post - translational histone modifications , 
 which include lysine or arginine methylation , play a pivotal role 
 in maintaining chromatin architecture and regulating transcription .
histone lysine methylation is a reversible process ; 
 the removal of methyl groups is catalyzed by two classes of histone 
 lysine demethylases ( kdms ) : the flavin - dependent lysine specific demethylase 
 ( lsd / kdm1 ) family that demethylate mono- and dimethyl lysine substrates , 
 and the larger class of fe(ii ) and 2-oxoglutarate ( 2og)-dependent 
 enzymes which can additionally convert trimethyl lysine substrates 
 to their dimethyl forms .
the 2og - dependent kdms belong 
 to the jumonji c ( jmjc ) domain containing subfamily of such enzymes 
 and have a catalytic site wherein methylated lysine substrates can 
 be oxidized to hemiaminal intermediates , which subsequently decompose 
 to the corresponding demethylated lysine and formaldehyde .
the kdm4 ( jmjd2 ) subfamily consists of six members kdm4a  f ; kdm4e and kdm4f are believed to be pseudogenes 
 although demethylase activity has been demonstrated when kdm4e is 
 ectopically expressed .
kdm4a  c contain phd and 
 tudor domains as well as a single jmjc domain that catalyzes demethylation 
 of histone substrates h3k9me3/me2 , h3k36me3/me2 , and h1.4k26me3 .
kdm4d is distinguished 
 from kdm4a  c in terms of its domains ( it lacks both the phd 
 and tudor domains ) , its selectivity for demethylation of h3k9me3/me2 over h3k36me3/me2 , and also its ability to demethylate both the dimethylated and trimethylated 
 forms of h1.4k26 .
emerging evidence suggests 
 that kdm4 subfamily members play an important role in cancer initiation 
 and progression ; various studies have shown overexpression of kdm4a  c in a range of human malignancies .
for example , kdm4b and kdm4c are reported to be overexpressed 
 in breast cancer ; kdm4b knockdown in er - positive 
 zr-75 - 1 breast cancer cells impaired growth in a human tumor xenograft 
 model , and kdm4b knockdown in human gastric 
 cancer cells also suppressed growth in a human tumor xenograft model.kdm4b gene expression is high 
 in rhabdomyosarcomas and has been associated with poor prognosis in 
 neuroblastoma .
a recent report demonstrates 
 a role for kdm4a in site - specific copy gain of chromosomal domains 
 ( e.g. , 1q12 ) and rereplication of regions amplified in tumors ; furthermore , a coding single - nucleotide polymorphism 
 ( snp ) in kdm4a was shown to associate with worse 
 outcome in non - small cell lung cancer patients and with increased sensitivity 
 to mtor inhibitors .
the kdm5 subfamily are 
 histone h3 lysine 4 ( h3k4 ) demethylases and have been implicated in 
 cancer progression in several tumor types . along with members of the kdm4 family ,
kdm5b and kdm5c have 
 been shown to play a role in breast and prostate cancer , respectively . 

kdm5b has been implicated in several subtypes of breast cancer , while kdm5c has been shown to be an independent prognostic factor 
 in prostate cancer .
jmjc kdm activity 
 is also associated with diseases other than cancer ; for example , kdm6b / a ( jmjd3/utx)-mediated demethylation 
 of h3k27 has been reported as a critical determinant of pro - inflammatory 
 gene activation in human primary macrophages .
thus , jmjc kdms have emerged as important therapeutic targets , particularly 
 for the treatment human malignancies .
a range of small - molecule inhibitors of jmjc 
 kdms has been reported , the majority of which are 2og - competitive 
 and coordinate to fe(ii ) in the catalytic site , for example , compounds 13 ( figure 1 ) .
many 
 of these early histone demethylase modulators lack human 2og oxygenase 
 specificity and possess a carboxylic 
 acid moiety which may limit cell permeability . as a result , such compounds 
 are suboptimal chemical tools with which to better understand the 
 biological role of specific kdm4 subfamily enzymes in disease and 
 normal development , in particular , to study the importance of jmjc 
 domain kdm activity within the context of full length kdm4 proteins 
 wherein the noncatalytic domains may play a role in chromatin localization 
 or scaffolding functions . in an attempt to overcome the poor cell 
 permeability of inhibitors bearing a carboxylic acid ,
corresponding 
 ester prodrugs have been investigated in some cases ; for example , 
 for the kdm6-selective inhibitor 4 ( gsk - j1 , figure 1 ) where the corresponding 
 ethyl ester prodrug found utility in a cellular context .
extensive interest in the kdm4/5 subfamilies in 
 the progression of human cancers , and the well - defined catalytic mechanism 
 for jmjc kdms , prompted us to initiate a program to identify histone 
 kdm inhibitors , and in particular potent , cell permeable , and selective 
 inhibitors of the kdm4 subfamily with which to further explore the 
 potential of such compounds as anticancer agents .
herein , we report 
 the identification of a 4-(pyridin-2-yl)thiazol-2-amine series as 
 kdm inhibitors from a high throughput screening ( hts ) campaign and 
 their subsequent structure - based optimization into potent , cell permeable 
 dual inhibitors of the kdm4 and kdm5 subfamilies .
synthesis of the hts - derived series of n-(4-(pyridin-2-yl)thiazol-2-yl)benzamides 7a  d ( scheme 1a , table 1 ) was achieved by edci / hobt - mediated 
 coupling of commercially available benzoic acids 6 with 
 4-(pyridin-2-yl)thiazol-2-amine ( 5 ) .
this procedure has 
 recently been reported for the preparation of 2-aminothiazole derivatives 
 as antimycobacterial and antiplasmodial agents .
4-(pyridin-3-yl)thiazole derivatives 9a , b ( scheme 1b , table 2 ) were obtained from commercially available 4-(pyridin-3-yl)thiazol-2-amine 
 ( 8) by a procedure analogous to that described for the 
 synthesis of 7a  d .
reagents 
 and conditions : ( a ) edci , 1-hydroxybenzotriazole , ch2cl2 or ch2cl2/dmf , room temp , 3.536 
 h , ( 7a = 33% , 7b = 25% , 7c = 
 40% , 7d = 50% , 9a = 28% , 9b = 16% ) .
results are mean values of two independent determinations or mean 
 ( sd ) for n > 2 unless specified otherwise .
. syntheses of 
 2-aminothiazoles 15 and 16 and 2-benzamidothiazole 19 are shown in scheme 2 .
treatment of methyl 2-bromoisonicotinate ( 10 ) with stannane 11 under pd - catalyzed conditions gave 
 methyl 2-(1-ethoxyvinyl)isonicotinate ( 12 ) ; subsequent 
 reaction with n - bromosuccinimide afforded bromide 13 .
thiazole ring formation to give the 2,4-disubstituted 
 thiazole 14 was effected by reacting 13 with n - boc - thiourea in the presence of trimethylamine , and subsequent 
 removal of the protecting groups provided 2-aminothiazole derivative 15 .
treatment of 2,4-disubstituted thiazole 14 with ammonia in methanol at 80 c followed by acid - mediated 
 removal of the n - boc protecting group provided the 
 primary amide 16 .
access to 2-benzamidothiazole 19 was achieved by coupling of benzoyl chloride with 2-aminothiazole 17 followed by ester hydrolysis under alkaline conditions 
 ( scheme 2 ) .
reagents 
 and conditions : ( a ) pd(pph3)4 , 1,4-dioxane , 
 reflux , 18 h , 65% ; ( b ) n - bromosuccinimide , 10% water 
 in thf , room temp , 1 h , 61% ; ( c ) n - boc - thiourea , 
 et3n , etoh , reflux , 1 h , 56% ; ( d ) ( i ) 4 m hcl in dioxane , 
 room temp , 1 h , ( ii ) thf / h2o , naoh , room temp , 1 h , 55% ; 
 ( e ) ( i ) 7 m nh3 in meoh , 80 c , 18 h , ( ii ) 4 m hcl 
 in dioxane , room temp , 1 h , 91% ; ( f ) 4 m hcl in dioxane , room temp , 
 1 h , 83% ; ( g ) benzoyl chloride , ch2cl2 , et3n , room temp , 2 h , 35% ; ( h ) thf / meoh , h2o , naoh , 
 room temp , 1 h , 40% . preparation of 22 followed an alternative approach whereby the thiazole ring 
 was introduced directly via a stille cross - coupling reaction ( scheme 3 ) .
access to 24 , a key intermediate for the preparation of 25 , was achieved by bromination of 23 followed by 2-methylthiazole 
 ring formation in a manner analogous to the preparation of 14 ( scheme 3 ) .
reagents 
 and conditions : ( a ) pd(pph3)4 , ag2o , dmf , 100 c , 1 h , 64% ; ( b ) meoh / h2o , naoh , 1 h , 
 46% ; ( c ) acoh , 48% aqueous hbr , br2 , 0 c to room 
 temp for 1 h , then 75 c for 1.5 h , 38% ; ( d ) ( i ) thioacetamide , 
 etoh , et3n , reflux , 1 h , ( ii ) etoh , conc hcl ( 2 drops ) , 
 reflux , 20 min , 23% ; ( e ) meoh / h2o , 1 m naoh , room temp , 
 50 min , 45% .
2-benzylaminomethylpyridine ( 30b ) and 2-furanylmethylamino derivative ( 30a ) were prepared from pyridine-2,4-dicarboxylate 
 ( 26 ) in a four - step sequence ( scheme 4 ) .
esterification of 26 , followed 
 by dibal - mediated selective reduction of the carboxylic acid ester 
 in the 2-position of 27 afforded aldehyde 28 .
reductive amination of aldehyde 28 with either benzylamine 
 or 2-aminomethylfuran in the presence of sodium triacetoxyborohydride 
 led to ester derivatives 29a , b , which were 
 subsequently hydrolyzed to the corresponding carboxylates 30a , b with lithium hydroxide ( scheme 4 ) .
reagents and conditions : 
 ( a ) ptsa , etoh , 80 c , 79% ; ( b ) dibal , thf , phme , 78 
 c , 39% ; ( c ) benzylamine or 2-aminomethylfuran , nabh(oac)3 , acoh , ch2cl2 , ( 29a = 
 99% , 29b = 93% ) ; ( d ) lioh ( aq ) , mecn , ( 30a = quant , 30b = quant ) . 8-chloropyrido[3,4-d]pyrimidin-4(3h)-one ( 34 ) , 
 the key intermediate for the synthesis of thiazole - c4 substituted 
 derivative 37 , 2,4-disubstituted thiazole 40 and subsequent sar exploration ( vide infra ) , was prepared in three 
 steps from methyl-3-amino-2-chloroisonicotinate ( 31 ) 
 ( scheme 5 ) .
ester hydrolysis 
 afforded 32 which was converted into the primary amide 33 via the corresponding acid chloride ; subsequent ring formation 
 was effected upon treatment with triethyl orthoformate ( scheme 5 ) .
a sem - protecting group was 
 introduced by reacting 34 with sem - cl in the presence 
 of potassium carbonate to give protected pyridopyrimidinone 35 .
bromide 39 , a key intermediate for the preparation 
 of 40 , was obtained from 34 in a manner 
 analogous to that described for 13 ( scheme 2 ) .
2-aminothiazole formation 
 to afford 40 was achieved by reacting 39 with thiourea in the presence of et3n .
compound 37 was prepared from 34 by a stille cross - coupling 
 reaction to introduce the thiazole ring followed by removal of the 
 sem protecting group under acidic conditions ( scheme 5 ) .
reagents and conditions : 
 ( a ) meoh / h2o , 1 m naoh , room temp , 45 min , 77% ; ( b ) ( i ) 
 socl2 , dmf , reflux , 2 h , ( ii ) ammonium hydroxide , thf , 
 room temp , 1.5 h , 89% ; ( c ) ch(oet)3 , reflux 24 h , 82% ; 
 ( d ) sem - cl , k2co3 , dmf , 60 c , 4 h , 84% ; 
 ( e ) 4-(tributylstannyl)thiazole , pd(pph3)4 , 
 ag2o , dmf , 100 c , 2 h , 32% ; ( f ) thf , 6 m hcl , 50 
 c , 4 h , 94% ; ( g ) pd(pph3)4 , 1,4-dioxane , 
 reflux , 2 h , 64% ; ( h ) 10% h2o in thf , n - bromosuccinimide , room temp , 1 h , 72% ; ( i ) thiourea , et3n , etoh , reflux , 1 h , 13% . 8-chloropyrido[3,4-d]pyrimidin-4(3h)-one ( 34 ) 
 was also employed for the synthesis of pyrazolyl derivatives 41a , b by a suzuki cross - coupling reaction with 
 ( 1h - pyrazol-3-yl)boronic acid or ( ( n - methyl)pyrazol-3-yl)boronic acid pinacol ester , respectively ( scheme 6 ) .
reagents 
 and conditions : ( a ) pd(pph3)4 , na2co3 , dma , ( 1h - pyrazol-3-yl)boronic acid 
 hydrochloride or ( ( n - methyl)pyrazol-3-yl)boronic 
 pinacol ester , 100 c , 1 h , ( 41a = 12% , 41b = 21% ) . suzuki cross - coupling reaction of 34 with trimethylboroxine afforded the c8-methyl derivative 42 which was oxidized to 4-oxo-3,4-dihydropyrido[3,4-d]pyrimidine-8-carbaldehyde ( 43 ) ; subsequent 
 reductive amination with the requisite primary or secondary amines 
 gave the 8-aminomethyl derivatives 44a  g ( scheme 7 , table 5 ) . reagents 
 and conditions : ( a ) pd(pph3)4 , k2co3 , trimethylboroxine , dme , 90 c , 1 h , 59% ; ( b ) 
 seo2 , 1,4-dioxane , 90 c , 8 h , 14% ; ( c ) nabh(oac)3 , r1r2nh , meoh / ch2cl2 , room temp , 13 h , 1255% . compounds 52a  g
( table 6 ) , 53a  i ( table 7 ) , 
 and 54a  n ( table 8) were prepared either from primary alcohol 47 by mesylate formation followed by sn2 displacement 
 or from carboxaldehyde 48 by reductive amination ( routes 
 a and b , respectively , scheme 8) .
the pyrazole moiety at c8 of the pyrido[3,4-d]pyrimidin-4(3h)-one scaffold was introduced using 
 an snar reaction on key intermediate 35 .
compound 59 ( table 6 ) bearing a longer linker at 
 c4 of the pyrazole moiety was also prepared according to route a described 
 in scheme 8 .
reagents 
 and conditions : ( a ) cs2co3 , anhydrous mecn , 
 reflux , 18 h , 71% ; ( b ) 1 m hcl , meoh , 0 c , 5 min , 99% ; ( c ) dess 
martin 
 periodinane , anhydrous ch2cl2 , room temp ; ( d ) 
 ( i ) methanesulfonic anhydride , et3n , anhydrous ch2cl2 , 0 c , 15 min , workup , ( ii ) cs2co3 , r1r2nh , anhydrous dmf , 90 c , 
 15 h or et3n , anhydrous dmf , 50 c , 15 h , 1784% ; 
 ( e ) r1r2nh , anhydrous 1,2-dichloroethane , nabh(oac)3 , room temp , overnight , 2469% ; ( f ) 6 m hcl , thf , 5060 
 c , 3 to 8 h or 4 m hcl in dioxane , dioxane / h2o , 50 
 c , 1697% ; ( g ) pyrazole , cs2co3 , anhydrous mecn , reflux , 3 h , 80% ; ( h ) thf , 6 m hcl , 50 c , 
 4 h , ( 56 = 70% , 57 = 75% ) .
the discovery of chemical starting 
 points for jmjc histone kdm inhibition was based upon an hts campaign 
 of a 150k compound collection tested at 30 m versus human recombinant 
 kdm4b ( amino acid residues 1500 with an n - terminal gst tag ) . 

the format of the hts was the same kdm4b alphascreen assay described 
 in the methods section ; this assay was also used to monitor the activity 
 of compounds reported herein .
this screening campaign provided n - substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives 
 exemplified by 7c as promising hits ( scheme 1 , table 1 ) . the kdm4b inhibitory activity of 7c was confirmed with a resynthesized sample ( ic50 = 10.9 m ) and triggered a medicinal chemistry program to 
 identify more potent , cell permeable inhibitors of the kdm4 subfamily . 

the methylsulfonyl analogue 7a , p - so2et isomer 7b , and benzamido analogue 7d displayed similar kdm4 inhibitory activities to 7c ( table 1 ) ; all four analogues 
 ( 7a  d ) were more potent inhibitors 
 of kdm4b relative to kdm5b by at least 3-fold under the standard assay 
 conditions ( table 1 ) .
however , the commercially available fragment - like scaffold 4-(pyridin-2-yl)thiazol-2-amine 
 ( 5 ) demonstrated very weak inhibition of kdm4b ( 30% inhibition 
 at 100 m ) and no activity against kdm5b at 100 m , indicating 
 an important role for the primary amine n - substituent .
we set out to establish the binding mode of the 4-(pyridin-2-yl)thiazol-2-amine 
 series to facilitate further medicinal chemistry design .
we postulated 
 that the pyridine nitrogen in 7a  d ( table 1 ) would be 
 a major contributor to kdm4a / b binding via coordination to the metal 
 in the jmjc - domain catalytic site analogous to the binding mode of 
 previously reported pyridine - based kdm inhibitors such as 14 ( figure 1 ) .
consistent 
 with this notion , and also with previous reports that suggested a 
 significant drop in potency for pyridin-3-yl regioisomers of active 
 compounds , the 4-(pyridin-3-yl)thiazole 
 derivatives 8 , 9a , and 9b were 
 considerably less potent or inactive relative to their pyridin-2-yl 
 counterparts ( tables 1 and 2 ) .
the commercially available phenyl 
 analogue of 5 , lacking the pyridyl nitrogen ( 4-phenylthiazol-2-amine ) , 
 was also inactive against kdm4a and kdm4b at 100 m compound 
 concentration ( data not tabulated ) .
attempts to elucidate the 
 binding mode of 7a or close analogues by cocrystallization 
 with kdm4a or by soaking of apo - kdm4a crystals with 7a were unsuccessful .
docking studies of the fragment - like scaffold 5 using the cocrystal structure of 2 in kdm4a 
 ( pdb code 3pdq ) suggested that 5 could 
 bind to the active site metal through bidentate coordination of the 
 pyridyl and thiazolyl nitrogen atoms ( figure 2a , b ) .
furthermore , the docked pose of 5 overlaid well with the binding mode of the bipyridyl - based 
 analogue 2-bound kdm4a cocrystal structure ( pdb code 3pdq ) .
this observation prompted the introduction of a carboxylate 
 moiety para to the pyridine nitrogen in 5 to interact with k206 and y132 ( kdm4a residue numbering ) in a manner 
 analogous to that observed for the cosubstrate 2og , nog , and literature 
 pyridyl - based analogues 1 and 2 .
pleasingly , 15 ( table 3 ) demonstrated potent inhibition of kdm4a and kdm4b ( ic50 = 0.200 and 0.083 m respectively , table 3 ) , kdm5b ( ic50 = 
 0.012 m , table 3 ) as well as kdm3a ( ic50 = 0.057 m ) while inhibiting 
 kdm2a and kdm6b to a lesser extent ( ic50 = 1.2 and 1.0 
 m , respectively ) .
interestingly , introduction of a carboxylate 
 at the equivalent position para to the pyridine nitrogen 
 in the n-(thiazol-2-yl)benzamido derivative 7d to give compound 19 ( table 3 ) resulted in demethylase activity similar 
 to 7d ( kdm4b ic50 = 17.0 m , table 3 ) consistent with 
 the docking predictions that binding of extended analogues at the 
 c2 thiazole position could be compromised due to the steric clash 
 of the benzamido substituent with the protein ( figure 2b ) .
overlay of a view from crystal structure of 2 ( light - blue ball - and - sticks ) bound to kdm4a ( displayed in 
 gray lines or gray surface ; pdb code 3pdq ) and a model 
 of 5 ( magenta ) docked into the protein structure from 3pdq .
results are mean values of two independent 
 determinations or mean ( sd ) for n > 2 unless 
 specified otherwise .
ligand 
 crystallographic analysis was mainly performed using kdm4a which has 
 a similar jmjc catalytic site to that of kdm4b .
a crystal structure of 15 bound to kdm4a confirmed 
 the proposed binding mode involving interactions of the carboxylate 
 moiety with k206 and y132 and bidentate coordination of both the pyridyl 
 and thiazolyl nitrogens to the metal . a deviation from coplanarity 
 of the pyridyl and thiazolyl aromatic ring systems ( torsional angle 
 = 17.4 ) and a rotation of the plane of the carboxylate relative 
 to the plane of the pyridine ring
these observations 
 are broadly consistent with the binding mode of an analogue of the 
 triazolopyridine 3 ( figure 1 ) and of aminomethyl 
 substituted pyridines 30a(36 ) and 30b ( table 4 ) . the carboxylic acid in 30b interacts with k206 and y132 of kdm4a and the carboxylic 
 acid in 30a with corresponding residues of kdm4d ; furthermore , 
 both ligands exhibit bidentate metal coordination ( figure 3d , e ) . the aminomethyl substituent 
 in both 30a and 30b is rotated away from 
 the plane of the biaryl ring scaffold in 15 to interact 
 with y175 in kdm4a and the equivalent residue in kdm4d ( y179 ) ( figure 3d , e ) ; additionally , 
 the furan oxygen of 30a interacts with the hydroxyl moiety 
 of y181 in kdm4d ( equivalent residue y177 in kdm4a ) ( figure 3e ) . views from crystal structures 
 of 15 , 16 , and 30b bound to 
 kdm4a and of 30a bound to kdm4d .
( a ) kdm4a - bound 15 ( gray ) , showing a close - up view of ligand  metal 
 coordination and interaction with y132 and k206 .
( b ) overlay of kdm4a - bound 15 ( gray ) and 16 ( green ) .
( d ) overlay of 
 kdm4a - bound 15 ( gray ) and 30b ( purple for 
 chain a , cyan for chain b of the asymmetric unit ) .
( e ) overlay of 
 kdm4a - bound 15 ( gray ) and kdm4d - bound 30a ( orange ) . indicated residues correspond to kdm4a numbering .
results are mean values of two independent determinations or mean 
 ( sd ) for n > 2 unless specified otherwise
the 15-bound kdm4a structure also revealed 
 -stacking of the pyridyl ring with the side chain of residue 
 f185 , an interaction of the thiazole c2-nh2 with e190 , 
 and interaction of this exocyclic nh2 to both d191 and 
 n290 via a mediating water molecule ( figure 3b , c ) .
consistent with the latter observation , 
 the desamino analogue 22 was a less potent kdm4 inhibitor 
 compared to 15 ( kdm4a ic50 = 0.90 m , 
 kdm4b ic50 = 0.35 m ; table 3 ) , and a further drop in potency was observed 
 with the thiazole c2-me analogue 25 ( kdm4a ic50 = 16.5 m , kdm4b ic50 = 2.3 m ; table 3 ) .
conversion of the 
 carboxylic acid in 15 to the corresponding primary amide 16 led to a significant drop in kdm4a and kdm4b potency ( 500- 
 and 400-fold , respectively ) , highlighting the importance of the carboxylate 
a crystal structure 
 of 16 bound to kdm4a showed a very similar binding mode 
 to that of 15 .
interestingly , the primary amide of 16 and the carboxylic acid of 15 overlay well , 
 further reinforcing the significant benefit of the carboxylate  k206 
 charge interaction ( figure 3b ) .
although the ionizable carboxylic acid appeared beneficial 
 for ligand affinity , we considered it a significant contributing factor 
 to the observed poor cellular permeability for 15 ( caco-2 
 a to b flux < 0.8  10 cm / s ) . therefore , 
 we focused on pyridine-4-carboxylic acid isosteres with the potential 
 to coordinate to the active site metal , make strong interactions with 
 k206/y132 ( kdm4a ) and to be only partially ionized at physiological 
 ph to improve cellular permeability while maintaining potent enzyme 
 binding .
our attention was drawn to the pyrido[3,4-d]pyrimidin-4(3h)-one scaffold ( 58 , table 5 , figure 4 ) 
 kindly disclosed to us by gsk and also 
 reported in the patent literature as a template for jmjc kdm inhibition .
the bicyclic aromatic scaffold 58 is a highly efficient ligand for both kdm4 and kdm5 subfamily members 
 ( kdm4b ic50 = 1.0 m , le = 0.76 , and kdm5b ic50 = 1.3 m , le = 0.75 ; table 5 ) while a less potent inhibitor of kdm3a 
 ( ic50 = 26.2 m ) , kdm2a ( 29% inhibition at 100 m ) , 
 and kdm6b ( 28% inhibition at 100 m ) .
pleasingly , 58 also displayed high cellular permeability ( caco-2 a to b flux = 
 42.61  10 cm / s ) with a measured pka for the pyridopyrimidinone amide moiety of 
 8.23 .
comparison of binding modes of 15 , 37 , 40 , and 58 to kdm4a 
 and 44a bound to kdm4d .
( a ) overlay of kdm4a - bound 15 ( gray ) and 58 ( yellow ) .
( b ) overlay of kdm4a - bound 15 ( gray ) , 37 ( light - green ) , and 40 ( orange ) .
( c ) overlay of kdm4a - bound 15 ( gray ) and 
 kdm4d - bound 44a ( light blue ) .
zn(ii ) and ni(ii ) are shown in place 
 of the active site fe(ii ) .
results 
 are mean values of two independent determinations or mean ( sd ) 
 for n > 2 unless specified otherwise .  
the 58-bound kdm4a 
 structure demonstrates simultaneous coordination of the pyrido[3,4-d]pyrimidin-4(3h)-one scaffold to the active 
 site metal via the pyridine nitrogen ( position 7 ) and interaction 
 of the pyrimidinone conh moiety with k206/y132 ; in addition , n1 of 58 is positioned to interact with the 
 side chain of k241 .
this network of interactions is consistent with 
 the highly ligand efficient inhibition profile of 58 versus 
 kdm4a and kdm4b , which are very similar in their jmjc catalytic sites . 

superposition of the 15-bound and 58-bound 
 kdm4a structures ( figure 4a ) suggested that the pyrido[3,4-d]pyrimidin-4(3h)-one scaffold 58 may provide a suitable replacement 
 for the pyridine-4-carboxylate , whereby derivatization at the c8 position 
 of 58 could lead to bidentate metal coordination and 
 access to the histone peptide binding site ( vide infra ) . to test this 
 hypothesis ,
the hybrid structures 37 and 40 were synthesized ( scheme 5 , table 5 ) . 

compound 40 displayed lower kdm4b inhibitory activity 
 ( ic50 = 5.0 m , table 5 ) compared to 15 and 58 . 

a similar trend was observed with the 2-desamino analogue 37 ( kdm4b ic50 = 3.1 m , table 5 ) .
compound 37 proved a more 
 potent inhibitor of kdm3a ( ic50 = 0.89 m ) and kdm5b 
 ( ic50 = 0.31 m ; table 5 ) and showed weaker inhibition of kdm2a ( 59% 
 inhibition at 100 m ) and kdm6b ( 31% inhibition at 100 m ) , 
 consistent with the profile of compound 15 .
crystal structures 
 of 40 and 37 bound to kdm4a show bidentate 
 coordination to the metal as well as the predicted interactions with 
 k206/y132 ( figure 4b ) .
however , interaction of k241 with n1 of the 
 pyridopyrimidinone scaffold is no longer observed for c8-substituted 
 pyridopyrimidinones 37 and 40 , which may 
 contribute to their lower potency compared to 58 ( figure 4a , b ) .
we postulate 
 that steric bulk of the thiazole c8 substituent in 37 and 40 hinders interaction of the scaffold with k241 . 

furthermore , we note that metal coordination of the thiazole nitrogen 
 in 37 and 40 may result in suboptimal interactions 
 of the pyridopyrimidinone conh moiety with k206/y132 owing to the 
 planar bicyclic scaffold which precludes the 28.6 dihedral angle 
 between the pyridine and carboxylate observed in our 15-bound kdm4a structure ( figures 3a , 4a , b ) .
interestingly , the 
 torsion angles between the planes of the pyridopyrimidinone and the 
 thiazole ring systems in 37 and 40 are similar 
 to that observed between the pyridine and the thiazole in 15 ( 4.7 and 7.2 difference with respect to 15 , respectively ) , rendering the metal - coordination geometry similar 
 for 15 , 37 , and 40 ( figure 4b ) .
although introduction 
 of a c8-thiazole substituent led to a slight decrease in kdm4a and 
 kdm4b potency compared to 58 , we envisaged that potency 
 for this class of compounds could be improved by introducing favorable 
 interactions with d135 and other residues in the histone peptide binding 
 site , a tactic previously reported to yield potent jmjc histone demethylase 
 modulators in the bipyridyl scaffold exemplified by 2 ( figure 2a ) .
access to the histone peptide binding pocket 
 required derivatization through a position equivalent to the sulfur 
 atom in 37 ( see figures 2a , b and 4a , b ) and necessitated the introduction of alternative 
 heterocyclic rings at the c8 position of the pyrido[3,4-d]pyrimidin-4(3h)-one scaffold ( table 5 ) .
we introduced a variety 
 of five - membered heterocycles at c8 of the pyrido[3,4-d]pyrimidin-4(3h)-one scaffold 58 , including 
 pyrazol-3-yl and pyrazol-1-yl moieties ( 41a and 56 , respectively , table 5 ) which exhibited similar potency versus kdm4b and 
 kdm5b .
we also investigated whether methylamino substituents at c8 
 could provide access to the pocket occupied by the trimethylated lysine 
 of the h3k9me3 peptide substrate , as previously described 
 for 30a and 30b in the pyridine-4-carboxylic 
 acid series ( table 4 ; figure 3d , e ) .
compounds 44a and 44b were both less potent inhibitors 
 of kdm4b and kdm5b compared to their pyridine-4-carboxylate counterparts 30b and 30a , respectively ( tables 4 , 5 ) .
we confirmed 
 the binding mode of 44a to kdm4d by cocrystal structure 
 determination , which proved consistent with that of the parent compound 
 ( compare figure 3d , e 
 and figure 4c ) .
interestingly , 
 both aminomethyl - substituted pyridopyrimidinones 44a and 44b display a preference for kdm5b / c inhibition over kdm4a / b . 

tertiary amine analogues 44c  g were 
 designed to rescue kdm4 potency by maintaining bidentate metal chelation 
 while also extending into space occupied by the c8-thiazole of 37 or 40 and benefiting from interactions with 
 y175 and y177 in kdm4a ( figure 4 ) . at best , the tertiary aminomethyl compounds were comparable 
 in potency to simple c8-heterocyclic substituted derivatives 41a , b and 56 ( table 5 ) .
considering the data presented in table 5 , and cognizant of 
 synthetic tractability , we focused our effort on elaboration of the 
 pyrazol-1-yl analogue 56 by targeting residues in the 
 histone substrate binding site including d135 ( kdm4a ) ( table 6 ) .
not determined results ( ic50s ) are mean values of two 
 independent determinations or mean ( sd ) for n > 2 unless specified otherwise .
substitution at the pyrazole - c4 position with a hydroxyethyl 
 group ( 57 ) was tolerated and resulted in a similar kdm4/5 
 inhibitory profile to 56 ( tables 5 and 6 ) .
introduction 
 of a basic nitrogen at the pyrazole - c4 substituent , exemplified by 
 compounds 52a , 52b , and 52c , resulted in at least a 4-fold improvement in kdm4a / b inhibitory 
 activity ( table 6 ) ; 
 notably , all three compounds were more potent inhibitors of kdm5b / c 
 compared to kdm4a / b .
introduction of a p - fluorobenzyl 
 group ( 52d ) , gave a more potent inhibitor of kdm4b , kdm5b , 
 and kdm5c ( ic50 = 0.39 , 0.042 , and 0.078 m , respectively , table 6 ) .
pleasingly , 52d displayed a weaker inhibitory activity against kdm2a ( ic50 = 13.4 m ) , kdm3a ( ic50 = 13.9 m ) , 
 and kdm6b ( ic50 = 26.3 m ) relative to kdm4/5 subfamily 
 members .
a crystal structure of 52d bound to kdm4a revealed 
 that the compound bound in multiple conformations , with the two most 
 predominant binding modes depicted in figure 5 . in conformation
a , the pyrazole - c4 substituent 
 adopts an extended conformation with the p - fluorobenzyl 
 group oriented toward the solvent accessible area ; this induces movement 
 of the side chain of y175 and displaces the nearby loop ( residues 
 165175 ) .
however , no interaction between 52d and 
 the induced side chain conformation of y175 is observed ( minimal distance 
 of 4.7  between a carbon of the y175 aromatic ring and a carbon 
 of the p - fluorophenyl group of 52d ) . 
 in conformation b , the p - fluorobenzyl group of the 
 pyrazole - c4 substituent oriented toward an asparagine / glutamine - rich 
 pocket ( residues 73 , 84 , and 86 , figure 5 ) . in conformation b ,
the basic nitrogen 
 of the pyrazole - c4 substituent is proximal to d135 with a minimum 
 interaction distance of 2.9  ( distances between 2.9 and 3.2 
  depending upon the chain in the asymmetric unit ) , slightly 
 shorter compared with the equivalent distance in conformation a ( 3.44.3 
  ) .
attempts to cocrystallize compounds presented in this study 
 with kdm5b proved more challenging relative to kdm4a crystallography ; 
 however , we were successful in resolving a crystal structure of 52d bound to kdm5b ( figure 5b ) , which shows coordination of both the pyridyl and 
 pyrazole n2 nitrogen to the active site metal and 
 interaction of the pyridopyrimidinone conh moiety with k517/y425 ( equivalent 
 to residues k206/y132 in kdm4a ) .
the electron density of the p - fluorobenzyl group in this crystal structure was poor , 
 precluding accurate positioning of this moiety .
however , the binding 
 mode of the well - defined ligand scaffold to kdm5b was similar to that 
 observed in the structure of 52d bound 
 to kdm4a .
( a ) overlay of views from crystal structure of kdm4a37 ( light - green ) and 52d ( two main conformations : 
 dark - brown for conformation a and light - brown for conformation b ) . 

( b ) overlay of 52d bound to kdm4a ( brown ) and kdm5b ( cyan ) , 
 the indicated residue numbers correspond to kdm5b numbering .
zn ( ii ) 
 or ni(ii ) are shown in place of the active site fe ( ii ) .
pleasingly , 52d displayed cellular 
 permeability ( caco-2 a to b flux = 9.98  10 cm / s , table 6 ) and 
 moderate hlm and mlm stability ( 40% and 42% metabolized after a 30 
 min incubation , respectively ) .
surprisingly , we found that 52d had high in vivo plasma clearance ( iv clearance in mouse of 1640 
 ml / min / kg , following a 6 mg / kg iv compound administration ) , which 
 we suspected may be due to aldehyde oxidase ( ao)-mediated metabolism .
we subsequently identified the c2 position of 
 the pyrido[3,4-d]pyrimidin-4(3h)-one 
 template as the major site of ao - mediated metabolism .
the 3,4-dichlorobenzyl analogue 52e also 
 demonstrated cellular permeability ( caco-2 a to b flux = 21.21  
 10 cm / s , table 6 ) ; however , attempts to gain additional kdm4a / b affinity 
 by introduction of hydrogen bond interactions with n86 , q73 , or q84 
 ( 52f and 52 g ; figure 5 ) were unsuccessful ( table 6 ) .
homologation of the pyrazole - c4 substituent 
 by one methylene unit ( 59 ) also failed to improve kdm4a 
 or kdm4b inhibition ( table 6 ) .
the multiple ligand conformations observed in the 52d - bound kdm4a structure suggested that no single binding 
 mode was favored and is consistent with multiple degrees of freedom 
 in the ligand conformation .
thus , we next attempted to constrain the 
 pyrazole - c4 substituent by introduction of a piperidine ring .
this 
 approach led to the synthesis of 53a , a potent and cell 
 permeable ( caco-2 a to b flux = 6.86  10 cm / s ) kdm4a and kdm4b inhibitor ( ic50 = 0.126 and 0.050 
 m , respectively ) which also strongly inhibited kdm5b and kdm5c 
 ( ic50 = 0.014 and 0.023 m , respectively ; table 7 ) .
this kdm4 and kdm5 subfamily profile differentiates 53a from 52d , which is approximately 9-fold more 
 potent in inhibiting kdm5b compared to kdm4b ( table 6 ) .
pleasingly , 53a maintained 
 selectivity for kdm4 and kdm5 subfamily inhibition versus kdm2a ( ic50 = 8.6 m ) , kdm3a ( ic50 = 4.8 m ) , 
 and kdm6b ( ic50 = 48.5 m ) .
the crystal structure 
 of 53a bound to kdm4a confirmed a single binding mode 
 in which the ligand extends toward the histone peptide binding site 
 similar to conformation a of 52d ( figures 5 and 6 ) .
the bulky 
 pyrazole - c4 substituent of 53a induces movement of the 
 y175 side chain and the nearby loop ( residues 165175 ) as observed 
 in conformation a of 52d .
we also observed a hydrophobic 
 interaction between the piperidine ring of 53a with the 
 side chain of y175 ( 3.5  between proximal piperidine carbon 
 atoms and the aromatic ring of y175 , figure 6 ) .
notably , the distance between the piperidine 
 nitrogen and the carboxylate of d135 is 4  , longer than the 
 equivalent distance observed in a crystal structure of 52d ( conformation b ) bound to kdm4a ( figures 5 and 6 )
. additionally , 
 the p - chlorobenzyl group points toward the histone 
 peptide binding site and the solvent accessible area creating a new 
 hydrophobic interaction with v313 ( 3.9  between the p - chlorobenzyl of 53a and the nearest side 
 chain carbon of v313 )
. results 
 ( ic50s ) are mean values of two independent determinations 
 unless specified otherwise . binding mode 
 of 53a in kdm4a .
zn(ii ) is shown in place of the active 
 site fe(ii ) . considering these 
 structural observations , including the single observed conformation 
 of the pyrazole - c4 substituent in 53a
replacement of p - cl with 
 h ( 53b ) , f ( 53c ) , cf3 ( 53d ) , or introduction of an additional meta substituent ( 53e and 53 g ) resulted in 
 a kdm4a / b and kdm5b / c inhibitory profile broadly similar to that of 53a ( table 7 ) . the m - methoxybenzyl analogue ( 53f ) was a less potent inhibitor of kdm4a relative to 53a although kdm5b / c potency was maintained ( table 7 ) .
replacement of the p - chlorophenyl 
 in 53a with heterocyclic moieties failed to improve kdm4a / b 
 or kdm5b / c inhibitory activities ( compounds 53h and 53i , table 7 ) . in a further attempt to restrain conformational freedom 
 in the pyrazole side chain of 53a ,
we attached the p - chlorophenyl moiety directly to the piperidine 4 position 
 ( 54a ) ; this tactic broadly maintained kdm4a / b and kdm5b / c 
 inhibitory activities ( kdm4b ic50 = 0.086 m , kdm5b 
 ic50 = 0.030 m ; table 8) .
notably , 54a demonstrated selectivity for kdm4b and kdm5b versus kdm2a 
 ( ic50 = 6.74 m ) and also displayed cellular permeability 
 ( caco-2 a to b flux = 2.66  10 cm / s , table 8) .
replacement of 
 the p - cl substituent in 54a with f ( 54b ) , ome ( 54c ) , so2me ( 54d ) , or cn ( 54e ) resulted in a similar kdm4a / b inhibitory 
 profile to that of 54a .
interestingly , balanced inhibition 
 of kdm4 and kdm5 subfamily members was observed for 54a , 54b , and 54c ( table 8) .
analogue 54f bearing an o - chlorophenyl substituent and the unsubstituted phenyl 
 derivative 54 g exhibited weaker kdm4a and kdm4b inhibition 
 compared to that of 54a ( table 8) .
heterocyclic replacements for the p - chlorophenyl moiety in 54a gave a similar 
 kdm4a / b and kdm5b / c inhibition profile ( see 54h and 54i , table 8) .
the thiophene derivative ( 54i ) was a less potent 
 inhibitor of kdm3a ( ic50 = 8.3 m ) , kdm6b ( 25% inhibition 
 at 100 m ) , and kdm2a ( ic50 = 2.4 m ) .
moderate 
 hlm and mlm stability was observed for 54i ( 49% and 62% 
 metabolized after a 30 min incubation , respectively ) , and cellular 
 permeability was maintained ( caco-2 a to b flux = 17.83  10 cm / s ) .
notably , introduction of a 3,5-dichlorophenyl , 
 compound 54j gave the most potent inhibitor of kdm4 and 
 kdm5 subfamily members in this series , with kdm4a / b and kdm5b / c potencies 
 indicating a balanced inhibition profile ( kdm4a / b ic50 = 
 0.080 and 0.017 m , kdm5b / c ic50 = 0.014 and 0.051 
 m , respectively , table 8) .
pleasingly , 54j showed cellular permeability 
 ( caco-2 a to b flux = 6.34  10 cm / s ) and 
 a selective inhibition profile versus kdm3a ( ic50 = 6.1 
 m ) , kdm6b ( 4% inhibition at 100 m ) , and kdm2a ( ic50 = 2.4 m ) .
the m - cl analogue 54k displayed a similar profile to that of 54j ( table 8) and benefited 
 from low mlm and hlm turnover ( 13% and 17% metabolized respectively 
 after a 30 min incubation ) .
compound 54k also displayed 
 a selective inhibition profile versus kdm2a ( ic50 = 12.9 
 m ) , kdm3a ( ic50 = 5.3 m ) , and kdm6b ( 15% 
 inhibition at 100 m ) .
furthermore , profiling of 54k in a 50-kinase panel , representative of the human kinome , at a concentration of 1 m revealed no 
 significant kinase inhibitory activity ( supporting information , table s2 ) .
analogues 54l  n also demonstrated a broadly balanced kdm4b and kdm5b inhibition 
 profile and showed good cellular permeability in the caco-2 assay , 
 reinforcing the sar trend in this 4-phenyl piperidine subseries ( table 8) .
= 
 not determined results ( ic50s ) are mean values of two independent determinations or mean 
 ( sd ) for n > 2 unless specified otherwise .
cellular activity of 54k was assessed in hela cells transiently overexpressing 
 wild - type ( wt ) kdm4a or kdm5b or the respective mutants ( mut ) lacking 
 catalytic activity .
cells were treated with the inhibitor , and changes 
 in the global methylation levels were quantified by high content immunofluorescence 
 assay after 24 h. global hypomethylation 
 was observed in cells overexpressing either wt kdm4a or wt kdm5b as 
 determined by reduction in the levels of methyl  lysine antibody 
 staining ( relative to cells overexpressing the corresponding mut demethylase 
 or nontransfected cells ) .
treatment with 54k resulted 
 in a concentration - dependent increase of the respective methyl mark 
 ( h3k9me3 for kdm4a , h3k4me3 for kdm5b ) in cells 
 overexpressing wt demethylase . at high concentrations of 54k ( > 50 m ) ,
h3k9me3 levels approached those in 
 hela cells overexpressing mut kdm4a , consistent with cell - based inhibition 
 of kdm4a demethylase activity ; similarly , at high concentrations of 54k , h3k4me3 levels approached those in hela cells 
 overexpressing mut kdm5b , consistent with cell - based inhibition of 
 kdm5b demethylase activity . occasionally , at the highest concentrations , 

an effect on the methylation mark was also observed in cells overexpressing 
 mut kdm , pointing to inhibition of either the endogenous enzyme(s ) 
 or an unspecific effect ( supporting information , figure s1 ) . in studies to assess the quality of promising 
 analogues as potential lead compounds , we noted chemical instability 
 of 54j and 54l when left in air at room 
 temperature for prolonged periods ; mass spectrometry analysis suggested 
 an oxidation - mediated degradation .
however , compound 54j was stable for > 8 weeks as solution in dmso when stored under 
 a nitrogen atmosphere .
these observations prompted us to resynthesize 54j and study its stability as a solution in dmso kept at 
 room temperature with no protection from air and also in solid form 
 kept at room temperature under a nitrogen atmosphere .
lcms and nmr 
 ( solid sample ) monitoring indicated no degradation for up to 8 weeks . 

given these observations , we recommend that , as a precaution , compounds 
 of this class are stored under a nitrogen atmosphere .
crystal 
 structures of 54a and 54j bound to kdm4a 
 showed that both compounds adopt an extended conformation consistent 
 with that observed for 52d and 53a ( figure 7a ) .
the orientation 
 and position of the piperidine ring in 54a and 54j is perpendicular to the aromatic ring of y175 in kdm4a 
 and is rotated by nearly 90 compared to the piperidine ring 
 of 53a ( compare figures 6 and 7a ) .
the piperidine ring 
 in 54a or 54j maintains hydrophobic interactions 
 with the side chain of y175 ( distances of 3.5 and 3.1  between 
 the nearest carbon atom of the piperidine and the aromatic ring of 
 y175 for 54a and 54j , respectively ) .
however , 
 in the 54a- and 54j - bound structures , the 
 piperidine nitrogen resides at a minimum distance of 3.9 and 3.8  
 from the carboxylate of d135 , respectively , which is longer than the 
 equivalent distance in the 52d 
notably , the p - chlorophenyl and m - dichlorophenyl groups of 54a and 54j both 
 engage in hydrophobic interactions with v313 of kdm4a , albeit with 
 a slightly different orientation of the aromatic ring ( figure 7 ) .
we observe a closer interaction 
 of one m - cl of 54j with the side chain 
 of v313 ( distance of 3.5  , figure 7b ) compared to the aromatic ring of 54a ( distance of 4  ) , potentially explaining the enhanced 
 kdm4a / b inhibition of 54j ; indeed , this hydrophobic v313 
 interaction may contribute to the balanced kdm4 and kdm5 activity 
 profile of these compounds through enhancing kdm4 subfamily affinity .
( a ) overlay 
 of kdm4a - bound 54a ( orange ) and 54j ( cyan ) . 

( b ) 2d interaction diagram of 54j showing interactions 
 with key residues in kdm4a .
an hts campaign versus 
 the jmjc histone lysine demethylase kdm4b resulted in the discovery 
 of n - substituted 4-(pyridin-2-yl)thiazole-2-amine 
 derivatives as promising hit compounds .
comparison with previously 
 reported pyridine - based jmjc kdm inhibitors generated a binding mode 
 hypothesis , and subsequent incorporation of an appropriately positioned 
 carboxylic acid moiety led to the discovery of 15 , a 
 potent inhibitor of the kdm3 , kdm4 , and kdm5 kdm subfamilies but which 
 lacked cellular permeability .
a crystal structure of 15 bound to kdm4a confirmed the proposed binding mode involving interactions 
 of the carboxylate moiety with k206 and y132 as well as bidentate 
 coordination of both the pyridyl and thiazolyl nitrogens to the active 
 site metal .
interestingly , the corresponding primary amide 16 maintained this binding mode ; however , loss of the carboxylate 
k206 
 interaction led to a > 400-fold reduction in potency , illustrating 
 the dominance of this ionic interaction in the buried kdm4 catalytic 
 site .
replacement of the pyridine-4-carboxylate scaffold in 15 with the less acidic , bicyclic pyrido[3,4-d]pyrimidin-4(3h)-one isostere 58 ( pka = 8.23 ) gave cell - permeable kdm inhibitors 
 with maintained affinity , presumably due to ionization of the weakly 
 acidic scaffold in the kdm4 active site .
a crystal structure of 58 bound to kdm4a demonstrated simultaneous coordination to 
 the active site metal via the pyridine nitrogen , interaction with 
 k206/y132 via the pyrimidinone conh moiety , and interaction with the 
 side chain of k241 through n1 of the scaffold .
subsequent 
 structure - guided optimization at c8 of the pyrido[3,4-d]pyrimidin-4(3h)-one allowed extension into the 
 histone peptide binding site by virtue of a c8-pyrazole moiety .
this 
 tactic favored bidentate coordination to the active site metal through 
 both the pyridine and pyrazole n2 nitrogen as well 
 as concomitant additional pyrazole substitution to gain beneficial 
 interactions with d135 and y175 ( kdm4a residue numbering ) as exemplified 
 by a crystal structure of 52d bound in kdm4a .
however , 
 this 52d - bound crystal structure also revealed alternative 
 bound conformations of the flexible pyrazole c4 substituent , suggesting 
 that conformational restriction may be required to further benefit 
 from interactions in the histone peptide binding site .
incorporation 
 of a rigidifying piperidine linker into pyrazole c4 substituent and 
 subsequent positioning of a meta - substituted phenyl 
 ring at the piperidine 4-position , resulted in the discovery of potent 
 jmjc histone demethylase inhibitors such as 54j , 54k , and 54i .
a crystal structure of 54j bound in kdm4a demonstrates a conserved bidentate binding mode of 
 the c8-pyrazolo - pyrido[3,4-d]pyrimidin-4(3h)-one scaffold to the active site metal and additional 
 beneficial interactions of the 4-phenylpiperidine substituent with 
 d135 and y175 as well as a further hydrophobic interaction with v313 
 of kdm4a ( kdm4a residue numbering ) , which we postulate is important 
 to the balanced kdm4 and kdm5 activity profile of these compounds . 

importantly , incorporation of the conformationally constrained 4-phenylpiperidine 
 in derivatives 54j and 54k results in selective 
 inhibition of the kdm4 and kdm5 subfamily demethylases over representative 
 exemplars of the kdm2 , kdm3 , and kdm6 subfamilies , cellular permeability 
 in the caco-2 assay , and , for 54k , we demonstrate inhibition 
 of h3k9me3 and h3k4me3 demethylation in a cell - based assay .
we propose 
 that compounds such as 54k could be utilized for the 
 study of kdm4/kdm5 subfamilies in a cellular context . as both kdm4 
 and kdm5
family members have been implicated in breast , prostate , 
 and other cancer contexts , such tool compounds may be particularly 
 useful in studying the merits of dual inhibition of these family members 
 in cell - based assays .
in addition , the sars and the ligand  protein 
 interactions presented in this study could provide valuable leads 
 for the further exploring the design and synthesis of kdm4 or kdm5 
 subfamily selective compounds with which to elucidate the role of 
 the jmjc catalytic domain activity of these subfamilies in human disease . 

we are continuing our research toward the identification of potent , 
 cell permeable , and selective inhibitors of the kdm4 subfamily with 
 which to study the role of kdm4 enzyme function in cancer biology 
 and to further explore the potential of such compounds as anticancer 
 agents .
protein production and purification : baculovirus 
 containing residues 1500 of kdm4b with an n - terminal gst tag 
 were generated according to bac - to - bac protocol . following infection 
 of sf9 insect cells
, protein was purified by gsh 
 affinity chromatography followed by gel filtration ( superdex 200 ) . 

purification of kdm4a is described in the crystallography section . 
 for the purpose of biochemical assays ,
enzyme activity was measured using an alphascreen assay that monitored 
 the demethylation of a biotinylated trimethylated h3k9 peptide using 
 a h3k9 dimethyl specific antibody and appropriate donor and acceptors 
 beads from perkinelmer life sciences . for each compound , a 10 mm stock concentration in 100% dmso was 
 used .
sunnyvale , ca , usa ) to generate 8 pt dilution curves 
 directly into 384-well proxiplates ( no .
6008289 , perkinelmer , waltham , 
 ma , usa ) to give final assay concentrations in the range 0.0005100 
 m in 2% ( v / v ) dmso as appropriate .
the enzyme reaction was 
 performed in assay buffer which consisted of hepes ( 50 mm , ph 7.5 ) , 
 bsa ( 0.1% w / v ) , sodium ascorbate ( 100 m ) , 2og ( 2 m for 
 kdm4b or 10 m for kdm4a ) , ammonium iron(ii ) sulfate hexahydrate 
 ( 1 m ) , and tween 20 ( 0.01% v / v ) .
compounds were preincubated 
 for 10 min with 2.5 l enzyme n - terminal gst kdm4b prepared 
 in - house ( 0.5 nm final concentration ) or his tagged kdm4a prepared in - house ( 1.5 
 nm final concentration ) before the addition of 2.5 l of peptide ( artkqtark(me3)stggkaprkqla - ggk - biotin , genecust , 30 nm final concentration ) .
the plate 
 was sealed and centrifuged at 1000 rpm for 1 min before being left 
 for 20 min at room temperature .
the reaction was stopped with the 
 addition of 2.5 l of edta ( 30 mm final concentration ) before the addition 
 of 2.5 l of alphascreen detection reagents ( no .
the detection reagents containing igg mouse acceptor 
 beads , streptavidin donor beads ( 20 g / ml final concentration ) , and an antidimethyl 
 h3k9 antibody ( no .
ab1220 , 0.5 nm final concentration ) were preincubated for 1 h 
 in detection buffer containing hepes ( 50 mm , ph 7.5 ) , bsa ( 0.1% w / v ) , 
 and tween 20 ( 0.01% v / v ) prior to addition to the plate .
plates were 
 incubated at room temperature for 1 h in the dark before being read 
 on an envision multilabel reader ( perkinelmer life sciences ) , using 
 an alphascreen format .
all data analysis was carried out using the studies package from dotmatics 
 ( bishops stortford , uk ) .
the percentage inhibition was calculated 
 relative to blank wells ( containing no enzyme and 2% dmso ) and total 
 wells ( containing all reagents and 2% dmso ) .
all buffer components were 
 from sigma - aldrich unless otherwise stated and were of the highest 
 purity available .
bovine serum albumin fraction v was essentially 
 free from fatty acids and globulins ( sigma - aldrich , code a7030 ) .
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic 
 acid ( hepes ) buffer was from life technologies ( code bi8181 ) .
the 
 biotinylated peptide ligand substrate used for the kdm5 assay ( h3(121)k4-me3-ggk biotin ) was from anaspec ( code 64192 ) .
anti h3k4me2 antibody was from cell signaling technology ( code 9725s ) , 
 and alphascreen general igg detection kit was from perkinelmer . alphascreen 
 assay buffer ( 50 mm hepes ph 7.5 , 0.1% bsa , 0.01% tween-20 ) was prepared 
 fresh each week , filter sterilized through a 0.2 m filter , 
 and stored at 4 c .
ferrous ammonium sulfate ( fas ) was dissolved 
 fresh each day to 400 mm ( 156.856 mg / ml ) in 20 mm hcl and diluted 
 to 1 mm in deionized water .
2og was prepared fresh each day by dissolving 
 to 10 mm ( 1.901 mg / ml ) in deionized water .
l - ascorbic acid 
 ( l - aa ) was dissolved to 50 mm ( 8.806 mg / ml ) in deionized water .
compound 
 dispensing was performed using an echo-550 acoustic dispenser ( labcyte ) , 
 and all reagent dispenses were performed using a multidrop combi reagent 
 dispenser ( thermo scientific ) equipped with a small tube dispensing 
 cassette ( thermo scientific ) unless otherwise stated .
all kdm5 alphascreen 
 assays were carried out in white 384-well proxiplates plus ( perkinelmer , 
 code 6008280 ) .
compounds for ic50 determination were dissolved 
 in 100% dmso to a concentration of 10 mm and an 11-point , 3-fold dilution 
 of each compound for ic50 was dry dispensed ( 100 nl ) into 
 384-well proxiplates using an echo-550 acoustic dispenser .
then 100 
 nl of dmso was dispensed into each well of column 12 and 2,4-pdca 
 ( 100 mm ) was dispensed into each well of column 24 .
kdm5b and kdm5c 
 were diluted to 4 nm in assay buffer , and 5 l of kdm5 enzyme 
 dispensed into each well of a 384-well proxiplate .
plates were sealed 
 with an aluminum foil , centrifuged at 1000 rpm for 5 s , and compound 
 allowed to preincubate with enzyme for 15 min at room temperature 
 before addition of peptide substrate .
substrate was prepared in assay 
 buffer ( 200 m l - aa , 20 m fas , 0.2 m h3k4me3 peptide , 10 m 2og ) and 5 l dispensed into each 
 well to initiate the enzyme reaction .
plates were sealed with an aluminum 
 foil centrifuged at 1000 rpm for 15 s , and the enzyme reaction incubated 
 at room temperature for 20 min .
enzyme reactions were stopped after 
 20 min by addition of 5 l of assay buffer containing edta ( 30 
 mm ) and nacl ( 1.2 m ) .
alphascreen detection reagent was prepared by 
 diluting alphascreen streptavidin donor and protein a acceptor beads 
 in assay buffer ( 0.08 mg / ml with respect to each bead ) containing 
 anti - h3k4me2 antibody ( 1:750 ) and preincubated for 1 h. 
 product h3k4me2 methyl mark was detected by addition of 
 5 l of detection reagent followed by 2 h incubation at room 
 temperature .
after 2 h , plates were read in a bmg labtech plate reader 
 equipped with an alphascreen 680 nm/570 nm optic module .
data were 
 normalized to the no enzyme ( 2,4-pdca ) control and ic50 determined from nonlinear regression curve fitting using graphpad 
 prism 5.0 .
an enzyme  substrate cocrystal structure of kdm4a ( pdb 3pdq ) was prepared for modeling using protein preparation wizard 
 in maestro , and all water molecules not 
 coordinating to the catalytic metal were removed . to propose predicted 
 binding modes of the hit series ,
glide ( grid - based ligand docking 
 with energetics ) was used for the docking 
 experiments .
the receptor grid was defined by a grid box of 30  
 30  30  with a default inner box ( 10  
 10  10  ) centered on the cocrystallized ligand 
 in pdb 3pdq . 

ligands were prepared using ligprep , applying 
 the opls_2005 force - field with possible tautomeric and ionization 
 states within ph range 5.09.0 generated using epik metal binding 
 state settings . using extra precision ( xp ) settings , flexible docking 
 of ligands
was constrained to the coordinates of the core , defined 
 as the terminal pyridine ring of the cocrystallized ligand in pdb 3pdq , with an rmsd tolerance 
 of 1.0  .
the docked poses with the lowest rmsd and state penalties 
 were selected as the preferred solutions . for kdm4a bound to compounds 15 , 16 , 37 , 40 ,
52d , 53a , 54a , 54j , and 58 : a previously established 
 n - terminally his - tagged kdm4a construct ( residues 1359 ) was produced in escherichia coli and purified by nickel affinity chromatography , followed by tag 
 removal with tev protease , reverse nickel affinity , and gel filtration . 

the protein was stored at 80 c at 10 mg / ml concentration 
 in a buffer containing 10 mm hepes ph 7.5 and 200 mm nacl .
purified 
 kdm4a was crystallized in the apo form at 18 c using the hanging - drop 
 vapor - diffusion method .
the crystallization drops were composed of 
 1.5 l of protein ( 7 mg / ml ) and 1.5 l of reservoir solution 
 containing 1216% ( w / v ) peg4000 and 0.1 m btp ph 7.5 , placed 
 over 800 l of reservoir solution .
plate - like crystals typically 
 grew in a week and were then soaked by addition of 0.75 l of 
 compound at 10200 mm in dmso directly to the drops , followed 
 by 448 h incubation at 18 c .
crystals were briefly transferred 
 to cryoprotectant solution containing 14% ( w / v ) peg4000 , 75 mm btp 
 ph 7.5 , and 25% ( v / v ) glycerol prior to freezing in liquid nitrogen .
for kdm4a bound to compound 30b : the same kdm4a protein 
 was produced , purified , and stored as described previously .
a complex of kdm4a with compound 30b was formed by mixing 30 mg / ml protein in 20 mm hepes ph 7.5 , 5% 
 ( v / v ) glycerol , 500 mm nacl , and 0.5 mm tcep with 100 mm compound 30b in dmso to a final concentration of 5 mm .
then , cocrystallization 
 was performed in sitting drop plates at 4 c by mixing 100 nl 
 of protein  ligand complex with 50 nl of reservoir solution 
 containing 0.1 m bis - tris ph 5.9 , 0.15 m ammonium sulfate , 13% ( w / v ) 
 peg3350 , placed over 20 l of reservoir solution . plate - like 
 crystals typically grew in a week . prior to data collection , crystals 
 were briefly transferred to a cryoprotectant solution consisting of 
 the precipitation buffer supplemented with 25% ( v / v ) ethylene glycol 
 and were subsequently flash cooled in liquid nitrogen . for structures 
 of kdm4d bound to compounds 30a and 44a : 
 dna encoding for kdm4d ( residues 1342 ) was cloned into an 
 in - house pet - based vector pnic28-bsa4 using ligation - independent cloning .
the resulting construct expressed 
 the desired proteins with an n - terminal his6-tag and tobacco 
 etch virus ( tev ) protease cleavage site ( extension mhhhhhhssgvdlgtenlyfq*sm ) . 

the protein was produced in e. coli and purified by nickel affinity chromatography , followed by tag 
 removal with tev protease , reverse nickel affinity , and gel filtration . 

the protein was stored at 80 c at 11 mg / ml in a buffer 
 containing 10 mm hepes ph 7.5 , 0.5 m nacl , 5% glycerol , and 0.5 m 
 tcep .
kdm4d was crystallized in sitting drop plates at 20 c 
 by mixing 100 nl of protein complex with 50 nl of reservoir solution 
 containing 0.1 m bis - tris ph 5.9 , 0.15 m ammonium sulfate , and 11% 
 ( w / v ) peg3350 , placed over 20 l of reservoir solution .
the 
 crystals were soaked with compound 30a or 44a by mixing 0.5 l of 10 mm compound ( in 9 parts ethylene glycol/1 
 part dmso ) with 1.5 l of reservoir solution and adding it to 
 the crystals .
the crystals were then flash frozen in liquid nitrogen 
 after incubating them for 3 h. for kdm5b bound to compound 52d : a kdm5b construct encoding regions phe26ile770 
 was amplified from an origene cdna clone and cloned into a pfastbac - derived 
 vector ( pfb - lic - bse ) containing a tobacco etch virus ( tev ) protease 
 cleavable n - terminal his6-tag .
the recombinant kdm5b ( residues 
 26770 ) construct was expressed in sf9 cells , with generation 
 of recombinant baculo viruses , insect cell culture , and infections 
 being performed according to the manufacturer s instructions 
 ( invitrogen ) .
the cells were harvested 72 h post infection , lysed 
 in a buffer containing 50 mm hepes ph 7.5 , 500 mm nacl , 10 mm imidazole , 
 5% glycerol , 0.5 mm tcep , and a protease inhibitor cocktail ( calbiochem ) 
 and purified using nickel affinity chromatography using a stepwise 
 gradient of imidazole . the eluted protein
was then incubated with 
 tev protease at 4 c overnight followed by size - exclusion chromatography 
 ( superdex 200 ) .
the tev protease and uncleaved protein were removed 
 using nickel affinity chromatography , and protein was concentrated 
 to 8.1 mg / ml and stored at 80 c .
kdm5b 
 was preincubated with 4 mm mncl2 before the protein was 
 transferred to crystallization plates .
crystals were obtained in drops 
 consisting of 100 nl of protein mix ( 8.1 mg / ml ) , 200 nl of a precipitant 
 consisting of 1.6 m na / k phosphate , 0.1 m hepes ph 7.5 , and 20 nl 
 of kdm5b seeds of crystals obtained from the same condition .
compound 
 was soaked into apo crystals of kdm5b for 5 min at a concentration 
 of 5 mm .
crystals were cryoprotected with mother liquor supplemented 
 with 25% ethylene glycol before they were flash - frozen in liquid nitrogen .
- ray data were 
 collected in - house at the institute of cancer research , london , uk , 
 on a rigaku frx - afc11-varimax cu - vhf - pilatus300 k , at the structural 
 genomics consortium , oxford , uk , on a bruker microstar generator equipped 
 with an apex ii detector , at diamond light source , oxfordshire , uk , 
 at beamlines i02 , i03 , i04 , and i04 - 1 , and at the european synchrotron 
 ray facility , grenoble , france , at beamline id23 . crystals of kdm4a 
 belonged to the space group p1211 ( or p21212 for kdm4a bound to compound 30b ) and diffracted to resolutions between 2.05 and 2.65  . 

crystals of kdm4d belonged to the space group p43212 and diffracted to resolutions between 1.41 
 and 1.88  .
the kdm5b crystal belonged to the space group p6522 and diffracted to a resolution of 2.35 
  .
all data sets were integrated with xds , scaled , and merged with aimless , except for data sets collected on the bruker system which were integrated 
 with saint ( version 8.3 , bruker axs inc . , 2013 ) and scaled with sadabs 
 ( version 2012/1 , bruker axs inc . , 2012 ) .
the structures were solved 
 by molecular replacement using phaser , and publicly 
 available kdm4a , kdm4d , and kdm5b structures ( pdb codes 2oq7 , 3dxt , and 5a1f , respectively ) with ligand and water molecules removed were 
 used as molecular replacement models .
ligand structures 
 were manually corrected and rebuilt in coot and refined with buster , phenix , or refmac in iterative 
 cycles .
the data collection and refinement statistics are presented in supporting 
 information , table s1 .
papp ( apparent permeability ) was determined 
 in the caco-2 human colon carcinoma cell line .
cells were maintained 
 ( dmem with 10% fetal bovine serum , penicillin , and streptomycin ) in 
 a humidified atmosphere with 5%co2/95% air for 10 days . 

cells were plated out onto a cell culture assembly plate ( millipore , 
 uk ) , and monolayer confluency was checked using a teer electrode prior 
 to the assay .
media was washed off and replaced in the appropriate 
 apical and basal wells with hbss buffer ( ph7.4 ) containing compound 
 ( 10 m , 1% dmso ) .
the caco-2 plate was incubated for 2 h at 
 37 c , and lucifer yellow was used to confirm membrane integrity 
 after the assay .
samples from the apical and basolateral chambers 
 were analyzed using waters tq - s lc - ms / ms .
full length cdna for kdm4a ( jmjd2a , 
 o75164 ) and kdm5b ( jarid1b , q9ugl1 ) were amplified by pcr and cloned 
 into the pdonr-221 vector using a gateway bp reaction . to produce 
 catalytically inactive kdms ,
residues involved in iron coordination 
 were mutated to alanine ( kdm5b , h499a / e501a ; kdm4a , h188a / e190a ) . 

mutations were introduced into the full length kdm gateway entry clones 
 using 15 cycles of the quikchange ii pcr protocol ( agilent technologies ) . 

mammalian expression constructs encoding n - terminal 
 3flag were constructed by a gateway lr recombination reaction between 
 pcdna5-frt / to-3flag destination vector ( pmid : 24412199 ) and the wild - type 
 or mutated kdm gateway entry clone .
the human cervical carcinoma 
 cell line hela was obtained from the american type cultures collection 
 and maintained in eagle s minimal essential media ( sigma - aldrich ) 
 supplemented with 10% heat - inactivated fbs ( life technologies ) .
cells 
 were transiently transfected with either the flag - tagged demethylase 
 or an inactive version containing mutations in the catalytic site 
 using lipofectamine 3000 ( life technologies ) .
four hours after transfection , 
 the cells were treated with serial dilutions of compound 54k for 24 h. the cells were then fixed and stained with a mouse monoclonal 
 anti - flag ( sigma f3165 ) and with either a rabbit anti - h3k9me3 1 h 
 at rt ( abcam ab8898 ) or a rabbit anti - h3k4me3 overnight at 4 c 
 ( diagenode c15410003 ) .
the secondary antibodies were goat antirabbit 
 alexa fluor 488 ( life technologies a11034 ) and goat antimouse alexa 
 fluor 568 ( life technologies a21124 ) .
image acquisition was conducted 
 using the operetta high content imaging system ( perkinelmer ) , and 
 image analysis was performed with the columbus software ( perkinelmer ) . 

after the dapi - stained nuclei were automatically identified , a minimum 
 threshold for anti - flag intensity was set so that only cells highly 
 expressing the demethylase were analyzed .
cyp450 and ao - mediated metabolism 
 of kdm4 inhibitor compounds were assessed by incubation of compound 
 ( 1 m concentration ) in mouse and human liver microsomes ( mlm , 
 hlm ) and mouse liver cytosol ( 0.5 mg / ml ) ( tebu - bio ) respectively in 
 0.1 m pbs at 37 c .
microsomal incubations 
 were initiated by addition of nadph and udpga for phase i and ii reactions . 

cytosol incubations were initiated by kdm4 inhibitor compounds in 
 the presence and absence of ao inhibitor raloxifene .
compound levels 
 were measured over time ( 0 , 5 , 10 , 15 , and 30 min ) to calculate clearance 
 by removing aliquots of the incubate at specific time points and quenching 
 into acetonitrile with internal standard ( is ) and centrifugation at 
 3000 rpm for 30 min at 4 c .
flash column chromatography was performed using merck silica 
 gel 60 ( 0.0250.04 mm ) .
thin layer chromatography was performed 
 using merck millipore tlc silica gel 60 f254 aluminum sheets 
 and visualized by uv ( 254 and 280 nm ) , iodine , and kmno4 .
column chromatography was also performed on a flashmaster personal 
 unit using isolute flash silica columns or a biotage isolera purification 
 system using biotage kp - snap cartridges .
ion exchange chromatography 
 was performed using acidic isolute flash scx - ii cartridges or basic 
 isolute flash nh2 cartridges .
h nmr spectra 
 were recorded on either a bruker avance-500 or bruker avance-400 nmr 
 machine .
samples were prepared as solutions in a deuterated solvent 
 and referenced to the appropriate internal nondeuterated solvent peak 
 or tetramethylsilane .
analysis was performed on a waters lct with a waters alliance 2795 
 separations module and waters 2487 dual wavelength absorbance detector 
 coupled to a waters / micromass lct time - of - flight mass spectrometer 
 with esi source .  
method a : analytical separation was carried 
 out at 30 c on a merck purospher star column ( rp-18e , 30 mm 
  4 mm ) using a flow rate of 1.5 ml / min in a 4 min gradient elution 
 with detection at 254 nm .
the mobile phase was a mixture of methanol 
 ( solvent a ) and water ( solvent b ) , both containing formic acid at 
 0.1% .
gradient elution was as follows : 10:90 ( a / b ) to 90:10 ( a / b ) 
 over 2.5 min , 90:10 ( a / b ) for 1 min , and then reversion back to 10:90 
 ( a / b ) over 0.3 min , finally 10:90 ( a / b ) for 0.2 min .
analysis was 
 also performed on an agilent 1200 series hplc and diode array detector 
 coupled to a 6210 time - of - flight mass spectrometer with dual multimode 
 apci / esi source .  
method b : analytical separation was carried 
 out at 30 c on a merck purospher star column ( rp-18e , 30 mm 
  4 mm ) using a flow rate of 1.5 ml / min in a 4 min gradient elution 
 with detection at 254 nm .
the mobile phase was a mixture of methanol 
 ( solvent a ) and water ( solvent b ) , both containing formic acid at 
 0.1% .
gradient elution was as follows : 10:90 ( a / b ) to 90:10 ( a / b ) 
 over 2.5 min , 90:10 ( a / b ) for 1 min , and then reversion back to 10:90 
 ( a / b ) over 0.3 min , finally 10:90 ( a / b ) for 0.2 min .  
method 
 c : analytical separation was carried out at 40 c on a merck 
 purospher star column ( rp-18e , 30 mm  4 mm ) using a flow rate 
 of 3 ml / min in a 2 min gradient elution with detection at 254 nm . 

the mobile phase was a mixture of methanol ( solvent a ) and water ( solvent 
 b ) , both containing formic acid at 0.1% .
gradient elution was as follows : 
 10:90 ( a / b ) to 90:10 ( a / b ) over 1.25 min , 90:10 ( a / b ) for 0.5 min , 
 and then reversion back to 10:90 ( a / b ) over 0.15 min , finally 10:90 
 ( a / b ) for 0.1 min .  
method d : analytical separation was carried 
 out at 40 c on a merck chromolith flash column ( rp-18e , 25 mm 
  2 mm ) using a flow rate of 1.5 ml / min in a 2 min gradient elution 
 with detection at 254 nm .
the mobile phase was a mixture of methanol 
 ( solvent a ) and water ( solvent b ) , both containing formic acid at 
 0.1% .
gradient elution was as follows : 5:95 ( a / b ) to 100:0 ( a / b ) over 
 1.25 min , 100:0 ( a / b ) for 0.5 min , and then reversion back to 5:95 
 ( a / b ) over 0.05 min , finally 5:95 ( a / b ) for 0.2 min .  
method 
 e : analytical separation was carried out at 30 c on a merck 
 chromolith flash column ( rp-18e , 25 mm  2 mm ) using a flow rate 
 of 0.75 ml / min in a 4 min gradient elution with detection at 254 nm . 

the mobile phase was a mixture of methanol ( solvent a ) and water ( solvent 
 b ) , both containing formic acid at 0.1% .
gradient elution was as follows : 
 5:95 ( a / b ) to 100:0 ( a / b ) over 2.5 min , 100:0 ( a / b ) for 1 min , and 
 then reversion back to 5:95 ( a / b ) over 0.1 min , finally 5:95 ( a / b ) 
 for 0.4 min .
analysis was also performed on a waters acquity uplc 
 and diode array detector coupled to a waters g2 qtof mass spectrometer 
 fitted with a multimode esi / apci source .  
method f : analytical 
 separation was carried out at 30 c on a phenomenex kinetex xb - c18 
 column ( 30 mm  2.1 mm , 1.7  , 100 a ) using a flow rate 
 of 0.5 ml / min in a 2 min gradient elution with detection at 254 nm . 
 the mobile phase was a mixture of methanol ( solvent a ) and water ( solvent 
 b ) , both containing formic acid at 0.1% .
gradient elution was as follows : 
 10:90 ( a / b ) to 90:10 ( a / b ) over 1.25 min , 90:10 ( a / b ) for 0.5 min , 
 and then reversion back to 10:90 ( a / b ) over 0.15 min , finally 10:90 
 ( a / b ) for 0.1 min .  
method g : analytical separation was carried 
 out at 30 c on a phenomenex kinetex xb - c18 column ( 30 mm  
 2.1 mm , 1.7  , 100 a ) using a flow rate of 0.3 ml / min in a 4 
 min gradient elution with detection at 254 nm .
the mobile phase was 
 a mixture of methanol ( solvent a ) and water ( solvent b ) , both containing 
 formic acid at 0.1% .
gradient elution was as follows : 10:90 ( a / b ) 
 to 90:10 ( a / b ) over 3 min , 90:10 ( a / b ) for 0.5 min , and then reversion 
 back to 10:90 ( a / b ) over 0.3 min , finally 10:90 ( a / b ) for 0.2 min .  
method h : analytical separation was carried out at 30 c on 
 a phenomenex kinetex c18 column ( 30 mm  2.1 mm , 2.6  , 
 100 a ) using a flow rate of 0.5 ml / min in a 2 min gradient elution 
 with detection at 254 nm . the mobile phase was a mixture of methanol 
 ( solvent a ) and water ( solvent b ) , both containing formic acid at 
 0.1% .
gradient elution was as follows : 10:90 ( a / b ) to 90:10 ( a / b ) 
 over 1.25 min , 90:10 ( a / b ) for 0.5 min , and then reversion back to 
 10:90 ( a / b ) over 0.15 min , finally 10:90 ( a / b ) for 0.1 min .  
method i : analytical separation was carried out at 30 c on 
 a phenomenex kinetex c18 column ( 30 mm  2.1 mm , 2.6  , 
 100 a ) using a flow rate of 0.3 ml / min in a 4 min gradient elution 
 with detection at 254 nm .
the mobile phase was a mixture of methanol 
 ( solvent a ) and water ( solvent b ) , both containing formic acid at 
 0.1% .
gradient elution was as follows : 10:90 ( a / b ) to 90:10 ( a / b ) 
 over 3 min , 90:10 ( a / b ) for 0.5 min , and then reversion back to 10:90 
 ( a / b ) over 0.3 min , finally 10:90 ( a / b ) for 0.2 min .
analysis was 
 also performed on a waters system equipped with a waters 2545 binary 
 gradient module , a waters sq detector 2 , waters 2489 uv / visible detector , 
 and a waters 2424 els detector .  
method j : analytical separation 
 was carried out on a kinetex 5u evo c18 column ( 100 mm  3.0 
 mm , 100 a ) using a flow rate of 2 ml / min in a 3 min gradient elution . 

the mobile phase was a mixture of 93% h2o , 5% acetonitrile , 
 and 2% of 0.5 m ammonium acetate adjusted to ph 6 with glacial acetic 
 acid ( solvent a ) and 18% h2o , 80% acetonitrile , and 2% 
 of 0.5 m ammonium acetate adjusted to ph 6 with glacial acetic acid 
 ( solvent b ) .
gradient elution was as follows : 95:5 ( a / b ) 0.35 min , 
 95:5 ( a / b ) to 5:95 ( a / b ) over 1 min , 5:95 ( a / b ) over 0.75 min , and 
 then reversion back to 95:5 ( a / b ) over 0.1 min and 95:5 ( a / b ) over 
 0.8 min .  
method k : analytical separation was carried out on 
 a chromolith performance rp-18e column ( 100 mm  2.0 mm ) using 
 a flow rate of 1.5 ml / min in a 5 min gradient elution .
the mobile 
 phase was a mixture of 93% h2o , 5% acetonitrile , and 2% 
 of 0.5 m ammonium acetate adjusted to ph 6 with glacial acetic acid 
 ( solvent a ) and 18% h2o , 80% acetonitrile and 2% of 0.5 
 m ammonium acetate adjusted to ph 6 with glacial acetic acid ( solvent 
 b ) .
gradient elution was as follows : 95:5 ( a / b ) over 0.5 min , 95:5 
 ( a / b ) to 5:95 ( a / b ) over 2 min , 5:95 ( a / b ) over 1 min , then reversion 
 back to 95:5 ( a / b ) over 0.1 min and 95:5 ( a / b ) over 1.4 min .  
method l : analytical separation was carried out on a waters sunfire 
 c18 column ( 4.6 mm  150  4.6 mm , 100 a ) using a flow rate 
 of 1 ml / min in a 20 min gradient run .
the mobile phase was a mixture 
 of 99.99% water and 0.01% cf3co2h ( solvent a ) 
 and 99.99% acetonitrile and 0.01% cf3co2h ( solvent 
 b ) .
gradient elution was as follows : 100:0 ( a / b ) over 2 min , 100:0 
 ( a / b ) to 0:100 ( a / b ) over 16 min and 0:100 ( a / b ) over 2 min .
all compounds 
 submitted for biological testing were determined to be > 95% pure 
 by methods a 
lc - hrms was performed using either an agilent 1200 series hplc 
 and diode array detector coupled to a 6210 time - of - flight mass spectrometer 
 with dual multimode apci / esi source ( methods b and e ) or a waters 
 acquity uplc and diode array detector coupled to a waters g2 qtof 
 mass spectrometer fitted with a multimode esi / apci source ( methods 
 g and i ) or on an agilent 1290 uhplc system coupled with an agilent 
 esi - qtof 6530 mass spectrometer ( method j ) .
lc - hrms methods b and 
 e referenced to caffeine [ m + h ] 195.087652 , reserpine 
 [ m + h ] 609.280657 ( method b ) , or hexakis ( 2,2-difluoroethoxy)phosphazene 
 [ m + h ] 622.02896 ( methods b and e ) and hexakis(1h,1h,3h - tetrafluoropentoxy)phosphazene 
 [ m + h ] 922.009798 .
lc - hrms methods g and i referenced 
 to leucine enkephalin fragment ion [ m + h ] 397.1876 .
lc - hrms 
 method j referenced to hexakis ( 2,2-difluoroethoxy)phosphazene [ m 
 + h ] 622.02896 and hexakis(1h,1h,3h - tetrafluoropentoxy)phosphazene [ m 
 + h ] 922.009798 .
cesium carbonate 
 ( 3 equiv ) was added to a mesylate intermediate freshly made from alcohol 47 ( 1 equiv ) and amine ( 2 equiv ) in 
 anhydrous dmf under n2 .
the reaction mixture was stirred 
 for 15 h at 90 c and monitored by lcms .
the combined 
 organic layers were washed with saturated brine solution , dried over 
 mgso4 , and concentrated in vacuo to give the crude material 
 which was purified by biotage column chromatography ( see individual 
 compounds for details of the eluent used ) .
triethylamine 
 ( 2 equiv ) was added to a solution of mesylate intermediate freshly made from alcohol 47 ( 1 equiv ) and amine [ or amine hydrochloride ] 
 ( 1.2 equiv ) in anhydrous dmf under n2 .
the reaction mixture 
 was heated at 50 c for 15 h and monitored by lcms . when the 
 reaction had gone to completion ,
the combined 
 organic layers were washed with saturated licl solution and saturated 
 brine solution , dried over mgso4 , and concentrated in vacuo 
 to give the crude material which was purified by biotage column chromatography 
 ( see individual compounds for details of the eluent used ) . to a microwave vial was 
 added the required aldehyde ( 1 equiv ) and corresponding amine ( 1.5 
 equiv ) and the flask purged with argon .
anhydrous 1,2-dichloroethane 
 ( 4 ml ) was then added , the mixture was stirred to allow dissolution 
 followed by the addition of sodium triacetoxyborohydride ( 1.6 equiv ) , 
 the vial was capped , and the mixture was stirred at room temperature 
 overnight .
once the reaction was deemed complete by lcms analysis , 
 the mixture was directly absorbed unto silica gel and purified by 
 flash column chromatography to afford the requisite amine ( workup 
 procedure a ) .
alternatively , the mixture was concentrated to afford 
 an amorphous solid and passed through a plug of silica eluting with 
 40% meoh / ch2cl2 to afford an oil which was used 
 without further purification ( workup procedure b ) .
hydrochloric acid ( 6 
 m , 6090 equiv ) was added to a solution of sem protected material 
 ( 1 equiv ) in thf ( 0.1 m ) .
the reaction mixture was stirred at 5060 
 c for between 3 and 8 h and monitored by lcms .
following completion 
 of the reaction , the reaction mixture was concentrated in vacuo and 
 purified by biotage column chromatography on a kp - nh snap column unless 
 otherwise stated ( see individual compounds for details of the eluent 
 used ) .
the product obtained from column chromatography was triturated 
 with et2o to give the pure product . to a microwave vial 

was added the sem protected starting material ( 1 equiv ) , 1,4-dioxane 
 ( 3 ml ) , and distilled water ( 1 ml ) followed by the dropwise addition 
 of hcl in dioxane ( 4 m , 25 equiv ) .
the vial was capped and the mixture 
 stirred at 50 c until analysis by lcms indicated complete conversion 
 to the product .
the mixture was then concentrated and passed through 
 an scx-2 cartridge washing initially with meoh and then nh3/meoh .
the basic wash was concentrated and triturated with et2o to afford a solid . if necessary , the solid was further purified 
 by flash column chromatography on a snap kp - nh column eluting with 
 040% etoh / ch2cl2 . to a mixture of 3-(ethylsulfonyl)benzoic acid ( 0.098 g , 
 0.46 mmol , 1 equiv ) , 1-hydroxybenzotriazole hydrate ( 0.068 g , 0.50 
 mmol , 1.1 equiv ) , edci ( 0.105 g
, 0.55 mmol , 1.2 equiv ) , and anhydrous 
 ch2cl2 ( 4 ml , 0.12 m ) was added 2-amino-4-(2-pyridyl)thiazole ( 0.085 g , 0.48 mmol , 1.05 equiv ) .
the reaction mixture was stirred 
 at room temperature for 4 h under argon , then diluted with ch2cl2 ( 50 ml ) and washed with a saturated nahco3 solution ( 2  20 ml ) and saturated brine solution ( 20 
 ml ) , dried ( na2so4 ) , and concentrated in vacuo . 

this residue was absorbed on silica gel and the free running powder 
 was placed on a 20 g isolute silica ii column which was eluted with 
 30% etoac in ch2cl2 , 70% etoac in ch2cl2 , and finally 2% meoh in etoac / ch2cl2 ( v / v ; 4:1 ) .
the title compound was obtained as a white solid 
 ( 0.068 g , 40% ) .
h nmr ( 500 mhz , dmso - d6 )  1.15 ( t , j = 7.2 hz , 3h ) , 
 3.42 ( q , j = 7.2 hz , 2h ) , 7.36 ( ddd , j = 1.2 , 4.8 , 7.5 hz , 1h ) , 7.87 ( t , j = 7.9 hz , 1h ) , 
 7.91 ( td , j = 1.9 , 7.8 hz , 1h ) , 7.94 ( s , 1h ) , 8.02 
 ( d , j = 7.8 hz , 1h ) , 8.15 ( dt , j = 1.3 , 7.8 hz , 1h ) , 8.44 ( dt , j = 1.3 , 7.8 hz , 
 1h ) , 8.618.64 ( m , 2h ) , 13.15 ( s , 1h ) .
lc - ms ( method b , esi , m / z ) tr = 2.50 
 min 374 ( m + h ) . hrms ( method b ) : found .
374.0610 ; 
 calculated for c17h16n3o3s2 ( m + h ) , 374.0628 . to a mixture of methyl 2-bromoisonicotinate 
 ( 0.80 g , 3.7 mmol , 1 equiv ) , tributyl(1-ethoxyvinyl)stannane ( 1.44 
 g , 4.0 mmol , 1.1 equiv ) , and 1,4-dioxane ( 20 ml , 0.19 m ) was added 
 tetrakis(triphenylphosphine)palladium ( 0.300 g , 0.173 mmol , 5 mol 
 % ) .
the reaction mixture was heated at reflux ( 120 c ) for 18 
 h under nitrogen , cooled to room temperature , and then concentrated 
 in vacuo .
the residue was purified by silica gel column chromatography 
 eluting with ch2cl2:hexane ( 2:1 ) to afford the 
 desired product as a yellow oil which solidified on standing ( 0.500 
 g , 65% ) .
h nmr ( 500 mhz , cdcl3 )  1.34 
 ( t , j = 7.0 hz , 3h ) , 3.95 ( s , 3h ) , 3.98 ( q , j = 7.0 hz , 2h ) , 4.41 ( d , j = 2.1 hz , 1h ) , 
 5.45 ( d , j = 2.1 hz , 1h ) , 7.73 ( dd , j = 1.6 , 5.0 hz , 1h ) , 8.19 ( t , j = 1.0 hz , 1h ) , 8.69 
 ( dd , j = 0.8 , 5.0 hz , 1h ) .
lc - ms ( method c ; esi , m / z ) tr = 1.4 
 min 208 ( m + h ) .
a mixture of methyl 2-(1-ethoxyvinyl)isonicotinate 
 ( 0.500 g , 2.41 mmol ) and 1-bromopyrrolidine-2,5-dione ( 0.429 g , 2.41 
 mmol ) was suspended in 10% water in thf : h2o ( 2:0.2 ml ) . 

the reaction was stirred at room temperature for 1 h. volatiles were 
 removed in vacuo and the residue purified by flash silica gel column 
 chromatography eluting with 30% hexane in dichloromethane .
the pure 
 fractions provided the desired product as a pale - brown oil which solidified 
 on standing ( 0.38 g , 61% ) .
h nmr ( 500 mhz , cdcl3 )  4.00 ( s , 3h ) , 4.84 ( s , 2h ) , 8.07 ( dd , j = 1.7 , 5.0 hz , 1h ) , 8.59 ( dd , j = 0.9 , 1.7 hz , 
 1h ) , 8.84 ( dd , j = 0.7 , 5.0 hz , 1h ) .
lc - ms ( method 
 c ; esi , m / z ) tr = 1.25 min 258 , 260 [ ( m + h ) , br isotopic 
 pattern ] . for the synthesis of compound 25 ,
this intermediate 
 was prepared from methyl 2-acetylpyridine-4-carboxylate as follows : 
 to a  paste like  mixture of methyl 2-acetylpyridine-4-carboxylate 
 ( 1.79 g , 10.0 mmol ) , acetic acid ( 12.5 ml ) , and 48% aqueous hbr ( 1.12 
 ml , 10.0 mmol ) cooled in an ice - bath , bromine ( 0.57 ml , 11.0 mmol ) 
 was added dropwise
. the ice - bath was removed , and stirring was continued 
 at room temperature for 1 h under argon , then at 75 c for 1.5 
 h under argon .
the reaction mixture was cooled to room temperature 
 with the aid of an ice - bath , diluted with thf ( 12 ml ) , and stirred 
 at room temperature for 20 min and allowed to stand overnight at room 
 temperature ; the volatiles were then removed under vacuo .
the residue 
 was diluted with etoac ( 130 ml ) , and to this solution a saturated 
 nahco3 solution ( 50 ml ) was carefully added .
the two layers 
 were separated , and the organic layer was carefully washed with more 
 saturated nahco3 solution ( 30 ml ) and water ( 20 ml ) , dried 
 ( na2so4 ) , and concentrated in vacuo . the liquid 
 residue was absorbed onto silica gel and placed on a 50 g isolute 
 silica ii column which was eluted with 5% , 10% , and finally 15% etoac 
 in hexane to provide in order of elution the dibrominated product , 
 and the title compound as an oily residue which solidified on standing 
 at room temperature ( 0.98 g , 38% ) .
h nmr and lc - ms as 
 reported above . a mixture of methyl 2-(2-bromoacetyl)isonicotinate 
 ( 0.100 g , 0.387 mmol , 1 equiv ) , n - boc - thiourea ( 0.068 
 g , 0.387 mmol , 1 equiv ) , and triethylamine ( 0.04 g , 0.4 mmol , 1.05 
 equiv ) was suspended in ethanol ( 10 ml , 0.04 m ) and heated under reflux 
 for 1 h. the reaction was concentrated in vacuo and the residue purified 
 by flash silica gel column chromatography eluting with 5% etoac in 
 ch2cl2 to give the product as a yellow oil which 
 solidified on standing ( 0.072 g , 56% ) .
h nmr ( 500 mhz , 
 cdcl3 )  1.50 ( s , 9h ) , 3.98 ( s , 3h ) , 7.76 ( s , 1h ) , 
 7.78 ( dd , j = 1.6 , 5.0 hz , 1h ) , 8.47 ( s , 1h ) , 8.62 
 ( br s , 1h ) , 8.76 ( d , j = 4.9 hz , 1h ) .
lc - ms ( method 
 c ; esi , m / z ) tr = 1.56 min 336 ( m + h ) .
a solution of 4 m hcl in dioxane ( 1 
 ml ) was added to methyl 2-(2-((tert - butoxycarbonyl)amino)thiazol-4-yl)isonicotinate 
 ( 0.022 g , 0.066 mmol ) .
the mixture was stirred at room temperature 
 for 1 h. volatiles were removed in vacuo , and the residue was passed 
 through an isolute - nh2 cartridge eluting with methanol 
 to afford a brown solid which was dissolved in thf ( 2 ml ) and naoh 
 solution ( 3.3 m , 0.3 ml ) . after stirring at room temperature for 1 
 h , acetic acid ( 0.06 g , 1.0 mmol ) was added and the solution was evaporated 
 to dryness .
water ( 1 ml ) was added to the residue and a white powder 
 separated out which was filtered and dried ( 8 mg , 55% ) .
h nmr ( 500 mhz , dmso - d6 )  7.18 
 ( s , 2h ) , 7.31 ( s , 1h ) , 7.66 ( dd , j = 1.6 , 5.0 hz , 
 1h ) , 8.30 ( s , 1h ) , 8.68 ( d , j = 5.0 hz , 1h ) , 14.00 
 ( br s , 1h ) .
lc - ms ( method c ) ; esi , m / z ) tr = 0.56 min 222 ( m + h ) .
hrms ( method b ) : found , 222.0333 ; calculated for c9h8n3o2s ( m + h ) , 222.0332 . to a 
 solution of methyl 3-amino-2-chloroisonicotinate ( 1.00 g , 5.36 mmol , 
 1 equiv ) in methanol ( 13 ml ,
0.4 m ) was added 1 m aqueous sodium hydroxide 
 ( 14.4 ml , 14.4 mmol ) , and the reaction was stirred at room temperature 
 for 45 min .
methanol was removed in vacuo , and the remaining aqueous 
 solution was acidified to ph 5.5 by addition of 1 m hcl . a white precipitate 
 formed which was filtered and dried ( 0.715 g , 77% ) .
h 
 nmr ( 500 mhz , dmso - d6 )  6.80 ( br 
 s , 2h ) , 7.58 ( d , j = 5.0 hz , 1h ) , 7.61 ( d , j = 5.0 hz , 1h ) , 13.80 ( br s , 1h ) .
lc - ms ( method c ; esi , m / z ) tr = 0.83 
 min 173 , 175 [ ( m + h ) cl isotopic pattern ] . to a stirred 
 solution of 3-amino-2-chloroisonicotinic acid ( 0.750 g , 4.35 mmol ) 
 in thionyl chloride ( 10 ml ) was added 2 drops of dmf and the reaction 
 heated at reflux under nitrogen for 2 h. volatiles were removed and 
 the residue was suspended in anhydrous thf ( 10 ml ) and was slowly 
 added to a solution of ammonium hydroxide ( 10 ml ) cooled in an ice 
 bath and the reaction stirred at room temperature for 1.5 h. etoac 
 ( 40 ml ) and ch2cl2 ( 10 ml ) were added and the 
 layers separated . the organic solution was dried and concentrated 
 in vacuo .
the residue obtained was purified by flash silica gel column 
 chromatography eluting with 20% hexane in ethyl acetate to afford 
 the desired product as a white powder ( 0.664 g , 89% ) .
h nmr ( 500 mhz , dmso - d6 )  6.76 
 ( br s , 2h ) , 7.50 ( d , j = 5.1 hz , 1h ) , 7.61 ( d , j = 5.1 hz , 1h ) , 7.67 ( br s , 1h ) , 8.18 ( br s , 1h ) .
lc - ms 
 ( method a ; esi , m / z ) tr = 0.52 min 172 , 174 [ ( m + h ) cl 
 isotopic pattern ] .
3-amino-2-chloroisonicotinamide ( 0.60 g , 3.5 mmol ) in anhydrous 
 triethoxymethane ( 15 ml ) was heated at reflux ( 160 c ) for 18 
 h. the reaction was cooled to room temperature , and the pale - white 
 powder was filtered and washed with et2o ( 2  5 ml ) 
 then dried to give the product as a white solid ( 0.52 g , 82% ) .
h nmr ( 500 mhz , dmso - d6 )  
 7.96 ( d , j = 5.1 hz , 1h ) , 8.32 ( s , 1h ) , 8.42 ( d , j = 5.1 hz , 1h ) , 12.85 ( br s , 1h ) .
lc - ms ( method h ; esi , m / z ) tr = 0.66 
 min 182 , 184 [ ( m + h ) cl isotopic pattern ] .
hrms 
 ( method i ) : found , 182.0125 ; calculated for c7h4n3clo ( m + h ) , 182.0121 .
8-chloropyrido[3,4-d]pyrimidin-4(3h)-one ( 1.87 g , 10.3 mmol , 1 equiv ) was dissolved in dry dmf ( 15 ml , 
 0.7 m ) .
the solution was heated to 60 c , and potassium carbonate 
 ( 2.85 g , 20.6 mmol , 2 equiv ) was added .
after 5 min of stirring , ( 2-(chloromethoxy)ethyl)trimethylsilane 
 ( 2.06 g , 12.36 mmol , 1.2 equiv ) was slowly added .
stirring was continued 
 at 60 c for 4 h , the reaction cooled to room temperature , and 
 etoac ( 60 ml ) was added .
the heterogeneous mixture was washed with 
 water ( 2  60 ml ) , dried ( na2so4 ) , and 
 concentrated in vacuo .
the crude product was purified by silica gel 
 column chromatography eluting with 3% etoac in ch2cl2 to provide the title compound as a white powder ( 2.7 g , 84% ) .
h nmr ( 500 mhz , cdcl3 )  0.01 ( s , 9h ) , 0.971.00 
 ( m , 2h ) , 3.673.71 ( m , 2h ) , 5.46 ( s , 2h ) , 8.06 ( d , j = 5.1 hz , 1h ) , 8.34 ( s , 1h ) , 8.49 ( d , j = 5.1 hz , 1 h ) . lc - ms ( method h ; esi , m / z ) tr = 1.4 min ( 100% purity ) 
 254 , 256 [ ( m  sem + tms ) , cl isotopic 
 pattern ) ] .
this compound was also prepared in lower yield ( 55 to 65% ) 
 by performing the reaction at room temperature .
tert - butylchlorodimethylsilane 
 ( 7.09 g , 47.0 mmol , 1.05 equiv ) was added to a solution of 1h - imidazole ( 3.35 g , 49.2 mmol , 1.1 equiv ) and 2-(1h - pyrazol-4-yl)ethanol ( 5.02 g , 44.8 mmol , 1 equiv ) in dmf 
 ( 50 ml , 0.9 m ) at room temperature .
the resultant solution was stirred 
 for 30 min at room temperature and monitored by lcms .
on completion , 
 the reaction mixture was diluted with water ( 100 ml ) and extracted 
 three times with etoac ( 3  100 ml ) .
the combined organic layers 
 were washed with saturated lithium chloride solution ( 150 ml ) and 
 saturated brine solution ( 150 ml ) and dried over mgso4 . 

the filtrate was concentrated in vacuo to give the product as a clear , 
 pale - yellow oil ( 9.63 g , 95% ) .
h nmr ( 500 mhz , cdcl3 )  0.04 ( s , 6h ) , 0.91 ( s , 9h ) , 2.73 ( t , j = 6.8 hz , 2h ) , 3.76 ( t , j = 6.8 hz , 2h ) , 7.47 ( s , 
 2h ) , nh signal not observed .
lc - ms ( method c ; esi , m / z ) tr = 1.46 min 227 
 [ ( m + h ) ] .
hrms ( method b ) : found , 227.1580 ; calculated 
 for c11h23n2osi ( m + h ) , 227.1574 .
note : when attempted at scale , these reactions were carried out 
 in batches , generally using 150 mg portions of 8-chloro-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3h)-one per microwave vial . 
 cesium carbonate ( 1.33 g , 4.09 mmol , 2 equiv ) and 4-(2-((tert - butyldimethylsilyl)oxy)ethyl)-1h - pyrazole ( 920 
 mg , 4.06 mmol , 2 equiv ) were added to a microwave vial equipped with 
 a stirrer bar .
anhydrous mecn ( 14 ml , 0.15 m ) was added to the vial and 
 the reaction mixture stirred for 20 min under n2 at room 
 temperature .
the cap was then removed , 8-chloro-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3h)-one ( 637 mg , 2.04 mmol , 
 1 equiv ) was added , the vial resealed , and evacuated and flushed with 
 n2 .
the reaction mixture was then stirred at reflux for 
 18 h. solids were removed by filtration and rinsed with ch2cl2 ( 3  15 ml ) .
the filtrate was concentrated in 
 vacuo and purified by biotage column chromatography on a kp - sil snap 
 cartridge ( 30% etoac in cyclohexane ) to give the title product as 
 a pale - yellow solid ( 724 mg , 71% ) .
h nmr ( 500 mhz , cdcl3 )  0.01 ( s , 9h ) , 0.06 ( s , 6h ) , 0.91 ( s , 9h ) , 0.951.00 
 ( m , 2h ) , 2.80 ( t , j = 6.7 hz , 2h ) , 3.673.71 
 ( m , 2h ) , 3.84 ( t , j = 6.7 hz , 2h ) , 5.47 ( s , 2h ) , 
 7.79 ( s , 1h ) , 8.05 ( d , j = 5.1 hz , 1h ) , 8.29 ( s , 
 1h ) , 8.61 ( s , 1h ) , 8.63 ( d , j = 5.1 hz , 1h ) .
lc - ms 
 ( method c ; esi , m / z ) tr = 1.79 min 502 [ ( m + h ) ] .
hrms ( method 
 b ) : found , 502.2672 ; calculated for c24h40n5o3si2 ( m + h ) , 502.2670 .
hydrochloric acid ( 1 m , 5.5 ml , 10 equiv ) was added to a solution 
 of 8-(4-(2-((tert - butyldimethylsilyl)oxy)ethyl)-1h - pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3h)-one ( 274 mg , 0.546 mmol , 
 1 equiv ) in meoh ( 5.5 ml , 0.1 m ) at 0 c .
the reaction was stirred 
 at 0 c for 5 min , then diluted with etoac ( 20 ml ) , washed with 
 saturated nahco3 solution ( 20 ml ) and saturated brine solution 
 ( 20 ml ) , dried over mgso4 , and concentrated in vacuo to 
 give the product as a white solid ( 210 mg , 99% ) .
h nmr 
 ( 500 mhz , cdcl3 )  0.01 ( s , 9h ) , 0.961.00 
 ( m , 2h ) , 2.86 ( t , j = 6.3 hz , 2h ) , 3.673.72 
 ( m , 2h ) , 3.88 ( t , j = 6.3 hz , 2h ) , 5.46 ( s , 2h ) , 
 7.81 ( s , 1h ) , 8.07 ( d , j = 5.1 hz , 1h ) , 8.30 ( s , 
 1h ) , 8.60 ( s , 1h ) , 8.62 ( d , j = 5.1 hz , 1h ) , oh signal 
 not observed .
lc - ms ( method h ; esi , m / z ) tr = 1.34 min 388 [ ( m + h ) ] .
hrms ( method i ) : found , 388.1798 ; calculated for c18h26n5o3si ( m + h ) , 388.1805 .
methanesulfonic anhydride ( 94 mg , 0.542 mmol , 1.5 equiv ) 
 was added in one portion to a solution of alcohol 47 ( 140 
 mg , 0.361 mmol , 1 equiv ) and triethylamine ( 0.1 ml , 0.717 mmol , 2 
 equiv ) in anhydrous ch2cl2 ( 4 ml , 0.09 m ) at 
 0 c under n2 . the reaction mixture was stirred for 
 15 min at 0 c and monitored by lcms .
when the reaction was complete , 
 the reaction mixture was quenched with saturated nahco3 ( 10 ml ) solution and extracted with ch2cl2 ( 3  15 ml ) .
the combined organic layers were washed with saturated 
 brine solution ( 30 ml ) , dried over mgso4 , and concentrated 
 in vacuo to give 2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1h - pyrazol-4-yl)ethylmethanesulfonate 
 as a pale - yellow oil .
h nmr ( 500 mhz , cdcl3 )  
 0.01 ( s , 9h ) , 0.961.01 ( m , 2h ) , 2.99 ( s , 3h ) , 3.07 ( t , j = 6.7 hz , 2h ) , 3.683.72 ( m , 2h ) , 4.44 ( t , j = 6.7 hz , 2h ) , 5.47 ( s , 2h ) , 7.81 ( s , 1h ) , 8.09 ( d , j = 5.1 hz , 1h ) , 8.31 ( s , 1h ) , 8.64 ( d , j = 5.1 hz , 1h ) , 8.68 ( s , 1h ) .
lc - ms ( method h ; esi , m / z ) tr = 1.34 min 466 
 [ ( m + h ) ] .
according to general procedure 1 , cesium carbonate 
 ( 340 mg , 1.044 mmol ) , 2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1h - pyrazol-4-yl)ethylmethanesulfonate 
 ( 162 mg , 0.348 mmol ) , and 1-(4-fluorophenyl)-n - methylmethanamine 
 ( 0.1 ml , 0.759 mmol ) were reacted together in anhydrous dmf ( 2 ml ) . 

purification on a kp - sil snap cartridge ( 2% etoh in ch2cl2 ) gave the title product as a pale - yellow oil ( 27.7 mg , 
 29% ) .
h nmr ( 500 mhz , cdcl3 )  0.01 ( s , 
 9h ) , 0.951.01 ( m , 2h ) , 2.28 ( s , 3h ) , 2.69 ( t , j = 7.4 hz , 2h ) , 2.80 ( t , j = 7.4 hz , 2h ) , 3.54 ( s , 
 2h ) , 3.683.72 ( m , 2h ) , 5.47 ( s , 2h ) , 6.967.01 ( m , 
 2h ) , 7.287.31 ( m , 2h ) , 7.78 ( s , 1h ) , 8.06 ( d , j = 5.1 hz , 1h ) , 8.30 ( s , 1h ) , 8.54 ( s , 1h ) , 8.63 ( d , j = 5.1 hz , 1h ) .
lc - ms ( method c ; esi , m / z ) tr = 1.19 min 509 
 [ ( m + h ) ] .
hrms ( method b ) : found , 509.2514 ; calculated 
 for c26h34fn6o2si ( m + 
 h ) , 509.2496 . according to general procedure 4 , 8-(4-(2-((4-fluorobenzyl ) 
 ( methyl)amino)ethyl)-1h - pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]pyrimidin-4(3h)-one ( 42.46 mg , 0.083 
 mmol ) and hydrochloric acid ( 6 m , 1 ml ) were reacted together in thf 
 ( 1 ml ) .
purification on a kp - nh snap cartridge ( 25% etoh in ch2cl2 ) gave the product as a white solid ( 23.6 mg , 75% ) .
h nmr ( 500 mhz , meoh - d4 )  
 2.35 ( s , 3h ) , 2.732.78 ( m , 2h ) , 2.832.88 ( m , 2h ) , 
 3.66 ( s , 2h ) , 7.027.08 ( m , 2h ) , 7.347.39 ( m , 2h ) , 
 7.75 ( s , 1h ) , 8.04 ( d , j = 5.2 hz , 1h ) , 8.23 ( s , 
 1h ) , 8.55 ( d , j = 5.2 hz , 1h ) , 8.70 ( s , 1h ) , nh signal 
 not observed .
lc - ms ( method c ; esi , m / z ) tr = 0.68 min 379 [ ( m + h ) ] .
hrms ( method b ) : found , 379.1665 ; calculated for c20h20fn6o ( m + h ) , 379.1677 . according to general procedure 2 , 2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1h - pyrazol-4-yl)ethylmethanesulfonate 
 ( 96 mg , 0.206 mmol ) ,
4-(4-chlorophenyl)piperidine ( 48 mg , 0.247 mmol ) , 
 and triethylamine ( 0.057 ml , 0.409 mmol ) in dmf were reacted at 50 
 c overnight .
after workup , the resulting oil was passed through 
 a plug of silica eluting with 30% meoh / dcm to afford a yellow oil 
 which was used without further purification .
h nmr ( 500 
 mhz , cdcl3 )  0.01 ( s , 9h ) , 0.961.02 ( m , 
 2h ) , 1.751.90 ( m , 4h ) , 2.122.19 ( m , 2h ) , 2.482.57 
 ( m , 1h ) , 2.672.73 ( m , 2h ) , 2.812.86 ( m , 2h ) , 3.14 
 ( br s , 1h ) , 3.16 ( br s , 1h ) , 3.683.73 ( m , 2h ) , 5.47 ( s , 2h ) , 
 7.11 ( d , j = 8.4 hz , 2h ) , 7.18 ( d , j = 8.4 hz , 2h ) , 7.80 ( s , 1h ) , 8.08 ( d , j = 5.1 hz , 
 1h ) , 8.30 ( s , 1h ) , 8.57 ( s , 1h ) , 8.64 ( d , j = 5.1 
 hz , 1h ) .
lc - hrms ( method c ) tr = 1.28 
 min , m / z 565.2515 cl isotopic 
 pattern [ ( m + h ) ] . according to general 
 procedure 5
, 8-(4-(2-(4-(4-chlorophenyl)piperidin-1-yl)ethyl)-1h - pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]pyrimidin-4(3h)-one ( 73 mg , 0.129 mmol ) 
 was reacted with hcl in 1,4-dioxane ( 4 m , 0.81 ml , 3.23 mmol ) in 1,4-dioxane / water 
 at 50 c overnight . after workup and trituration ,
the title product 
 was obtained ( 16 mg , 29% ) as an off - white solid .
h nmr 
 ( 500 mhz , dmso- d6 )  1.65 ( qd , j = 12.3 , 3.5 hz , 2h ) , 1.76 ( d , j = 11.2 
 hz , 2h ) , 2.11 ( t , j = 7.5 hz , 2h ) , 2.532.65 
 ( m , 3h ) , 2.72 ( t , j = 7.5 hz , 2h ) , 3.09 ( d , j = 11.2 hz , 2h ) , 7.29 ( d , j = 8.3 hz , 
 2h ) , 7.34 ( d , j = 8.3 hz , 2h ) , 7.73 ( s , 1h ) , 7.98 
 ( d , j = 5.1 hz , 1h ) , 8.29 ( s , 1h ) , 8.42 ( s , 1h ) , 
 8.56 ( d , j = 5.1 hz , 1h ) , 12.44 ( br s , 1h ) .
lc - hrms 
 ( method i ) tr = 2.01 min , m / z found , 435.1696 ; calculated for c23h24cln6o [ ( m + h ) ] , 435.1700 cl 
 isotopic pattern .
according to general procedure 2 , triethylamine ( 0.06 
 ml , 0.411 mmol ) , 2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1h - pyrazol-4-yl)ethylmethanesulfonate 
 ( 95.7 mg , 0.206 mmol ) , and 4-(3,5-dichlorophenyl)piperidine ( 95 mg , 
 0.411 mmol ) were reacted together in anhydrous dmf ( 1 ml ) and purified 
 on kp - sil snap cartridge ( 4% [ 0.2 m nh3 in meoh ] in ch2cl2 ) to give the product as a pale - yellow oil ( 104 
 mg , 84% ) .
h nmr ( 500 mhz , cdcl3 )  0.00 
 ( s , 9h ) , 0.951.00 ( m , 2h ) , 1.78 ( qd , j = 
 12.0 , 3.1 hz , 2h ) , 1.831.89 ( m , 2h ) , 2.14 ( td , j = 11.8 , 1.9 hz , 2h ) , 2.452.54 ( m , 1h ) , 2.662.71 
 ( m , 2h ) , 2.792.85 ( m , 2h ) , 3.14 ( br d , j = 
 11.4 hz , 2h ) , 3.663.72 ( m , 2h ) , 5.46 ( s , 2h ) , 7.11 ( d , j = 1.9 hz , 2h ) , 7.19 ( t , j = 1.9 hz , 1h ) , 
 7.80 ( s , 1h ) , 8.06 ( d , j = 5.1 hz , 1h ) , 8.30 ( s , 
 1h ) , 8.57 ( s , 1h ) , 8.62 ( d , j = 5.1 hz , 1h ) .
lc - ms 
 ( method h ; esi , m / z ) tr = 1.32 min 599 , 601 , 603 [ ( m + h ) , cl isotopic pattern ] . hrms ( method i ) : found , 599.2128 ; calculated 
 for c29h37cl2n6o2si ( m + h ) , 599.2124 . according to general 
 procedure 4
, 8-(4-(2-(4-(3,5-dichlorophenyl)piperidin-1-yl)ethyl)-1h - pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]pyrimidin-4(3h)-one ( 40.6 mg , 0.068 mmol ) 
 and hydrochloric acid ( 6 m , 1 ml ) were reacted together in thf ( 1 
 ml ) .
purification on a kp - nh snap cartridge ( 40% etoh in ch2cl2 ) gave the product as a white solid ( 16.4 mg , 52% ) .
h nmr ( 500 mhz , dmso - d6 )  
 1.66 ( qd , j = 12.4 , 3.5 hz , 2h ) , 1.731.80 
 ( m , 2h ) , 2.06 ( t , j = 11.6 , 1.6 hz , 2h ) , 2.542.61 
 ( m , 3h ) , 2.71 ( t , j = 7.5 hz , 2h ) , 3.06 ( br d , j = 11.3 hz , 2h ) , 7.33 ( d , j = 1.9 hz , 
 2h ) , 7.41 ( t , j = 1.9 hz , 1h ) , 7.73 ( s , 1h ) , 7.98 
 ( d , j = 5.1 hz , 1h ) , 8.29 ( s , 1h ) , 8.41 ( s , 1h ) , 
 8.55 ( d , j = 5.1 hz , 1h ) 12.79 ( br s , 1h ) .
lc - ms 
 ( method h ; esi , m / z ) tr = 1.02 min 469 , 471 , 473 [ ( m + h ) , cl isotopic pattern ] . hrms ( method i ) : found , 469.1316 ; calculated 
 for c23h23cl2n6o ( m + 
 h ) , 469.1310 . according to general procedure 2 , 2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1h - pyrazol-4-yl)ethylmethanesulfonate 
 ( 79 mg , 0.170 mmol ) ,
4-(3-chlorophenyl)piperidine hydrochloride ( 59 
 mg , 0.255 mmol ) , and triethylamine ( 0.057 ml , 0.409 mmol ) in dmf were 
 reacted at 50 c for 3 days .
after workup , the oil obtained was 
 purified by flash column chromatography to afford the product ( 23 
 mg , 24% ) as a yellow oil .
h nmr ( 500 mhz , cdcl3 )  0.02 ( s , 9h ) , 0.961.01 ( m , 2h ) , 1.811.93 
 ( m , 5h ) , 2.152.24 ( m , 2h ) , 2.492.60 ( m , 1h ) , 2.692.77 
 ( m , 2h ) , 2.832.91 ( m , 2h ) , 3.153.21 ( m , 2h ) , 3.673.73 
 ( m , 2h ) , 5.48 ( s , 2h ) , 7.127.15 ( m , 1h ) , 7.177.20 
 ( m , 1h ) , 7.237.27 ( m , 1h ) , 7.81 ( br s , 1h ) , 8.08 ( d , j = 5.1 hz , 1h ) , 8.32 ( s , 1h ) , 8.59 ( br s , 1h ) , 8.64 ( br 
 s , 1h ) .
hrms : found , 565.2480 ; calculated for c29h38cln6o2si [ ( m + h ) ] , 565.2514 cl 
 isotopic pattern . according to general 
 procedure 5
, 8-(4-(2-(4-(3-chlorophenyl)piperidin-1-yl)ethyl)-1h - pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]pyrimidin-4(3h)-one ( 23 mg , 0.041 mmol ) 
 was reacted with hcl in 1,4-dioxane ( 4 m , 0.1 ml , 0.407 mmol ) in 1,4-dioxane / water 
 at 50 c overnight . after workup and trituration with et2o ,
the title product was obtained ( 9 mg , 51% ) as a yellow 
 solid .
h nmr ( 500 mhz , dmso - d6 )  1.66 ( dq , j = 12.3 , 3.5 hz , 2h ) , 1.76 
 ( br s , 1h ) , 1.78 ( br s , 1h ) , 2.09 ( t , j = 10.4 hz , 
 2h ) , 2.532.57 ( m , 1h ) , 2.60 ( t , j = 7.3 hz , 
 2h ) , 2.72 ( t , j = 7.3 hz , 2h ) , 3.043.10 ( m , 
 2h ) , 7.227.26 ( m , 2h ) , 7.307.35 ( m , 2h ) , 7.73 ( s , 
 1h ) , 7.98 ( d , j = 5.3 hz , 1h ) , 8.29 ( s , 1h ) , 8.42 
 ( s , 1h ) , 8.56 ( d , j = 5.3 hz , 1h ) , 12.78 ( br s , 1h ) . 

hrms : 
 found , 435.1675 ; calculated for c23h24cln6o [ ( m + h ) ] , 435.1695 .
 we 
 report the discovery of n - substituted 4-(pyridin-2-yl)thiazole-2-amine 
 derivatives and their subsequent optimization , guided by structure - based 
 design , to give 8-(1h - pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3h)-ones , a series of potent 
 jmjc histone n - methyl lysine demethylase ( kdm ) inhibitors 
 which bind to fe(ii ) in the active site . 
 substitution from c4 of the 
 pyrazole moiety allows access to the histone peptide substrate binding 
 site ; incorporation of a conformationally constrained 4-phenylpiperidine 
 linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the kdm4 ( jmjd2 ) and 
 kdm5 ( jarid1 ) subfamily demethylases , selectivity over representative 
 exemplars of the kdm2 , kdm3 , and kdm6 subfamilies , cellular permeability 
 in the caco-2 assay , and , for 54k , inhibition of h3k9me3 and h3k4me3 demethylation in a cell - based assay . 
Introduction
Chemistry
Results and Discussion
Conclusion
Experimental Section