd for 3 days , 
 or vehicle ( peg 400/water 8:2 ) po qd for 3 days . after the treatment 
 period , tumor samples were immediately frozen in liquid nitrogen and 
 stored at 80 c .
frozen tumors were mechanically homogenized 
 using tissuelyser and stainless steel beads ( qiagen ) , and proteins 
 were extracted as described for the western blot method .
whole protein 
 lysate ( 50 g per sample ) was transferred with the dot - blot 
 system minifold-1 ( whatman ) onto a nitrocellulose membrane ( invitrogen ) . 

membrane were blocked in 5% milk pbs - t and immunoprobed with the sy46 
 methylation antibody .
the secondary antibody used was goat antirabbit 
 irdye 800 cw ( li - cor , no .
bands were detected 
 and quantified with odyssey fc software ( li - cor biosciences ) .
pharmacokinetic properties of ( s)-4 were determined by administering the test compound 
 as indicated .
blood samples were withdrawn at different time points , 
 and plasma was separated by centrifugation .
liver cells were distributed in williams medium e containing 
 5% fetal calf serum ( fcs ) to glass vials at a density of 1.0  
 10 vital cells / ml . the test compound was added to a final 
 concentration of 1 m . during incubation ,
the hepatocyte suspensions 
 were continuously shaken and aliquots were taken at 2 , 8 , 16 , 30 , 
 45 , and 90 min , to which an equal volume of cold meoh was immediately 
 added .
samples were frozen at 20 c overnight , then centrifuged 
 ( 15 min , 3000 rpm ) , and the supernatant was analyzed with an agilent 
 1200 hplc system with lc - ms / ms detection .
the half - life of a test 
 compound was determined from the concentration  time plot .
the 
 intrinsic clearances were calculated from the half - life , together 
 with additional parameters ( liver blood flow , quantity of liver cells 
 in vivo and in vitro ) .
the hepatic in vivo blood clearance ( cl ) and 
 the maximal oral bioavailability ( fmax ) were calculated using the following parameters : liver blood flow , 
 4.2 l / h / kg rat ; specific liver weight , 32 g / kg rat body weight ; liver 
 cells in vivo , 1.1  10 cells / g liver ; liver cells 
 in vitro , 0.5  10/ml .
caco-2 cells ( purchased from dsmz braunschweig , germany ) 
 were seeded at a density of 4.5  10 cell per well 
 on 24-well insert plates , 0.4 m pore size , and grown for 15 
 days in dmem medium supplemented with 10% fetal bovine serum , 1% glutamax 
 ( 100 , gibco ) , 100 g / ml penicillin , 100 g / ml streptomycin 
 ( gibco ) , and 1% nonessential amino acids ( 100 ) .
medium was 
 changed every second to third day . before running the permeation assay , 

the culture medium was replaced by an fcs - free hepes - carbonate transport 
 buffer ( ph 7.2 ) . for assessment of monolayer integrity , the transepithelial 
 electrical resistance ( teer )
test compounds were dissolved 
 in dmso and added either to the apical or basolateral compartment 
 in a final concentration of 2 m . before and after incubation 
 at 37 c , samples were taken from both compartments .
analysis 
 of compound content was performed following precipitation with methanol 
 and lc / ms / ms analysis .
permeability ( papp ) was calculated in the apical to basolateral ( a  b ) and 
 basolateral to apical ( b  a ) directions . the apparent permeability 
 was calculated using following equation : where vr = volume of medium 
 in the receiver chamber , p0 = measured 
 peak area of the test drug in the donor chamber at t = 0 , s = surface area of the monolayer , p2 = measured peak area of the test drug in the 
 acceptor chamber after incubation for 2 h , and t = 
 incubation time .
the efflux ratio basolateral ( b ) to apical ( a ) was 
 calculated by dividing papp ( b  a ) 
 by papp ( a  b ) .
 protein 
 lysine methyltransferases have recently emerged as a new target class 
 for the development of inhibitors that modulate gene transcription 
 or signaling pathways . 
 set and mynd domain containing protein 2 ( smyd2 ) 
 is a catalytic set domain containing methyltransferase reported to 
 monomethylate lysine residues on histone and nonhistone proteins . 
 
 although several studies have uncovered an important role of smyd2 
 in promoting cancer by protein methylation , the biology of smyd2 is 
 far from being fully understood . 
 utilization of highly potent and 
 selective chemical probes for target validation has emerged as a concept 
 which circumvents possible limitations of knockdown experiments and , 
 in particular , could result in an improved exploration of drug targets 
 with a complex underlying biology . here , 
 we report the development 
 of a potent , selective , and cell - active , substrate - competitive inhibitor 
 of smyd2 , which is the first reported inhibitor suitable for in vivo 
 target validation studies in rodents . 
Introduction
Results and Discussion
Conclusion
Experimental
Section