ubiquitin is a small , 
 highly conserved protein of 76 amino acids 
 that is post - translationally conjugated to substrate proteins , including 
 itself , via a three - step enzymatic reaction . the initial covalent 
 attachment primarily occurs between the c - terminal glycine of ubiquitin 
 and the -amino group of lysine residue(s ) of the target protein . additional ubiquitin molecules can be ligated 
 to one of the seven internal lysines of ubiquitin , resulting in diverse 
 ubiquitin chain topologies . the biological outcome of ubiquitination 
 is determined by both the length and linkage topology . for example , lys48-linked polyubiquitin chains are almost 
 exclusively associated with allocating proteins for proteasome - dependent 
 degradation , while monoubiquitination or chains linked through different 
 lysines have been shown to serve multiple nonproteolytic functions . similar to other types of post - translational modifications , ubiquitination 
 is a reversible process counter - regulated by enzymes known as deubiquitinases 
 ( dubs ) , which catalyze the removal of ubiquitin from modified proteins . more importantly , dysfunction in ubiquitin - dependent 
 signaling pathways has been linked to various human diseases , suggesting 
 inhibition of ubiquitin pathway components as novel therapeutic targets 
 for drug discovery . proteasome 
 system has been well established by the successful use of proteasome 
 inhibitors for anticancer treatment . nevertheless , 
 the ubiquitin  proteasome system offers additional opportunities 
 for therapeutic intervention that could afford increased specificity 
 and the possibility for improved clinical efficacy , which proteasome 
 inhibitors are currently lacking . the 
 most apparent targets include enzymes involved in ubiquitin conjugation 
 and deconjugation ( i.e. , ubiquitin ligases and dubs ) , processes upstream 
 of proteasome - mediated protein degradation . among the dubs , ubiquitin - specific 
 protease 1 ( usp1 ) has become an attractive anticancer target because 
 of its involvement in regulating dna damage response pathways . usp1 
 associates with uaf1 ( usp1-associated factor 1 ) , resulting in the 
 heterodimeric usp1/uaf1 complex that is required for deubiquitinase 
 activity . the usp1/uaf1 complex has been 
 shown to regulate the tolerance of dna damage induced by dna cross - linking 
 agents through deubiquitination of pcna ( proliferating cell nuclear 
 antigen ) and fancd2 ( fanconi anemia complementation 
 group d2 ) , which are proteins that function 
 in translesion synthesis and the fanconi anemia pathway , respectively . also recently reported that usp1/uaf1 deubiquitinates 
 and prevents proteasomal degradation of i d ( inhibitor of dna - binding ) 
 proteins , which have been shown to activate 
 multiple pathways involved in tumor progression , including preservation 
 of the cancer stem cell phenotype . moreover , 
 evidence for aberrant overexpression of usp1 in several tumor types 
 suggests that inhibitors of usp1 are likely to provide a therapeutic 
 benefit . several small - molecule inhibitors 
 of usp1/uaf1 have been identified 
 by high - throughput screening . we initially identified the first usp1/uaf1 
 inhibitors , pimozide and gw7647 , from a collection of bioactive molecules . although gw7647 and pimozide display reasonable 
 selectivity over related proteases and demonstrate cellular inhibition 
 of human usp1 , both compounds show moderate pubchem promiscuity with 
 a 9.5% and 11.4% hit rate , respectively , and have annotated activity 
 against unrelated targets . . reported 
 c527 and related analogues , which exhibit submicromolar inhibition 
 ( ic50 = 0.88 m ) of usp1/uaf1 activity , promote id1 
 degradation , and cause cytotoxicity in several leukemic cell lines . we recently described the discovery and biological 
 activity of ml323 ( 70 ) ( figure 1 ) , a novel small - molecule inhibitor of usp1/uaf1 . compound 70 was found to exhibit selective , nanomolar inhibition of 
 usp1/uaf1 deubiquitinase activity , leading to an increase in monoubiquitinated 
 forms of pcna and fancd2 and synergistic interaction with cisplatin 
 in human tumor cell lines . herein , we 
 describe the medicinal chemistry efforts that led to the selection 
 of 70 as a chemical probe and provide biological activity 
 and in vitro adme data for additional analogues . to initiate our medicinal chemistry efforts , we 
 began by resynthesizing 
 the hts hit molecule ( 1 ) , which was prepared as described 
 in scheme 1a / b . the synthesis of related analogues ( 221 ) commenced with an edc - mediated amide coupling between 
 2-aminobenzamide ( i ) and 2 - 9(trifluoromethyl)benzoic 
 acid as shown in scheme 1a . the resulting diamide was heated to reflux with sodium 
 hydroxide for 3 h to provide the cyclized quinazoline scaffold , which 
 upon treatment with pocl3 gave the key intermediate 4-chloro-2-(2-(trifluoromethyl)phenyl)quinazoline 
 ( ii ) . this intermediate was then reacted with a variety 
 of amines to provide analogues 221 ( table 1 ) . notably , 
 compound 1 could also be synthesized in this manner using 
 thiophen-2-ylmethanamine in step d. reagents and conditions : ( a ) 
 2-(trifluoromethyl)benzoic acid , edc ( 1.2 equiv ) , hobt ( 1.2 equiv ) , 
 et3n ( 3.0 equiv ) , ch2cl2 , 72 h ; ( b ) 
 5% naoh , reflux 3 h ; ( c ) n , n - dimethylaniline , 
 pocl3 , toluene , reflux 1 h ; ( d ) rnh2 , i - pr2net , dmf , 18 h , 60 c ; ( e ) 
 for analogue 1 , [ thiophen-2-ylmethanamine ] ; for analogues 2253 and 7175 , [ ( 4-(pyridin-3-yl)phenyl)methanamine ] , et3n , 
 chcl3 , 18 h , 60 c ; ( f ) for analogues 2233 , 3753 , 
 and 7175 , rb(oh)2 ( 3 equiv ) , 2 m na2co3 ( 4 equiv ) , dpp - pd silica 
 bound ( 0.3 equiv ) , or pd(pph3)4 ( 0.2 equiv ) , 
 dme , mw , 150 c , 3045 min ; ( g ) for analogues 3436 , rnh2 ( 3.0 
 equiv ) , thf , sealed tube 100 c , 24 h. the synthesis of analogues 2253 and 7175 were all achieved in 
 a similar manner using ( 4-(pyridin-3-yl)phenyl)methanamine for the 
 amino side chain but differed in the dichloropyridimide - based scaffold 
 starting material as shown in scheme 1b . the 
 preparation of analogues 2236 involved 
 addition of ( 4-(pyridin-3-yl)phenyl)methanamine to 2,4-dichloroquinazoline 
 to obtain the desired chloropyridimide intermediate ( iv ) . suzuki coupling of iv with various boronic acids 
 using pd(pph3)4 in a microwave reactor for 30 
 min at 150 c gave analogues 2233 . for subsequent analogues , we changed to a silica bound dpp - palladium 
 catalyst in order to alleviate any palladium contamination concerns . however , instead of a suzuki coupling , 
 intermediate iv was heated in a sealed tube for 24 h 
 at 100 c with an excess of requisite amine . the synthetic route 
 for compounds 3753 ( table 3 ) was performed as described above starting from 
 other commercially available 2,4-dichloropyrimidine heterocycles , 
 and 2-isopropylphenyl boronic acid was utilized in the cross coupling 
 reaction . compounds 5469 were 
 synthesized 
 using 2,4-dichloro-5-methylpyrimidine ( v ) as the starting 
 material along with various commercially available heterocyclic and 
 aromatic methanamines , followed by a suzuki coupling with 2-isopropylphenyl 
 boronic acid ( scheme 2 ) . the tetrazole analogue 
 ( 68 ) was obtained via reaction of the 4-cn - ph moiety 
 with sodium azide in dmf . compounds 6267 were synthesized by reacting commercially available tert - butyl 4-(aminomethyl)piperidine-1-carboxylate ( for 6265 ) , tert - butyl-3-(aminomethyl)pyrrolidine-1-carboxylate 
 ( for 67 ) , or tert - butyl 3-(aminomethyl)azetidine-1-carboxylate 
 ( for 66 ) , with 2,4-dichloro-5-methylpymidine , followed 
 by the aforementioned suzuki coupling conditions . the elevated temperatures 
 used in the suzuki coupling resulted in concomitant removal of the n - boc group to provide intermediates viii . buchwald - hartwig conditions for the cross coupling of the requisite 
 amine and the heteroaryl bromides gave the desired products 6367 . initially , 
 we utilized the davephos ligand , however , this was met with limited 
 success . subsequent analysis of other ligands revealed that the ruphos 
 palladacycle proved optimal for compounds 6364 , whereas compounds 6567 worked best with davephos . oxetane analogue ( 62 ) was 
 obtained via a reductive amination of intermediate viii ( n = 2 ) and commercially available oxetan-3-one , 
 with sodium triacetoxyborohydride . reagents and conditions : 
 ( a ) 
 4-r - amine ( 1.1 equiv ) , et3n ( 3.0 equiv ) , dmf , 
 100 c 18 h ; ( b ) 2-isopropylphenyl boronic acid ( 3.0 equiv ) , 
 2 m nahco3 ( 4.0 equiv ) , dpp - pd silica bound silicycle , 
 dme , mw , 150 c , 30 min ; ( c ) for r = 4-cn - ph , nan3 ( 8.0 equiv ) , nh4cl ( 8.0 equiv ) , dmf 130 c , 30 min ; 
 ( d ) t - butyl 4-(aminomethyl)-r-1-carboxylate 
 ( 1.1 equiv ) , et3n ( 3.0 equiv ) , dmf , 100 c 18 h ; ( e ) 
 2-isoproylphenyl boronic acid ( 3.0 equiv ) , 2 m nahco3 ( 4.0 
 equiv ) , dpp - pd silica bound silicycle , dme , mw , 150 c , 30 min ; 
 ( f ) for analogues 6364 , rbr ( 1.2 equiv ) , cs2co3 , dicyclohexyl(2,4,6-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine , 
 ruphos palladacycle , toluene , sealed tube , 100 c , 18 h ; ( g ) 
 for analogues 6567 , rbr ( 1.2 equiv ) , naobu , pd2(dba)3 ( 0.02 equiv ) , davephos ( 0.02 equiv ) xylenes , sealed 
 tube , 100 c , 1 h ; ( h ) for analogue 62 , oxetan-3-one , 
 sodium triacetoxyborohydride ( 10 equiv ) , dce , 2 h. compounds 70 ( ml323 , table 4 ) and 7684 ( table 5 ) , were synthesized starting with 4-amino - benzonitrile 
 as a tfa salt , trimethylsilyl azide , and t - butyl 
 nitrite at 0 c to give the desired azide in 98% yield ( scheme 3 ) . the resulting azide 
 was dissolved in a mixture of dmso / h2o , treated with copper 
 sulfate , ethynyltrimethylsilane , sodium ascorbate , and heated for 
 24 h at 80 c to give the desired 1,2,3-triazole . for the synthesis 
 of the deuterated benzyl amine derivatives , we initially tried using 
 the h - cube flow - reactor with d2o but incomplete reduction 
 and deuterium enrichment was observed . as such , the reduction was 
 then carried out with lithium aluminum deuteride ( lad ) to give the 
 desired product in moderate yield ( 40% ) . our initial attempts for the nitrile reduction on the h - cube with 
 a 70 mm catcart ( 10% pd / c ) at 50 c and 40 bar returned just 
 trace amounts of product . however , the addition of tfa and changing 
 to a meoh / dmf mixture resulted in complete conversion to the amine 
 ( by lc - ms analysis ) . given the water solubility of the 4-triazole - benzyl 
 amine intermediates ( x ) , it was used directly in the 
 next step without further purification or characterization . the resulting 
 mixture was heated for 18 h with 2,4-dichloro-5-methylpyrimidine and 
 et3n to give derivative xi in moderate yields 
 of ( 40% ) over the two steps . compound xi was 
 then coupled to the 2-isopropylphenyl boronic acid as described previously 
 to give the final compound 70 . compounds 7684 were synthesized as described above , however , 
 several of the required boronic acids were not commercially available 
 and had to be synthesized separately . for the deuterated analogues 8082 and 84 , the requisite d6 and d7 - 2-isopropylphenyl 
 boronic acids were prepared in four steps starting from 2-bromoacetophenone 
 ( see supporting information for details ) . ( a ) tfa ( 1.0 equiv ) , rt , 5 min 
 then t - butyl nitrite ( 1.5 equiv ) , azidotrimethylsilane 
 ( 1.4 equiv ) , 0 c , 30 min , 98% yield ; ( b ) ethynyltrimethylsilane 
 ( 3.0 equiv ) , sodium ascorbate ( 0.8 equiv ) , cu(ii)so4 ( 0.07 
 equiv ) , dmso / h2o ( ratio 4/1 ) 80 c 24 h ; ( c ) tfa ( 1 
 equiv ) , acetonitrile , reflux , 1.5 h ( 57% ) ; ( d ) h - cube pro , 10% pd / c , 
 50 c , 40 bar , tfa ( 1.2 equiv ) , meoh / dmf ( ratio 10/1 ) ( 98% ) ; 
 ( e ) lithium aluminum deuteride ( lad ) ( 3.0 equiv ) , thf , 0 c , 
 1.5 h ( 40% ) ; ( f ) 2,4-dichloro-5-methylpyrimidine , et3n 
 ( 3 equiv ) , dmf , 100 c , 18 h ; ( g ) ( 2-isopropylphenyl)boronic 
 acid ( 3.0 equiv ) , 2 m nahco3 ( 4.0 equiv ) , dpp - pd silica 
 bound silicycle , dme , 150 c , 30 min , 3570% yield . the synthesis of the oxetane containing analogues 72 and 77 involved preparation of 3-(2-bromophenyl)oxetane 
 ( xii ) using a method described by jacobsen et al . for 
 a comparable phenyl oxetane derivative . formation of the ( 2-(oxetan-3-yl)phenyl)boronic acid ( xiii ) was achieved in situ via treatment of xii with n - butyllithium and triisopropyl borate in thf at 78 
 c and subsequently used as - is with vi or xi to provide compounds 72 and 77 , respectively ( scheme 4 ) . ( a ) n - butyllithium 
 ( 2.0 equiv ) , triisopropyl borate ( 2.0 equiv ) thf , 78 c , 
 18 h ; ( b ) , 2 m na2co3 ( 4.0 equiv ) , dpp - pd silica 
 bound silicycle 0.26 mmol / g ( 20 mol % ) , dme , mw , 150 c , 30 min , 
 3550% yield . after resynthesis of the thiophene - containing hts hit ( 1 ) and confirmation of its activity in both the primary ub - rhodamine 
 qhts assay and orthogonal gel - based diubiquitin assay , we decided 
 to focus our initial sar exploration efforts around the thiophene 
 moiety ( northern portion ) . despite the modest activity against usp1/uaf1 ( table 1 ; ic50 = 7.9 m ) , we were encouraged by the selectivity of 
 this compound , with no significant activity against a small panel 
 of dubs and appreciable inhibition of only one of 451 kinases . initial changes to the thiophene functionality 
 were generally well tolerated , with only slight changes in potency 
 being observed . replacing the thiophene moiety with a simple phenyl 
 ring ( 3 ) led to a modest improvement in potency ( 3.1 
 m ) , however , most substituted ( electron - withdrawing and electron - donating 
 groups ) phenyl rings ( analogues 410 ) demonstrated fairly flat sar . we were encouraged that substitution 
 at the 4-position with a phenyl ring ( e.g. , 4-phenyl benzyl amine 12 ) was well tolerated with a comparable ic50 value 
 of 3.7 m . this allowed for a variety of different analogues 
 to be synthesized with representative examples ( analogues 1321 ) shown in table 1 . potencies approached 1 m with both the 4-pyridine ( 16 ) and 3-pyridine ( 17 ) with ic50 values of 
 1.9 and 1.1 m , respectively . given the comparable potency of 
 these two analogues and the propensity of 4-pyridine derivatives to 
 interact with cyps , we decided to proceed with the 3-pyridine group 
 for the next round of analogues which were aimed at modifying the 
 2-cf3-phenyl group and to investigate core scaffold changes . ic50 values represent 
 the half - maximal ( 50% ) inhibitory concentration as determined in the 
 hts assay ( n  3 ) . exploration of the sar around the 2-cf3-phenyl group 
 indicated that substitution at the 2-position was greatly favored 
 compared to the 3- and 4-positions ( table 2 ) . this finding is exemplified 
 by the inactivity of the 3-cf3-phenyl ( 22 ) 
 and 4-cf3-phenyl ( 23 ) derivatives . given these 
 results , we decided to focus on exploring sar at the 2-position by 
 replacing the cf3 group with various electron - donating / withdrawing 
 groups as well as varying the steric bulk in this portion of the molecule . incorporation of the electron - withdrawing group ; e.g. , 2-no2 ( 24 ) led to a 7-fold loss in activity , whereas the 
 electron - donating group , e.g. , 2-ome ( 25 ) , provided comparable 
 activity ( ic50 = 0.94 m ) . replacement with the simple 
 alkyl substituents ( r = 2-me , 26 or r = 2-et , 27 ) also provided comparable activity to the 2-cf3 group . our biggest potency improvement ( 6-fold ) occurred when we replaced 
 the 2-cf3 with an isopropyl group ( 28 ) , which 
 had an ic50 of 180 nm . changing the methyl groups of the 
 isopropyl moiety to fluoro groups ( 29 ) led to an appreciable 
 decrease in potency . other electron - withdrawing groups were prepared 
 ( analogues 3032 ) , yet none of these 
 had improved activity . finally , modification of the phenyl ring to 
 a cyclopentyl group ( 33 ) or nitrogen containing heterocycles 
 ( 3436 ) resulted in inactive compounds . having already improved the potency over the original hts hit compound 1 from 4.7 to 0.18 m ( 26-fold ) , we decided to turn 
 our attention toward modification of the quinazoline core . as determined in the 
 hts assay ( n  3 ) ; inactive denotes an ic50 > 57 m . initial data from analogues in our qhts collection suggested that 
 replacement of the quinazoline core with a pyrimidine would be tolerated . 
 this change would be beneficial in that it would reduce the molecular 
 weight and lipophilicity of the lead compound . gratifyingly , this 
 modification was in fact tolerated and resulted in a compound with 
 comparable potency ( 37 , table 3 ) . introduction of a 
 5-methyl group ( 38 ) resulted in a 2-fold increase 
 in potency with an ic50 value of 70 nm . interestingly , 
 moving the methyl group to the 6-position ( 39 ) resulted 
 in a 3-fold decrease in potency ( 210 nm ) . the 5,6-dimethyl derivative 
 ( 40 ) was also well tolerated as was the cyclopentylpyrimidine 
 analogue 45 , with ic50 values of 120 and 160 
 nm , respectively . incorporation of other heteroaromatic core scaffolds 
 ( 4144 and 4648 ) provided compounds with good potency , with the most potent 
 being the furan derivative 48 . other groups such as ome 
 ( 49 ) , f ( 50 ) , nh2 ( 51 ) , nme2 ( 52 ) , and sme ( 53 ) provided 
 good potency with ic50 values of 70 , 110 , 310 , 190 , and 
 110 nm , respectively . however , as stated above , our interest in these 
 structural modifications was to improve or maintain potency while 
 reducing molecular weight . therefore , we decided to continue our sar 
 explorations with the 5-methyl - pyridimine ( 38 ) as the 
 core scaffold given the potent inhibition ( 70 nm ) and reduced size . ic50 values represent 
 the half - maximal ( 50% ) inhibitory concentration as determined in the 
 hts assay ( n  3 ) . having established the 5-methyl pyrimidine core and 
 the 2-isopropyl 
 group as optimal , we then decided to return to exploration of the 
  northern portion  sar . despite the potency of the terminal 
 3-pyridine group , some initial adme data suggested that this group 
 may be a metabolic liability . replacement of the 4-(3-pyridine)-benzyl 
 amine with a simple 3-methyl - phenyl ( 54 ) , 3-pyridine 
 ( 55 ) , or thiophene ( 56 ) generally showed 
 good potency in the ub - rho assay ( 130 , 1300 , and 270 nm , respectively ) , 
 however , none of these had comparable potency to 38 in 
 the orthogonal diubiquitin assay ( data not shown ) . in an attempt to 
 increase the hydrophilicity of the compound , a branched hydroxymethyl 
 group ( 57 ) replacement of the phenyl group with an n - me - piperidine 
 ( 58 ) led to complete loss of activity . given these data , 
 we returned our focus to the two ring analogues ( 5970 ) . adding a fluoro ( 59 ) or methyl ( 60 ) group ,  to the pyridine nitrogen , was well tolerated ( 59 , ic50 = 80 nm ; 60 , ic50 = 50 nm ) , which could help alleviate potential cyp interactions 
 if necessary . however , these changes increase lipophilicity , which 
 is something we were trying to avoid if possible . looking to increase 
 the sp character of this side chain , we replaced the central 
 phenyl group with a piperidine ( analogues 6265 ) , which generally resulted in comparable potency with exception 
 of oxetane derivative 62 . however , the corresponding 
 four - membered ( 66 ) and five - membered ( 67 ) rings resulted in a loss of potency with ic50 values 
 of 21.7 and 0.8 m , respectively . these data seem to indicate 
 a size preference for six - membered ring with the potency rank order 
 being phenyl  piperidine > pyrollidine  azetidine . 
 in an attempt to attenuate the potential metabolic liability of the 
 3-pyridine moiety , the imidazole derivative ( 69 ) demonstrated moderate 
 potency ( 300 nm ) , whereas the tetrazole ( 68 ) resulted 
 in a significant decrease in potency . the latter result is unsurprising 
 given the pka differences in these heterocycles 
 and the general preference for basic moieties in this position ( e.g. , 
 pyridine ) . however , we were particularly intrigued by the 1,2,3-triazole 
 derivative ( 70 , ic50 = 76 nm ) , given its improved 
 metabolic stability , cell permeability , and activity in other biological 
 assays ( vide infra ) . ic50 values represent 
 the half - maximal ( 50% ) inhibitory concentration as determined in the 
 hts assay ( n  3 ) ; inactive denotes an ic50 > 57 m . compound 70 demonstrated modest metabolic stability 
 ( t1/2 = 15 min ) as shown in table 5 , and met i d studies 
 ( see supporting information , figure s1 ) 
 indicated that the two major metabolites involving oxidative removal 
 of the n - benzyl group and hydroxylation of the isopropyl 
 group . as such , in an effort to improve the half - life we first focused 
 on the benzylic position and synthesized gem - dimethyl 
 analogues 79 and 82 and deuterated analogues 83 and 84 . direct hydrogen abstraction is thought 
 to be the rate - limiting step for cyp - mediated hydroxylation of aliphatic 
 compounds , and c  h bond cleavage is approximately 610 
 times faster than the corresponding c  d bond ( kinetic isotope 
 effect ) . thus , replacement of metabolically labile 
 hydrogen atoms with deuterium has been an attractive concept in drug 
 discovery , as the change should have a negligible effect on the physicochemical 
 properties yet improve metabolic stability . accordingly , we sought to utilize this strategy at both the benzylic 
 position and 2-isopropyl moiety in effort to obviate metabolic instability . we thought this approach was particularly attractive for the 2-isopropyl 
 group because our sar efforts revealed this to be critical for activity 
 and not easily replaced . unfortunately , despite thorough investigation 
 of various deuterated analogues ( 8084 ) , we did not notice any appreciable improvement in the metabolic 
 half - life of this series . analogue 79 , which possesses 
 the gem - dimethyl group , did show a moderate improvement 
 in our studies , however , it remained below the desired stability of 
 > 30 min . in an attempt to address the undesired aliphatic hydroxylation 
 at isopropyl group , we synthesized the cyclopropyl ( 71 ) , cyclobutyl ( 76 ) , oxetane ( 72 and 77 ) , and difluoro ( 78 ) derivatives . while the 
 cyclopropyl ( 71 ) and cyclobutyl ( 76 ) analogues 
 demonstrated potent inhibition ( ic50 = 180 nm ) , the metabolic 
 stability was not improved , with t1/2 = 
 6.2 and 5.1 min , respectively . replacement of lipophilic moieties 
 with an oxetane group has been shown to improve the metabolic stability 
 and solubility of the parent compound . interestingly , this result was realized in our case , as both oxetane 
 derivatives ( 72 and 77 ) had favorable metabolic 
 stability ( t1/2 > 30 min ) and solubility 
 ( data not shown ) , however , they had lost all activity toward usp1/uaf1 . 
 while disappointed by this result , we decided to use compound 77 as our inactive control in subsequent biological studies , 
 making it a vital analogue . the preference for having lipophilic moieties 
 at the 2-position of the phenyl ring was further solidified with the 
 testing of the alcohol ( 7374 ) and 
 ketone ( 75 ) analogues , which exhibited potencies in the 
 micromolar range . ic50 values represent 
 the half - maximal ( 50% ) inhibitory concentration as determined in the 
 hts assay ( n  3 ) ; inactive denotes an ic50 > 57 m . represents the half - life in minutes 
 of the compounds in the presence of rat liver microsomes ( with nadph ) . our detailed sar explorations 
 around compound 1 led 
 to a sufficient understanding of the structural features that are 
 required for usp1/uaf1 inhibition . thus , we sought to further evaluate 
 the cellular activity of a number of selected compounds . we initially 
 assessed the ability of the compounds to modulate usp1/uaf1 activity 
 in cells by monitoring the level of monoubiquitinated pcna ( ub - pcna ) . 
 upon treatment of h1299 nonsmall cell lung cancer cells with select 
 compounds ( 1 , 64 , 70 , and 77 ) , we observed a dose - dependent increase in ub - pcna compared 
 to both the untreated control and the inactive oxetane analogue ( 77 ) ( see supporting information , figure 
 s2 ) . importantly , an increase in ub - pcna was observed at concentrations 
 as low as 1 m for top compounds ( e.g. , 64 and 70 ) . to ensure that a robust increase in ub - pcna was observed , additional compounds were tested at a fixed concentration of 20 m . 
 as shown in figure 2a , b , more importantly , we 
 observed a linear correlation between the logarithm of ic50 values obtained in the hts assay and fold change in ub - pcna ( r = 0.98 , figure 2c ) . in addition to validating the reliability of the hts assay , these 
 results further support the on - target effects of this compound series 
 in preventing the usp1/uaf1-catalyzed deubiquitination of pcna in 
 human cells . disruption of usp1/uaf1-catalyzed 
 deubiquitination of pcna in h1299 
 cells . ( a ) h1299 cells were treated with the indicated compounds at 
 20 m for 4 h. whole cell extracts were separated on 412% 
 gradient sds - page and subjected to western blotting with antibodies 
 against pcna and gapdh ( loading control ) . ( b ) fold changes in monoubiquitinated 
 pcna ( ub - pcna ) were calculated by normalizing to unmodified pcna and 
 gapdh . ( c ) correlation between mean ic50 values obtained in the 
 hts assay and fold change in ub - pcna in h1299 cells ( pearson r = 0.988 ; p < 0.0001 ) . we next examined the effects of 
 these compounds on cell survival 
 using a colony formation assay . as shown in figure 3a , several of the compounds markedly inhibited the colony - forming 
 capacity of h1299 cells relative to dmso - treated control . five compounds 
 ( 48 , 49 , 64 , 69 , and 70 ) caused greater than 70% reduction of colony 
 formation at 5 m , whereas the original hts hit ( 1 ) and the inactive oxetane analogue ( 77 ) had minimal 
 or no effect on cell survival , respectively . a direct correlation 
 between ic50 values for usp1/uaf1 inhibition and cell survival 
 was identified ( r = 0.93 , figure 3b ) . as anticipated , the fold change in ub - pcna also 
 correlated with cell survival ( r = 0.91 , 
 figure 3c ) , suggesting pcna monoubiquitination 
 as a candidate pharmacodynamic biomarker predictive of efficacy . compound 70 was further evaluated in dose  response and demonstrated 
 to have an ec50 of 3.0 m , while the inactive analogue 77 had no effect on cell survival up to 25 m ( figure 3d ) . these results suggest that usp1/uaf1 inhibitors 
 could show promise as a monotherapy despite being initially envisioned 
 as a combination therapy with dna damaging agents . ( a ) representative colony formation assays ( cfa ) in h1299 cells after 
 treatment with indicated compounds . cells were grown in the absence 
 or presence of 5 m of compound for 7 days and stained with 
 crystal violet . ( b ) correlation between mean ic50 values 
 obtained in the hts assay and percent survival at 5 m in h1299 
 cells ( pearson r = 0.967 ; p = 0.0004 ) . ( c ) correlation between fold change in ub - pcna ( see figure 2a , b ) and percent survival at 5 m in h1299 
 cells ( pearson r = 0.954 ; p = 0.0008 ) . ( d ) survival curve using cfa in h1299 cells treated with 
 increasing concentrations of 70 or 25 m of 77 . having already evaluated the 
 selectivity of this molecule against numerous dubs , human proteases , 
 and kinases , we next wanted to determine 
 ancillary pharmacology against gpcr and ion channel and transporter 
 targets . to do so , we tested compound 70 at 10 m 
 in the cerep leadprofilingscreen2 , which contains 80 different grcrs , 
 ion channels , and transporters . once again , compound 70 demonstrated an encouraging selectivity profile , with only 7 targets 
 showing inhibition of > 50% at 10 m ( 5-ht2a,2b,2c , a3 , ca channel , mt1 , and ppar ) ( see supporting information , 
 figure s4 ) . a selection of the in vitro adme attributes of 
 compound 70 is highlighted in table 6 . notably , compound 70 possesses good kinetic solubility in pbs buffer ( ph 7.4 ) 
 of 86 m , and the experimental log d ( ph 7.4 ) 
 of 1.97 is well within the ideal range for orally bioavailable drug - like 
 molecules . as anticipated , compound 70 is stable in pbs buffer , assay buffer , ph 2 , ph 9 ( see supporting information , figure s3 , and mouse 
 plasma ( table 6 ) . compound 70 also 
 exhibits excellent caco-2 permeability [ papp = 23 ( 10 cm / s ) ] and shows no signs of efflux 
 ( ratio = 0.9 ) , which seems to indicate the compound is not subject 
 to the action of transporters , such as pgp . one remaining liability 
 is the less than optimal microsomal stability , with an observed t1/2 of 15 min ( rat and mouse liver microsomes ) 
 and 26 min ( human liver microsome ) . while our efforts to improve this 
 liability have thus far been met with limited success , we fully expect 
 that through further medicinal chemistry optimization this metabolic 
 liability can be attenuated . finally , preliminary in vivo pk studies 
 of this compound indicate favorable bioavailability ( > 80% ) yet 
 rapid 
 clearance ( > 70 ml / min / kg ) , which is likely a result of the marginal 
 phase i metabolic stability discussed above . aqueous 
 solubility ( pbs buffer ) , 
 mouse liver microsome ( mlm ) human liver microsome stability ( hlm ) , 
 caco-2 , and plasma stability studies were conducted at pharmaron inc . the log d data was conducted by analiza inc . using 
 a miniaturized shake - flask methodology . dubs 
 have been implicated in cancer and other human diseases and 
 thus have become increasingly regarded as prospective targets for 
 therapeutic intervention . here , we summarize the medicinal 
 chemistry optimization efforts of a series of n - benzyl-2-phenylpyrimidin-4-amine 
 based usp1/uaf1 deubiquitinase inhibitors . we succeeded in generating 
 several inhibitors with nanomolar potency , which corresponds to 100-fold 
 improved activity compared to the original hts hit ( 1 ) . in addition , we demonstrate activity in cells with selected compounds 
 as single agents , specifically increasing the level of monoubiquitinated - pcna 
 and decreasing cell survival in nonsmall cell lung cancer cells ( figure 2 and 3 ) . it has recently 
 been demonstrated that small - molecule inhibitors of usp1/uaf1 are 
 cytotoxic to both established leukemic cell lines and patient - derived 
 leukemic cells . the data presented here 
 also supports the use of usp1/uaf1 inhibitors as a monotherapy in 
 nonsmall cell lung cancer ( figure 3 ) . in addition 
 to further medicinal chemistry optimization to improve adme properties , 
 we are currently attempting to identify cancer subtypes or patient 
 populations that may benefit from usp1/uaf1-targeted therapy . all air or moisture sensitive 
 reactions were performed under positive pressure of nitrogen with 
 oven - dried glassware . chemical reagents and anhydrous solvents were 
 obtained from commercial sources and used as - is . the column used was 
 a phenomenex luna c18 ( 5 m , 30 mm  75 mm ) at a flow rate 
 of 45 ml / min . the mobile phase consisted of acetonitrile and water 
 ( each containing 0.1% trifluoroacetic acid ) . analytical analysis 
 for purity was determined by two different methods denoted as final 
 qc methods 1 and 2 . method 1 : analysis was performed on an agilent 
 1290 infinity series hplc uhplc long gradient equivalent 4100% 
 acetonitrile ( 0.05% trifluoroacetic acid ) in water over 3.5 min run 
 time of 4 min with a flow rate of 0.8 ml / min . a phenomenex kinetex 
 1.7 m c18 column ( 2.1 mm  100 mm ) was used at a temperature 
 of 50 c . method 2 : analysis was performed on an agilent 1260 
 with a 7 min gradient of 4100% acetonitrile ( containing 0.025% 
 trifluoroacetic acid ) in water ( containing 0.05% trifluoroacetic acid ) 
 over 8 min run time at a flow rate of 1 ml / min . a phenomenex luna 
 c18 column ( 3 m , 3 mm  75 mm ) was used at a temperature 
 of 50 c . purity determination was performed using an agilent 
 diode array detector for both method 1 and method 2 . mass determination 
 was performed using an agilent 6130 mass spectrometer with electrospray 
 ionization in the positive mode . all of the analogues for assay have 
 purity greater than 95% based on both analytical methods . h and c nmr spectra were recorded on a varian 400 ( 100 ) 
 mhz spectrometer . high resolution mass spectrometry was recorded on 
 agilent 6210 time - of - flight lc - ms system . 2,4-dichloroquinazoline 
 ( 0.50 g , 2.51 mmol ) , thiophen-2-ylmethanamine ( 0.34 g , 3.0 mmol ) , 
 and triethylamine ( et3n ) ( 1.0 ml , 7.54 mmol ) was stirred 
 overnight in ch2cl2 ( 6.0 ml ) at room temperature . the reaction mixture was poured into water and extracted ( 3 ) 
 with ch2cl2 , and the organic layers were combined , 
 washed ( 1 ) with brine , dried over na2so4 , filtered , and concentrated to provide 2-chloro - n-(thiophen-2-ylmethyl)quinazolin-4-amine , which was used without 
 further purification in the next reaction . lc - ms retention time a 5 ml microwave reaction vessel was charged 
 with a mixture of the above - mentioned 2-chloro - n-(thiophen-2-ylmethyl)quinazolin-4-amine 
 ( 0.09 g , 0.31 mmol ) , 2-(trifluoromethyl)phenylboronic acid ( 0.18 g , 
 0.96 mmol ) , pd(pph3)4 ( 0.06 g , 0.05 mmol ) , sodium 
 carbonate ( 0.40 m in water , 1.2 ml , 0.48 mmol ) , and acetonitrile ( 1.2 
 ml ) . the vessel was sealed and heated , with stirring , at 150 c 
 for 10 min via microwave irradiation . the organic portion was concentrated 
 under reduced pressure , and the residue was purified by silica gel 
 chromatography using ( 80% hexanes , 20% ethyl acetate ) to give the 
 desired product . h nmr ( 400 mhz , dmso - d6 )  8.478.37 ( m , 1 h ) , 8.037.75 
 ( m , 7 h ) , 7.71 ( ddd , j = 1.25 , 7.10 , and 8.34 hz , 
 1 h ) , 7.38 ( dd , j = 1.27 , and 5.12 hz , 1 h ) , 7.07 
 ( dd , j = 1.22 , and 3.43 hz , 1 h ) , 6.95 ( dd , j = 3.46 , and 5.09 hz , 1 h ) and 5.03 ( d , j = 5.70 hz , 2 h ) . c nmr ( 101 mhz , dmso - d6 )  160.09 , 159.72 , 158.66 , 158.33 , 140.73 , 135.43 , 
 132.43 , 131.94 , 130.96 , 128.01 , 127.70 , 127.40 , 126.24 , 125.67 , 123.93 , 
 122.95 , 118.24 , 115.30 , 112.89 , and 40.60 . lc - ms retention time ( method 
 2 ) = 4.808 min and ( method 1 ) = 3.128 min . hrms : m / z ( m + h ) ( calculated for c20h15f3n3s 386.0933 ) found , 386.0942 . 2,4-dichlorofuro[3,2-d]pyrimidine ( 0.10 g , 0.53 mmol ) , ( 4-(pyridin-3-yl)phenyl)methanamine 
 ( 0.10 g , 0.53 mmol ) , and triethylamine ( 0.22 ml , 1.59 mmol ) were heated 
 in chloroform to 50 c for 18 h. the mixture was poured into 
 saturated nahco3 and extracted with chloroform ( 2 ) . the organic layers were combined , dried over na2so4 , filtered , and concentrated to give an oil which was used 
 in the next step without further purification . 2-chloro - n-(4-(pyridin-3-yl)benzyl)furo[3,2-d]pyrimidin-4-amine ( 72 mg , 0.21 mmol ) , ( 2-isopropylphenyl)boronic 
 acid ( 42 mg , 0.26 mmol ) , pd(pph3)4 ( 25 mg , 0.02 
 mmol ) , and sodium carbonate ( 2 m , 0.21 ml , 0.43 mmol ) in 1,2-dimethoxyethane 
 ( 2 ml ) was sealed in a microwave tube and heated to 150 c for 
 30 min in a biotage microwave reactor . the reaction was filtered through 
 a pad of celite and washed with ethyl acetate , concentrated , and purified 
 on reversed phase to give the desired compound . h nmr 
 ( 400 mhz , dmso - d6 )  9.46 ( br s , 
 1 h ) , 8.95 ( d , j = 1.96 hz , 1 h ) , 8.63 ( d , j = 5.09 hz , 1 h ) , 8.47 ( s , 1 h ) , 8.23 ( d , j = 7.83 hz , 1 h ) , 7.73 ( d , j = 8.22 hz , 2 h ) , 7.62 
 ( dd , j = 5.09 , and 7.83 hz , 1 h ) , 7.407.53 
 ( m , 5 h ) , 7.247.33 ( m , 1 h ) , 7.12 ( d , j = 
 1.96 hz , 1 h ) , 4.86 ( d , j = 5.87 hz , 2 h ) , 3.31 ( ddd , j = 6.85 , 7.04 , and 13.50 hz , 1 h ) , 1.05 ( s , 3 h ) , and 1.03 
 ( s , 3 h ) . lc - ms retention time ( method 1 ) : 2.706 min ; ( method 2 ) : 
 4.214 min . hrms : m / z ( m + h ) ( calculated for c27h26n4o 421.2023 ) found , 421.2011 . follow the synthesis for compound 48 and substitute 2,4-dichloro-5-methoxypyrimidine for 2,4-dichlorofuro[3,2-d]pyrimidine to give the desired product . h nmr ( 400 mhz , dmso - d6 )  9.519.67 
 ( m , 1 h ) , 8.888.97 ( m , 1 h ) , 8.63 ( d , j = 
 4.70 hz , 1 h ) , 8.168.25 ( m , 1 h ) , 8.11 ( s , 1 h ) , 7.71 ( d , j = 8.22 hz , 2 h ) , 7.327.64 ( m , 7 h ) , 4.78 ( d , j = 5.87 hz , 2 h ) , 3.974.08 ( m , 3 h ) , 2.983.10 
 ( tddd , j = 6.46 , 6.60 , 6.73 , and 13.52 hz , 1 h ) , 
 1.02 ( s , 3 h ) , and 1.01 ( s , 3 h ) . c nmr ( 151 mhz , dmso - d6 )  155.72 , 155.29 , 147.79 , 146.91 , 146.03 , 
 139.07 , 138.69 , 136.71 , 136.44 , 135.48 , 131.79 , 131.63 , 129.87 , 128.21 , 
 127.46 , 126.66 , 126.22 , 125.19 , 57.72 , 43.90 , 29.42 , and 24.12 . lc - ms 
 retention time ( method 1 ) , 2.738 min ; ( method 2 ) , 1.489 min . hrms : m / z ( m + h ) ( calculated for 
 c26h27n4o 411.2179 ) found , 411.2181 . the synthetic method for compound 48 was followed and 2,4-dichloro-5-methylpyrimidine was substituted 
 for 2,4-dichlorofuro[3,2-d]pyrimidine and tert - butyl 4-(aminomethyl)piperidine-1-carboxylate for ( 4-(pyridin-3-yl)phenyl)methanamine 
 to give the desired product . the cross coupling step removed the tert - butyl carbamate to give 2-(2-isopropylphenyl)-5-methyl - n-(piperidin-4-ylmethyl)pyrimidin-4-amine , which was purified 
 on silica gel with ch2cl2/meoh(saturated with 
 nh3 ) . this 
 compound was used in the next step with no further characterization . 
 to degassed xylene ( 5 ml ) , 2-(2-isopropylphenyl)-5-methyl - n-(piperidin-4-ylmethyl)pyrimidin-4-amine ( 0.52 g , 1.60 
 mmol ) , 2-(dicyclohexylphosphino)-n , n - dimethylbiphenyl-2-amine ( davephos ) ( 0.01 g , 0.02 mmol ) , 
 sodium tert - butoxide ( 0.22 g , 1.4 mmol ) , pd2(dba)3 ( 0.02 g , 0.02 mmol ) , and 3-bromopyridine ( 0.30 g , 
 1.92 mmol ) were added and heated in a sealed tube to 100 c for 
 18 h. the reaction material was loaded directly onto silica gel and 
 purified using ( 010% meoh / dcm ) . h nmr ( 400 mhz , 
 dmso - d6 )  8.678.79 ( m , 
 1 h ) , 8.37 ( d , j = 2.74 hz , 1 h ) , 8.23 ( s , 1 h ) , 
 8.08 ( d , j = 5.09 hz , 1 h ) , 7.727.83 ( m , 
 1 h ) , 7.537.62 ( m , 3 h ) , 7.457.52 ( m , 1 h ) , 7.347.43 
 ( m , 1 h ) , 3.813.94 ( m , 2 h ) , 3.193.28 ( m , 1 h ) , 2.80 
 ( t , j = 11.74 hz , 2 h ) , 2.18 ( s , 3 h ) , 1.89 ( dd , j = 3.72 , and 7.14 hz , 1 h ) , 1.78 ( d , j = 12.13 hz , 2 h ) , 1.211.32 ( m , 3 h ) , 1.20 ( s , 3 h ) , and 
 1.18 ( s , 3 h ) . c nmr ( 151 mhz , dmso - d6 )  161.99 , 159.42 , 148.29 , 147.84 , 141.13 , 131.89 , 
 129.83 , 127.98 , 126.91 , 126.36 , 118.30 , 116.32 , 114.19 , 47.12 , 46.61 , 
 35.19 , 29.51 , 28.96 , 24.40 , 13.87 . lc - ms retention time ( method 1 ) , 
 2.683 min ; ( method 2 ) , 3.717 min . hrms : m / z ( m + h ) ( calculated for c25h32n5 402.2653 ) found , 402.2655 . ( 0.50 g , 2.60 mmol ) and potassium phthalimide ( 0.58 g , 
 3.1 mmol ) were heated in dmf ( 2.5 ml ) to 120 c for 18 h. the 
 reaction mixture was cooled to 0 c , poured into water , and the 
 solids were filtered and washed with water to give 2-(4-(1h - imidazol-1-yl)benzyl)isoindoline-1,3-dione . the dried 
 2-(4-(1h - imidazol-1-yl)benzyl)isoindoline-1,3-dione 
 ( 0.40 g , 1.32 mmol ) was heated to 95 c in a mixture of etoh 
 and hydrazine ( 4:1 ) for 1 h , the mixture was cooled , and the solids 
 filtered . ( 4-(1h - imidazol-1-yl)phenyl)methanamine 0.20 g , 
 1.17 mmol ) , 2,4-dichloro-5-methylpyrimidine ( 0.19 g , 1.17 mmol ) , and 
 et3n ( 0.33 ml , 2.34 mmol ) in dmf ( 4 ml ) were heated to 
 100 c for 18 h. the reaction mixture was monitored by lc - ms ; 
 when complete , the mixture was poured into water and extracted with 
 ethyl acetate ( 3 ) , and the organic layers were combined , dried 
 over na2so4 , filtered , and concentrated to give 
 the desired intermediate . lc - ms retention time ( method 1 ) : 2.460 min . the cross coupling used the method as described in compound 48 , with a yield of 40% . h nmr ( 400 mhz , dmso - d6 )  9.21 ( d , j = 2.52 
 hz , 1 h ) , 9.14 ( s , 1 h ) , 8.29 ( d , j = 1.26 hz , 1 
 h ) , 8.07 ( t , j = 1.64 hz , 1 h ) , 7.737.64 
 ( m , 3 h ) , 7.577.39 ( m , 5 h ) , 7.33 ( ddd , j = 1.44 , 6.97 , and 7.67 hz , 1 h ) , 4.81 ( d , j = 5.97 
 hz , 2 h ) , 3.10 ( td , j = 7.44 , 13.64 , and 14.23 hz , 
 1 h ) , 2.22 ( d , j = 1.02 hz , 3 h ) , and 1.00 ( d , j = 6.80 hz , 6 h ) . c nmr ( 151 mhz , dmso - d6 )  162.03 , 159.83 , 147.80 , 142.20 , 139.50 , 
 135.11 , 134.63 , 131.75 , 129.85 , 128.75 , 126.73 , 126.26 , 123.10 , 122.07 , 
 120.73 , 118.26 , 116.28 , 114.36 , 44.07 , 29.31 , 24.17 , and 13.86 . lc - ms 
 retention time ( method 2 ) : 3.428 min . hrms : m / z ( m + h ) ( calculated for c24h26n5 384.2183 ) found , 384.2169 . 4-aminobenzonitrile 
 ( 1.00 g , 8.46 mmol ) and trifluoroacetic acid ( tfa ) ( 0.65 ml , 8.46 
 mmol ) in acetonitrile ( 70 ml ) were stirred at room temperature for 
 5 min . the reaction mixture was cooled to 0 c in a salt ice 
 bath before the dropwise addition of tert - butyl nitrite 
 ( 1.51 ml , 12.70 mmol ) , followed by azidotrimethylsilane ( 1.35 ml , 
 10.16 mmol ) . this reaction mixture was stirred for 30 min at 0 c 
 and allowed to warm to room temperature ( rt ) before pouring into ethyl 
 acetate ( 50 ml ) and water ( 75 ml ) . the water layer was extracted ( 2 ) 
 with ethyl acetate , and the organic layers were combine and washed 
 ( 1 ) with brine . the combined organic layer was dried over na2so4 , filtered , and concentrated under reduced pressure 
 to give 1.22 g of the product as a reddish - brown solid . the compound 
 was used as - is in the next reaction . lc - ms retention time ( method 
 2:3 min ) = 3.331 min . 4-azidobenzonitrile ( 1.22 g , 8.33 mmol ) , 
 sodium ascorbate ( 1.32 g , 6.66 mmol ) , copper(ii ) sulfate ( 93 mg , 0.06 
 mmol ) , and ethynyltrimethylsilane ( 6.20 ml , 50.0 mmol ) was heated 
 in dmso / water ( 80 ml/40 ml ) to 80 c in a sealed tube for 24 
 h. the reaction was allowed to cool to rt , poured into ethyl acetate , 
 and washed ( 3 ) with 100 ml of water . the combined organic layer 
 was dried over na2so4 , filtered , and concentrated 
 under reduced pressure to give a pale - yellow residue . the residue 
 was taken up in acetonitrile ( 16 ml ) and tfa ( 0.64 ml , 8.33 mmol ) 
 and heated to reflux for 3 h. after this time , the reaction was cooled 
 to rt and poured in to ethyl acetate ( 30 ml ) , washed ( 2 ) with 
 saturated sodium bicarbonate , dried over na2so4 , filtered , and concentrated and placed on a reverse phase flash 
 system for purification ( gradient 20100% acetonitrile w/0.1% 
 tfa in water w/0.1% tfa ) . h nmr ( 400 mhz , dmso - d6 )  8.98 ( d , j = 1.26 
 hz , 1 h ) , and 8.228.00 ( m , 5 h ) . c nmr ( 101 mhz , 
 cdcl3 )  139.81 , 135.13 , 133.94 , 133.14 , 121.64 , 
 120.71 , 120.69 , 117.68 , and 112.40 ; m / z ( m + h ) = 171.1 . 4-(1h-1,2,3-triazol-1-yl)benzonitrile ( 1.2 g , 7.05 mmol ) and 
 tfa ( 0.60 ml , 7.80 mmol ) was dissolved in methanol ( 100 ml)/dmf ( 10 
 ml ) and passed through a h - cube pro flow reactor using a 10% pd / c 
 70 mm catcart at 40 bar and 50 c . once the reaction was complete 
 by lc - ms , the meoh was concentrated and the crude used in the next 
 reaction sequence . lc - ms retention time ( method 2:3 min ) = 1.386 min 
 ( m / z ( m + h ) = 174.2 ) . ( 4-(1h-1,2,3-triazol-1-yl)phenyl)methanamine , tfa 
 ( 7.05 mmol ) , 2,4-dichloro-5-methylpyrimidine ( 1.16 g , 7.05 mmol ) , 
 and triethylamine ( 3.0 ml , 21.3 mmol ) was heated overnight to 100 
 c in dmf ( 25 ml ) . the completed reaction was poured into water 
 ( 30 ml ) and extracted with ethyl acetate . the ethyl acetate layer 
 was washed ( 2 ) with water ( 1 ) with saturated sodium bicarbonate , 
 dried over na2so4 , filtered , and concentrated . the residue was purified on a reverse phase flash system ( gradient 
 10100% acetonitrile w/0.1% tfa in water w/0.1% tfa ) to give 
 0.19 g of desired product . h nmr ( 400 mhz , dmso - d6 )  8.76 ( d , j = 1.17 
 hz , 1 h ) , 7.977.90 ( m , 2h ) , 7.867.80 ( m , 3 h ) , 7.547.48 
 ( m , 2 h ) , 4.63 ( d , j = 5.98 hz , 2 h ) , and 2.181.75 
 ( m , 3 h ) . c nmr ( 101 mhz , dmso - d6 )  162.60 , 157.80 , 154.97 , 140.20 , 135.90 , 134.78 , 
 129.01 , 123.61 , 120.61 , 113.64 , 43.54 , and 13.50 . lc - ms retention 
 time ( method 2:3 min ) = 2.770 min n-(4-(1h-1,2,3-triazol-1-yl)benzyl)-2-chloro-5-methylpyrimidin-4-amine ( 0.19 
 g , 0.63 mmol ) was combined with ( 2-isopropylphenyl)boronic acid ( 0.31 
 g , 1.90 mmol ) , sodium carbonate ( 2.0 m in water , 1.23 ml , 2.53 mmol ) , 
 and dpp - pd silicycle 0.26 mmol / g ( 0.30 g ) in dmf ( 4.50 ml ) . the reaction 
 was sealed and heated at 150 c for 30 min in a biotage initiator 
 microwave reactor . the resulting mixture was filtered over celite 
 and purified by hplc ( gradient 20100% acetonitrile w/0.1% 
 tfa in water w/0.1% tfa ) to give , after lyophilization , n-(4-(1h-1,2,3-triazol-1-yl)benzyl)-5-methyl-2-(2-isopropylphenyl)-pyrimidin-4-amine 
 ( 70 ) as a tfa salt ( 0.01 g , 0.02 mmol , 30% ) . h nmr ( 400 mhz , cdcl3 )  8.82 ( dd , j = 3.4 , and 7.9 hz , 1 h ) , 8.027.93 ( m , 2 h ) , 7.76 ( d , j = 1.4 hz , 1 h ) , 7.647.56 ( m , 2 h ) , 7.517.33 
 ( m , 5 h ) , 7.257.17 ( m , 1 h ) , 4.88 ( d , j = 
 5.9 hz , 2 h ) , 3.19 ( p , j = 6.8 hz , 1h ) , 2.22 ( s , 
 3 h ) , 1.10 ( d , j = 1.2 hz , 3 h ) and 1.091.07 
 ( m , 3 h ) . c nmr ( 100 mhz , cdcl3 )  162.02 , 
 160.18 , 148.00 , 140.63 , 138.03 , 136.14 , 134.35 , 134.28 , 131.89 , 130.34 , 
 129.25 , 129.00 , 126.61 , 126.04 , 122.04 , 121.99 , 120.74 , 113.81 , 44.62 , 
 29.43 , 23.95 , 13.71 and 13.67 . lc - ms retention time ( method 1 ) = 2.938 
 min and ( method 2 ) = 1.756 min . hrms : ( esi ) m / z ( m + h ) ( calculated for c23h25n6 385.2135 ) found , 385.2146 . al , except diethyl 2-(2-bromophenyl)malonate was 
 used as the starting material instead of diethyl 2-(2-(benzyloxy)phenyl)malonate . this synthetic sequence was carried out without further purification / characterization 
 of the intermediates . in an oven - dried 50 ml round - bottom flask , the above - mentioned 3-(2-bromophenyl)oxetane ( 0.14 g , 0.66 mmol ) and 
 thf ( 4 ml ) was cooled to 78 c . the dropwise addition 
 of n - butyllithium ( 0.85 ml , 1.36 mmol ) by syringe 
 occurred over a 15 min period . after this time , the reaction mixture 
 was allowed to stir at 78 c for an additional 2 h. the 
 contents were transferred via cannula to a second 50 ml oven - dried 
 round - bottom flask containing a stirred solution of tripropyl borate 
 ( 0.29 ml , 1.32 mmol ) and thf ( 2.0 ml ) and cooled to 78 c . 
 once transferred ( by cannula ) , the mixture was allowed to stir at 
 78 c for 10 min , after which time the ice bath was removed 
 and the reaction mixture stirred overnight . one n hcl was used to 
 adjust the ph to 1 , and the acidic mixture was stirred for 
 45 min , poured into water , and extracted with diethyl ether ( 3 ) . the organic layers were combined , dried over mgso4 , concentrated , 
 and once the solvent was evaporated , a 1 m solution of the 
 crude ( 2-(oxetan-3-yl)phenyl)boronic acid in 1,2-dme was made and 
 used in next step with no further purification . in a sealed tube n-(4-(1h-1,2,3-triazol-1-yl)benzyl)-2-chloro-5-methylpyrimidin-4-amine 
 ( 0.10 g , 0.33 mmol ) was combined with ( 2-oxetainphenyl)boronic acid 
 as a 1 m solution in dme sodium carbonate ( 2.0 m in water , 0.33 ml , 
 0.67 mmol ) and dpp - pd silicycle 0.26 mmol / g ( 0.20 g ) in dme ( 2.0 ml ) , 
 and heated at 150 c for 30 min in a biotage initiator microwave 
 reactor . the resulting mixture was filtered over celite and purified 
 by reversed - phase hplc ( gradient 20100% acetonitrile w/0.1% 
 tfa in water w/0.1% tfa ) to give the desired product 77 . h nmr ( 400 mhz , dmso - d6 )  ppm 9.369.47 ( m , 1 h ) , 8.80 ( s , 1 h ) , 8.35 ( s , 
 1 h ) , 8.29 ( d , j = 7.83 hz , 1 h ) , 7.97 ( s , 1 h ) , 
 7.90 ( d , j = 8.22 hz , 2 h ) , 7.68 ( d , j = 8.61 hz , 3 h ) , 7.52 ( s , 2 h ) , 5.12 ( br s , 1 h ) , 4.935.00 
 ( m , 2 h ) , 4.414.50 ( m , 2 h ) , 3.61 ( dd , j = 
 4.30 , and 10.56 hz , 2 h ) , and 2.22 ( s , 3 h ) . nmr ( 151 
 mhz , cdcl3 )  161.40 , 153.48 , 144.27 , 138.36 , 137.52 , 
 136.20 , 134.57 , 134.28 , 129.62 , 128.71 , 128.52 , 128.14 , 126.14 , 121.96 , 
 120.88 , 115.47 , 109.98 , 62.49 , 51.61 , 44.94 , 39.85 , and 14.15 . lc - ms : 
 method 2 ( short ) , retention time : 2.526 min . hrms : m / z ( m + h ) ( calculated for c23h23n6o 399.1928 ) found , 399.1924 . 2-(4-nitrophenyl)propan-2-amine , hcl ( 0.60 g , 2.77 mmol ) , 
 2,4-dichloro-5-methylpyrimidine ( 0.45 g , 2.77 mmol ) , and et3n ( 1.15 ml , 8.31 mmol ) in dmf ( 8.2 ml ) was sealed and heated in a 
 microwave vial to 150 c for 1 h. the reaction was cooled and 
 poured into etoac and washed with water ( 2 ) , dried over na2so4 , filtered , and concentrated . the residue was 
 purified using reverse phase over 30 min and gradient of 10100 
 water / ch3cn 0.01% tfa to give the desired compound in a 
 49% yield . lc - ms retention time ( method 1 ) : 3.478 min . 2-chloro-5-methyl - n-(2-(4-nitrophenyl)propan-2-yl)pyrimidin-4-amine , 
 tfa ( 0.19 g , 0.45 mmol ) , ( 2-isopropylphenyl)boronic acid ( 0.22 g , 
 1.26 mmol ) , sodium carbonate ( 2 m , 0.90 ml , 1.81 mmol ) , and dpp - pd 
 silica bound ( 0.20 g , 0.26 mmol / g ) in dimetoxyethane ( 3 ml ) was sealed 
 and heated in a microwave reactor to 150 c for 45 min . the reaction 
 was filtered and concentrated immediately placed on a reverse phase 
 column to give 75% of the desired intermediate , which was used without 
 further characterization . 2-(2-isopropylphenyl)-5-methyl - n-(2-(4-nitrophenyl)propan-2-yl)pyrimidin-4-amine , 
 tfa ( 0.28 g , 0.55 mmol ) , acetic acid ( 0,37 ml , 6.54 mmol ) , and zinc 
 ( 0.18 g , 2.73 mmol ) was stirred in ch3oh ( 5.50 ml ) for 
 1 h. the reaction mixture was filtered through a pad of celite , rinsed 
 with ch3oh , concentrated , and used as in with no further 
 purification or characterization . lc - ms retention time ( method 1 ) : 
 2.670 min . n-(2-(4-aminophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-methylpyrimidin-4-amine 
 ( 0.12 g , 0.33 mmol ) and trifluoroacetic acid ( tfa ) ( 0.04 ml , 0.33 
 mmol ) in acetonitrile ( 2 ml ) was stirred at room temperature for 5 
 min . the reaction mixture was cooled to 0 c in an ice bath before 
 the dropwise addition of tert - butyl nitrite ( 0.06 
 ml , 0.50 mmol ) followed by azidotrimethylsilane ( 0.05 ml , 0.40 mmol ) . this mixture was stirred for 30 min at 0 c , allowed to warm 
 to room temperature ( rt ) , and poured into ethyl acetate ( 10 ml ) and 
 water ( 15 ml ) . the water layer was extracted with ethyl acetate ( 2 ) 
 and organic layers were combined and washed with brine ( 1 ) , 
 dried over na2so4 , filtered , and concentrated 
 under reduced pressure to give to give the n-(2-(4-azidophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-methylpyrimidin-4-amine , 
 which was used as - is in the next reaction without further purification . 
 lc - ms retention time ( method 1 ) : 3.331 min . n-(2-(4-azidophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-methylpyrimidin-4-amine 
 ( 33 mg , 0.09 mmol ) , ethynyltrimethylsilane ( 0.13 ml , 1.03 mmol ) , sodium 
 ascorbate ( 14 mg , 0.07 mmol ) , and copper(ii)sulfate ( 1 mg , 0.006 mmol ) 
 in dmso / h2o ( 1 ml/0.5 ml ) was sealed in a tube and heated 
 to 80 c for 24 h. the cooled reaction mixture was poured into 
 water and extracted with ethyl acetate ( 3 ) , dried over na2so4 , filtered , concentrated , and taken up in acetonitrile 
 ( 3 ml ) and tfa ( 0.11 ml , 0.92 mmol ) , heated to reflux for 3 h , then 
 the reaction was cooled and concentrated under reduced pressure . h nmr ( 400 mhz , dmso - d6 )  8.75 ( d , j = 1.17 hz , 1h ) , 8.23 ( s , 1h ) , 
 7.94 ( d , j = 1.13 hz , 1h ) , 7.817.73 ( m , 2h ) , 
 7.557.47 ( m , 2h ) , 7.40 ( t , j = 7.60 hz , 1h ) , 
 7.337.26 ( m , 1h ) , 7.16 ( t , j = 7.54 hz , 1h ) , 
 7.02 ( d , j = 7.64 hz , 1h ) , 3.053.17 ( m , 1 
 h ) , 2.33 ( d , j = 1.06 hz , 3h ) , 1.78 ( s , 6h ) , and 
 0.72 ( d , j = 6.80 hz , 6h ) . hrms : m / z ( m + h ) : ( calculated for c25h29n6 413.2448 ) found , 413.2447 . h1299 cells were seeded in 12-well plates 
 at a density of 1  10 per well and allowed to attach 
 overnight . cells were treated with 20 m of the indicated compounds 
 or with an equal volume of dmso for 4 h. total protein extracts were 
 prepared in ripa lysis buffer ( 25 mm tris - hcl ( ph 7.6 ) , 150 mm nacl , 
 1% np-40 , 1% sodium deoxycholate , 0.1% sds ) containing protease and 
 phosphatase inhibitor cocktail ( thermo scientific ) . the concentration 
 of protein lysates was determined using the bio - rad dc protein assay 
 ( bio - rad ) . cell extracts were separated on 412% gradient sds - page 
 and transferred to nitrocellulose . the membranes were blotted with 
 pcna antibody ( pc10 , santa cruz ) or gapdh antibody ( d16h11 , cell signaling ) , 
 followed by incubation with hrp - conjugated antimouse or antirabbit 
 secondary antibody , respectively . blots were imaged using the bio - rad 
 chemidoctm xrs gel imager and quantified using imagequant tl ( ge ) . h1299 cells were seeded in 6-well 
 plates at a density of 200 cells per well and allowed to attach overnight . cells were treated with 5 m of the indicated compounds or with 
 an equal volume of dmso . seven days after compound treatment , plates 
 were fixed with 20% methanol and stained with 0.1% crystal violet . plates were images with the typhoon fla 7000 ( ge ) , and colonies were 
 counted using imagequant tl ( ge ) . cell survival was expressed in percentage 
 with dmso - treated cells set as 100% .
deregulation 
 of ubiquitin conjugation or deconjugation has been 
 implicated in the pathogenesis of many human diseases including cancer . 
 the deubiquitinating enzyme usp1 ( ubiquitin - specific protease 1 ) , 
 in association with uaf1 ( usp1-associated factor 1 ) , is a known regulator 
 of dna damage response and has been shown as a promising anticancer 
 target . to further evaluate usp1/uaf1 as a therapeutic target , we 
 conducted a quantitative high throughput screen of > 400000 compounds 
 and subsequent medicinal chemistry optimization of small molecules 
 that inhibit the deubiquitinating activity of usp1/uaf1 . 
 ultimately , 
 these efforts led to the identification of ml323 ( 70 ) 
 and related n - benzyl-2-phenylpyrimidin-4-amine derivatives , 
 which possess nanomolar usp1/uaf1 inhibitory potency . 
 moreover , we 
 demonstrate a strong correlation between compound ic50 values 
 for usp1/uaf1 inhibition and activity in nonsmall cell lung cancer 
 cells , specifically increased monoubiquitinated pcna ( ub - pcna ) levels 
 and decreased cell survival . 
 our results establish the druggability 
 of the usp1/uaf1 deubiquitinase complex and its potential as a molecular 
 target for anticancer therapies .