tuberculosis ( tb ) is 
 responsible for the death of 1.5 million people 
 annually . the development of antibiotic 
 resistant strains of the causal pathogen mycobacterium 
 tuberculosis ( mtb ) and comorbidity 
 with immunosuppressive conditions such as hiv and diabetes are major 
 contributors to the current crisis and there is an urgent need for 
 the development of new drugs with novel mechanisms of action . cytochrome p450 enzymes ( p450s or cyps ) are a currently unexploited 
 class of protein targets for tb therapeutics . the mtb genome encodes genes for 20 p450 isoforms , which is more than 200 
 times as many as humans for the relative size of the genome . the large proportion of genetic material dedicated 
 to p450s suggests that these enzymes might have important functions 
 for the survival and virulence of the pathogen . biochemical and genetic studies over the past 15 years 
 have provided evidence of essential roles for mtb p450s in bacterial viability , the catabolism of host cholesterol , 
 establishment of infection , persistence in the granuloma , cellular 
 stress response mechanisms , and drug susceptibility . a number of azole - containing antifungal compounds that have potent 
 antimycotic activity against mtb h37rv both in vitro 
 and in murine models of tb , have tight binding affinity ( kd ) for a number of the mtb p450s , which 
 provides further support for these enzymes as potential drug targets . there is correlation between 
 the minimum inhibitory concentration ( mic ) values of the most potent 
 azole compounds clotrimazole , econazole , and miconazole , which have 
 mics of 11 , 8 , and 8 g / ml , respectively , against mtb h37rv , and their kd s for the most strongly inhibited p450 in addition , evidence that the gene encoding cyp121 ( rv2276 ) is essential for mtb h37rv viability implies that this p450 is the major target responsible 
 for the efficacy of these compounds . while azole antifungal drugs 
 have promise as treatments for tb , the specific compounds that have 
 been shown to have efficacy against mtb suffer from 
 either low oral bioavailability or cause significant toxic side effects 
 because of their broad spectrum activity against human p450s or steroidogenic 
 inhibition . these properties make the azole 
 drugs incompatible with long - term dosing regimens required for tb 
 treatment . consequently , there is interest in developing more potent 
 and selective inhibitors of mtb p450s . cyp121 
 is a soluble , monomeric enzyme , which has limited similarity 
 ( 34% protein sequence identity ) to other mtb p450s . the enzyme catalyzes the cyclization 
 of the dipeptide cyclo-(l - tyr - l - tyr ) ( cyy ) to form 
 the diketopiperazine mycocyclosin . while 
 the function of mycocyclosin has not been determined , diketopiperazine 
 secondary metabolites often have antimicrobial or cytotoxic activity , 
 properties which might be of importance for mtb virulence . this combination of gene essentiality , low sequence 
 similarity to other p450s , and tight azole binding affinity has made 
 the development of cyp121 inhibitors an area of interest in the search 
 for new tb drugs and the focus of the research presented here . fragment - based drug discovery ( fbdd ) is an established technique 
 in both academia and industry that has been applied to a wide range 
 of molecular targets . the availability of high resolution crystal structures of cyp121 , 
 in both the substrate - free and substrate / ligand - bound forms , make 
 it amenable to a fragment - based approach . we have previously reported the development of low micromolar affinity 
 cyp121 inhibitors , developed from fragments that were identified in 
 a biophysical screen of our fragment library . six fragment hits were crystallized with cyp121 , one of which was 
 4-(1h-1,2,4-triazol-1-yl)phenol 1 ( kd = 1.7 mm ) ( figure 1 ) . fragment 1 bound in two different 
 overlapping orientations in the cyp121 active site , suggesting an 
 approach based on fragment merging . synthesis of a range of analogues , 
 guided by in silico screening to optimize the binding interactions 
 of the heterocyclic fragment core , resulted in the identification 
 of the triphenol pyrazole - amine 2 ( kd = 15 m ) . an x - ray crystal structure of 2 bound to cyp121 revealed that 2 maintained 
 the binding mode of the original fragment 1 . the optimized 
 hydrogen bonding interactions between the 5-aminopyrazole core of 2 and distal pocket residues gln385 , ala167 , and thr77 represented 
 a privileged binding mode for an azole - containing compound , which 
 generally bind directly to the heme cofactor of p450s using a heterocyclic 
 nitrogen atom . the combination of the low micromolar affinity of 2 and its unique binding mode , which was conserved with fragment 1 , led us to pursue further optimization of this compound 
 series . fragment merging and in silico directed optimization of fragment 1 ( green , pdb 4g47 ) led to the discovery of low micromolar cyp121 inhibitor 2 ( salmon , pdb 4ktl ) . fragment 1 and lead 2 retained 
 a conserved binding mode distal to the heme cofactor ( magenta ) . hydrogen 
 bonding interactions between the 5-aminopyrazole ring and cyp121 residues 
 gln385 and ala167 , and the phenol group with asn85 and active site 
 water molecules are shown as yellow dashed lines . figures prepared 
 using pymol v1.7.4 ( schrdinger , llc ) . despite a 100-fold improvement in binding affinity between 
 fragment 1 and lead 2 , the low ligand efficiency 
 ( le ) of 2 ( le = 0.23 ) led us to 
 undertake a deconstruction retrofragmentation of 2 to its component  fragments  
 was used to assess the binding contribution , or group efficiency ( ge ) , of the three aromatic rings that decorate the 
 amino - pyrazole core , hereafter referred to as ar1 , ar2 , and ar3 , as 
 defined in figure 3 . ge analysis provided an assessment of the change in binding 
 affinity 
 per heavy atom that could be attributed to each of the ar1 , ar2 , and 
 ar3 rings of lead 2 , as the compound was sequentially 
 rebuilt from a series of retrofragments 36 . x - ray crystal structures of 
 cyp121 in complex with retrofragments 36 were used to validate our ge analysis , enabled the identification 
 of conserved binding hotspots within the enzyme active site and was 
 used to guide our subsequent optimization of 2 using 
 fragment growing and merging strategies . the significant improvements 
 in binding affinity and le that can be achieved by the structure - guided 
 incorporation of a metal binding pharmacophore into the newly identified 
 minimal scaffolds is illustrated . in contrast , fragment growing toward 
 pockets in the distal active site produced only modest improvements 
 in binding affinity and resulted in the development of a second series 
 of compounds with a binding mode that was distinct from the metal - coordinating 
 ligands . investigation of the sar governing these interactions and 
 knowledge of binding hot - spots were subsequently exploited to improve 
 the isoform selectivity of inhibitors against human p450 enzymes , 
 and also identified regions of the cyp121 active site that were tolerant 
 to structural modification and which could be exploited during the 
 future optimization of these compounds . retrosynthetic fragmentation of 
 lead compound 2 indicated that the relative binding contributions 
 of ar1 , ar2 , and ar3 could be determined from a combination of monophenol 
 and biphenol fragments 3 , 4 , 5 , and 6 ( figure 3 ) . the sequential reconstruction 
 of 2 from these fragments enabled the ge of each aromatic 
 ring to be calculated from the difference in the free energy of binding 
 per heavy atom added at each stage of reconstruction ( ge = g/ha ) . retrofragments 3 and 5 were synthesized 
 according to the previously reported literature procedure , which was also used to access fragment 4 . biphenol 
 fragment 6 was synthesized according to the general route 
 for accessing 3,5-disubstituted aminopyrazoles described by johnson 
 et al . x - ray crystal structures of fragments 36 in complex 
 with cyp121 ( figure 2a c ) were obtained in order to aid 
 the interpretation of binding affinity and ge trends . retrofragments 36 each reproduced the privileged non - heme 
 binding mode of lead compound 2 , which is unusual for 
 p450 ligands containing azole motifs . analysis of the conserved and 
 variable binding interactions made by the retrofragments highlighted 
 structural features likely to be important for driving the binding 
 affinity of 2 . monophenol 4 bound in an orientation that overlapped with that of ar2 of the 
 lead compound 2 ( figure 2a ) . the water - bridged hydrogen bonding network between 
 asn85 , thr229 , and the heme propionate group with the 4-hydroxy substituent 
 of 4 was conserved , while the 5-aminopyrazole ring was 
 shifted toward the i - helix ( highlighted yellow in figure 2 ) to enable hydrogen bonding 
 interactions with val228 and aromatic interactions with phe168 and 
 trp182 , mimicking the function originally fulfilled by ar1 of lead 
 compound 2 . the small shift in position of the aminopyrazole 
 ring of 4 indicated that interactions made at the ar1 
 site might contribute more significantly to the affinity of lead compound 2 than do the hydrogen - bonding interactions made by 2 with gln385 . x - ray crystal structures of lead compound 2 and component 
 retrofragments 46 in complex with 
 cyp121 . the i- ( yellow ) , and f- , and g- ( blue ) helices are shown in 
 cartoon representation . ( a ) overlaid structures of retrofragments 4 ( purple ) ( pbd 4ktj ) , 5 ( yellow ) ( pdb 4ktf ) , and 6 ( cyan ) ( pdb 5ibj ) with lead compound 2 ( salmon ) ( pdb 4ktl ) , illustrating the 
 conserved binding mode distal to the heme cofactor ( magenta ) . ( b ) structures , binding affinity , and ligand 
 efficiency ( le ) of compound 2 and retrofragments 4 , 5 , and 6 . ( c  e ) x - ray 
 crystal structures of cyp121 in complex with retrofragments 4 , 5 , and 6 , respectively , indicating 
 polar interactions ( yellow dashes ) with active site residues ( blue ) 
 and solvent ( red spheres ) . co - crystallized sulfate ( yellow lines ) 
 is also indicated in ( d ) . the associated omit fofc electron density map of fragment 6 contoured to 3 has been provided in the supporting 
 information , figure s5 . a 1.65  x - ray crystal structure of retrofragment 3 in complex with cyp121 was obtained , however incomplete 
 ligand occupancy 
 prevented adequate refinement of the structure . the poorly resolved 
 density of 3 suggested the fragment likely acquired multiple 
 different binding orientations , as had been previously observed for 
 the original hit fragment 1 ( figure 1 ) . analysis of the ligand density suggested 
 that 3 bound cyp121 in an orientation that overlapped 
 with ar1 of lead 2 . however , the 5-aminopyrazole ring 
 was rotated 180 , further indicating that hydrogen bonding to 
 gln385 probably contributed less to binding affinity than other conserved 
 interactions ( figure s1 , supporting information ) . the fused ar1ar2 fragment 5 accurately recapitulated 
 the binding mode of 2 , satisfying the offset and edge - face 
 -stacking interactions with phe168 and trp182 that were identified 
 as important in the region occupied by ar1 , as well as the hydrogen 
 bonding networks formed by the ar2 phenol and between the aminopyrazole 
 ring and gln385 ( figure 2b ) . the conserved binding mode of 5 in the absence of 
 ar3 suggested a minimal contribution to binding from this motif and 
 was consistent with observations that ar3 resided in a large , water - filled 
 pocket in the x - ray structure of 2 ( figure 1 ) . this lack of binding contribution 
 was reflected in the low ge calculated for ar3 on reforming 2 from 5 ( figure 3 ) . the x - ray crystal structure of the ar1ar3 
 fused fragment 6 showed that the fragment bound cyp121 
 in an orientation 
 that was flipped 180 from that expected in lead 2 . the 5-amino - phenol ring that distinguished 6 from 
 retrofragment 3 was oriented toward the ar1 site , instead 
 of the ar3 pocket , and the original phenol of 3 was shifted 
 into the ar2 site ( figure 2c ) . in this orientation , the pyrazole core formed new hydrogen 
 bonding interactions with i - helix residue thr229 , similar to those 
 observed for fragment 4 . this change in binding mode 
 accounts for the good ge values calculated when elaborating fragment 3 into 6 ( vide infra ) . the 100-fold improvement 
 in binding affinity obtained for 6 was thus attributed 
 to optimizing interactions at the ar1 and ar2 sites , demonstrating 
 the importance of obtaining structural data for the accurate interpretation 
 of ge analysis . from these data , it was concluded that ar1 and ar2 
 formed highly conserved interactions that could be exploited in the 
 design of subsequent analogues . in contrast , ar3 contributed minimally 
 to binding and would likely be tolerant to structural modifications . as such , investigating the sar of ar3 was a priority in order to identify 
 more ge replacements for the original phenol ring of lead compound 2 . the binding affinity and le of the retrofragments 
 for cyp121 was 
 determined using itc ( figure 3 ) . fragments 3 ( kd = 1.3 mm , le = 0.30 ) and 4 ( kd = 1.9 mm , le = 0.29 ) were found 
 to have comparable binding affinities and le to that of the original 
 fragment hit 4-(1h-1,2,4-triazol-1-yl)phenol 1 , indicating that substitution of the original triazole core 
 of 1 for a 5-aminopyrazole ring resulted in no significant 
 difference in binding affinity . the combination of any two phenol 
 rings to satisfy binding interactions at the ar1 and ar2 sites , as 
 exemplified by biphenol retrofragment 5 ( kd = 40 m ) or 6 ( kd = 10.6 m ) , resulted in an almost additive improvement 
 in binding affinity . the small difference in the binding affinity 
 of 5 or 6 ( g  
 0.91.7 kj mol ) compared to that 
 calculated for the theoretical sum of 3 and 4 ( g3 + 4 ( theor ) 7.7 kj mol ) can be attributed in part 
 to binding contributions from the 5-aminopyrazole motif that is common 
 to the structures of the monophenol fragments . the additive binding 
 affinity was reflected in the good ge values ( ge = 0.290.41 ) 
 calculated for the addition of an aromatic ring to a monophenol scaffold 3 or 4 . reduced torsional strain between the 
 phenol rings was suggested to account for the slightly lower kd value of retrofragment 6 versus 5 . the addition of a third aromatic 
 ring to fragment 5 or 6 to give lead 2 had a negligible effect on binding affinity , as reflected 
 in the small ge values of 0.030.08 calculated for 
 occupation of the ar3 pocket . deconstruction of lead compound 2 into retrosynthetic 
 fragments 36 was used to assess 
 the binding affinity , ligand efficiency ( le ) , and group efficiency 
 ( ge ) contribution of the individual components . the three aromatic 
 rings decorating the aminopyrazole core of lead compound 2 have been labeled ar1 , ar2 , and ar3 as illustrated , which refers 
 to the orientation the rings occupy in the x - ray crystal structure 
 of 2 in complex with cyp121 ( figure 1 ) . the lower panel tabulates 
 the binding affinity ( kd ) and le and ge 
 properties of compounds from the sequential reconstruction of lead 2 from component monoaryl ( 3 and 4 ) and biaryl ( 5 and 6 ) retrofragments . le was calculated 
 from the gibbs free energy of binding divided by the number of heavy 
 atoms ( g / ha ) . ge contributions 
 of the three aromatic rings ar1 , ar2 , and ar3 were calculated from 
 the change in gibbs free energy per added heavy atom ( g/ha ) as the retrofragments were 
 progressively recombined to form lead compound 2.kd previously reported . the le of retrofragments ( le  0.3 ) was comparable 
 to that 
 of the original fragment hit 1 ( le = 0.32 ) , indicating 
 that both mono- and biphenol scaffolds represented better starting 
 points for the optimization of cyp121 inhibitors than did lead compound 2 ( le = 0.24 ) . retrofragments 4 and 5 were selected as the most favorable scaffolds for further optimization 
 because of the consistency of their binding modes relative to lead 
 compound 2 and because of the opportunities these scaffolds 
 provided for elaboration toward the heme iron from ar2 and from the 
 exocyclic pyrazole-5-amino group to explore the ar3 pocket . the change 
 in the binding mode of retrofragment 6 relative to lead 2 led us to deprioritize further optimization based on this 
 scaffold despite its slightly better binding affinity compared to 5 . this combination of ge analysis , assessment of le , and 
 interrogation of x - ray crystal structures facilitated a rational strategy 
 for the independent optimization of each aromatic ring ar1 , ar2 , and 
 ar3 of lead 2 , guided by insights into the key binding 
 interactions and activity hotspots . our approach to developing 
 potent cyp121 inhibitors focused on three independent series of analogues , 
 each optimizing the interactions of the ar1 , ar2 , and ar3 rings of 
 lead compound 2 , respectively ( figure 4a  c ) . analogues were designed as derivatives of either 
 the biaryl scaffold 5 , which had been identified as the 
 most le retrofragment that also retained the original binding mode 
 of 2 , or the monoaryl 4 scaffold , which 
 had been identified to participate in a conserved hydrogen - bonding 
 network and bound to cyp121 in close proximity to the heme cofactor . proposed 
 optimization strategies for ar1 , ar2 , and ar3 of lead 
 cyp121 ( a ) overlaid x - ray crystal structures 
 of biaryl fragment 5 ( yellow ) ( pdb 4ktf ) and benzamide fragment 7 ( green ) ( pdb 5edt ) bound to cyp121 . fc electron density 
 map of fragment 7 contoured to 3 can be found 
 in supporting information figure s5 . ( b ) 
 overlaid x - ray crystal structures of cyp121 in complex with retrofragment 5 ( yellow ) and with heme binding fragments 8 ( copper ) 
 ( pdb 4g44 ) and 9 ( cyan ) ( pdb 4g45 ) , indicating the proposed meta - attachment point for substituents and distance ( 6.5 
  ) to the heme coordinating position ( yellow dashed line ) . ( c ) 
 docking of ar3 analogues of lead 2 , elaborating from 
 the 5-aminopyrazole group of retrofragment 5 ( gray ) with 
 carbonyl ( orange ) or methylene ( green ) linkages to enhance binding 
 interactions with the distal active site pocket ( asn74 , thr65 , gray 
 sticks ) of cyp121 . analogues in the ar1 
 series were designed 
 to probe the effect of electronics and hydrogen bonding on the conserved 
 -stacking interactions formed between this motif and phe168 
 and trp182 and the potential to form polar interactions with thr77 
 and asp185 , residues residing on the substrate selective f / g - helices . the crystal structure of a substituted benzamide 
 fragment 7 ( kd = 3.4 mm ) , 
 which had been identified during our initial fragment screen on cyp121 
 ( pdb 5edt ) , indicated that meta - substitution 
 or elaboration from the para - position would be most 
 favorable ( figure 4a ) . ligand docking simulations were 
 used to support our design and indicated that changing the position 
 of the aromatic substituent was unlikely to cause conformational strain 
 or perturb the global geometry of the ligands ( figure s2 , supporting information ) . the proximity and orientation of ar2 
 in relation to the heme cofactor , and the conserved hydrogen bonding 
 interactions made by the 4-hydroxy substituent with asn85 and the 
 water - bridged heme propionate group , indicated that ar2 could provide 
 an ideal anchor for the incorporation of a metal - binding functional 
 group . compounds which interact with both active site residues and 
 the heme cofactor are generally considered to be more effective than 
 inhibitors that only utilize one form of interaction and it was proposed 
 that exploiting the high enthalpic contribution of metal coordination 
 could significantly improve the potency of cyp121 ligands . comparison of the x - ray crystal structures of 2 , 4 , and 5 with those of heme binding 
 fragments 8 and 9 , which had been identified 
 during our original fragment screen on cyp121 , indicated that extending from the meta - position of ar2 with substituents 5.56.5  in length 
 would provide the appropriate proximity for metal binding ( figure 4b ) . a range of substituents 
 containing aromatic or basic nitrogen atoms were selected as functional 
 groups commonly occurring in heme binding p450 inhibitors . primary 
 anilines were prioritized because of the prevalence of this chemotype 
 in fragment hits identified during our original fragment screen on 
 cyp121 and also from our subsequent optimization attempts which had 
 yielded several co - crystal structures of cyp121 with aniline ligands . analogues derived from both monoaryl 4 and biaryl 5 fragment scaffolds were proposed 
 to maximize le and to allow the importance of ar1 on the binding mode , 
 affinity , and selectivity of the compounds to be investigated ( figure 4b ) . a range of analogues 
 in which the 4-hydroxy group of ar2 was removed were also designed 
 in order to determine whether torsional strain resulting from interactions 
 between the 4-hydroxy group and metal - binding substituents introduced ortho to this might perturb the geometry required for metal 
 coordination . it was hypothesized that the loss of enthalpic contributions 
 from hydrogen bonds made by the 4-hydroxy group of ar2 was likely 
 to be small in comparison to that gained by metal - coordination . a fragment growing 
 approach was devised 
 to explore the sar of ar3 and to improve the ge of this motif by potentially 
 introducing binding interactions with residues in the back of the 
 active site . interrogation of the x - ray crystal structure of lead 2 and ligand docking studies were 
 used to select an appropriate linker - length for growing , with methylene 
 or carbonyl linkers being prioritized . a range of five- and six - membered 
 ( hetero)aromatic groups were selected to incorporate onto these linkers , 
 including phenols as direct mimics of lead 2 and the 
 natural cyy substrate , phenol bioisosteres , and substituents that 
 were predicted by docking simulations to form favorable cation 
 interactions with a nearby arg386 residue , or polar interactions with 
 amide groups of the protein backbone ( figure 4c ) . analogues varying at ar1 were synthesized 
 according the published procedure for biaryl retrofragment 5 ( scheme 1 ) . in brief , the claisen condensation of 4-methoxyphenylacetonitrile 11 with the appropriately substituted benzoate ester 10a  d afforded the -ketonitrile 
 intermediates 12a  d . reaction of 12a  d with hydrazine hydrate and concentrated 
 hydrochloric acid gave the 5-aminopyrazoles 13a  d in excellent yields ( 9197% ) . subsequent deprotection 
 of the aryl - methyl ether(s ) with bbr3 ( 1.0 m in dcm ) gave 
 the desired phenol analogues 5 and 14b  d in near quantitative yields . reagents and conditions : ( a ) 
 nah , thf , 60 c , 2048 h ; ( b ) hcl ( 37% ) , nh2nh2xh2o , etoh , 90 c , 14 h ; ( c ) 
 bbr3 ( 1.0 m in dcm ) , dcm , 0 c , 6 h. synthesis and 
 characterization of 14d previously described . the syntheses of analogues 
 incorporating a metal binding functional 
 group at the meta position of ar2 are summarized 
 in scheme 2 ( i ) and 
 ( ii ) for analogues based on the monoaryl 4 or biaryl 5 retrofragment scaffolds , respectively . analogues retaining 
 the 4-hydroxy substituent of ar2 were synthesized from the appropriately 
 protected 4-benzyloxy 15b or 4-methoxy 15c substituted 3-bromophenylacetonitrile starting materials , which 
 were either obtained directly from commercial sources or synthesized 
 in two steps by deprotection of the aryl methyl ether using bbr3 followed by benzyl protection under finkelstein conditions . synthesis of the common 5-aminopyrazole ring followed a similar route 
 to that employed to access ar1 analogues ( scheme 1 ) . claisen condensation of phenylacetonitriles 15a  c with either ethyl formate or methyl 
 4-methoxybenzoate afforded the 3-oxopropanenitrile 16a  c or -ketonitrile 21a  b intermediates , respectively . the crude products were treated 
 with hydrazine hydrate at acidic ph and heated under reflux to give 
 the 5-aminopyrazole compounds 17a  c and 22a  b in yields of 4867% 
 and 7795% , respectively , over two reaction steps . a microwave - assisted 
 suzuki  miyaura cross - coupling reaction was used to introduce 
 a range of nitrogen - containing heterocycles 18e  i or nitro - substituted aromatics 18a  d , 23a  b from their boronic 
 acid precursors . nitroarenes were subsequently reduced using tin(ii ) 
 chloride to reveal the heme binding anilines 19a  d , 24a  b in yields up to 
 77% . removal of the methyl or benzyl protecting groups using bbr3 afforded the 4-hydroxyphenyl ar2 analogues 20a and 25a  b , which were purified 
 by flash chromatography to give 4650% yields . however , the 
 presence of inorganic contaminants in 20a prevented the 
 testing of this compound . reagents and conditions : ( a ) 
 ethyl formate , naoet , etoh , 090 c , 22 h ; ( b ) acoh , nh2nh2xh2o , etoh , 75 c , 3 h ; 
 ( c ) aryl - boronic acid , pd(pph3)4 , na2co3 , 1,4-dioxane : h2o ( 1:1 v / v ) , w , 180 
 min ; ( d ) sncl22h2o , etoh , 75 c , 
 15 h ; ( e ) bbr3 ( 1.0 m in dcm ) , dcm , 0 c , 4 h ; ( g ) 
 methyl 4-methoxybenzoate , nah , thf , 60 c , 48 h ; ( h ) hcl ( 37% ) , 
 nh2nh2xh2o , etoh , 90 c , 
 16 h. ar3 analogues containing a methylamine - linked 
 substituent 26a  f were synthesized 
 from the reductive 
 alkylation of biaryl retrofragment 5 with the respective 
 aldehyde ( scheme 3 ) . 
 reaction conditions , including solvent polarity , acid catalyst , and 
 use of either sodium triacetoxyborohydride or sodium cyanoborohydride 
 reducing reagents were optimized for individual substrates . analogues 26 g and 26h were synthesized 
 in good yields ( 7895% ) under similar conditions by reductive 
 alkylation of the dimethoxy protected precursor 13a . subsequent deprotection of intermediates 27a  b with bbr3 revealed the diphenols 26g  h in 6168% yields . reagents and conditions : ( a ) 
 aldehyde ( 0.91.1 equiv ) , acoh * , na(oac)3bh , dmf , 
 dce , rt , 2448 h ( b ) aldehyde ( 1 equiv ) , acoh , nacnbh3 , meoh , rt , 1524 h ; ( c ) bbr3 ( 1.0 m in dcm ) , dcm , 
 0 c , 224 h. * no acoh with 26d . a series of 5-amidopyrazole analogues 29a  c containing a carbonyl linker were synthesized 
 from 5-aminopyrazole 13a and the respective benzoyl chloride 
 to give 28a  c ( scheme 4 ) . benzoyl chloride electrophiles were either 
 purchased 
 or synthesized in situ from the corresponding benzoic acid precursor 
 using oxalyl chloride and catalytic dmf . yields of the desired 5-amido 
 regioisomers were low ( 2734% ) , but significant polarity differences 
 enabled easy separation from endocyclic 1n - substituted byproducts 
 using column chromatography . subsequent deprotection of the aryl - ethers 
 with bbr3 afforded the analogues 29a  c . reagents and conditions : ( a ) 
 ( 4-ome)-benzoyl chloride , et3n , dcm , 0 c  rt , 
 3.5 h ; ( b ) nicotinic acid , oxalyl chloride , dmf , dcm , 0 c  rt , 
 2 h , then et3n , dcm , 0 c  rt , 14 h ; ( c ) bbr3 ( 1.0 m in dcm ) , dcm , 78 c  rt , 14 h. the binding mode of analogues was 
 initially characterized using uv  vis spectroscopy to allow 
 the identification of ligands that interacted with the ferric iron , 
 or with the axial water ligand of the heme cofactor , from changes 
 in the optical spectrum of cyp121 . p450 inhibitors which displace 
 the axial water ligand and directly coordinate to the ferric iron 
 of the heme cofactor , typically by the lone pair electrons of a nitrogen 
 atom , stabilize an inactive state of the enzyme by sterically blocking 
 the active site and usually lowering the reduction potential of the 
 enzyme . this binding mode ( type ii ) produces 
 a red - shift in the soret band of the p450 optical spectrum , the magnitude 
 ( max , nm ) of which is dependent on both the 
 affinity ( kd ) and concentrations of the 
 ligand and p450 under specific assay conditions . in contrast , ligands which interact with the heme cofactor 
 via water - bridged hydrogen bonding to the axial water ligand , or which 
 displace the axial water but do not directly coordinate to the ferric 
 iron , can produce reverse type i and type i spectra , respectively . a single shot uv  vis absorbance 
 assay was used to rapidly identify ar2 substituents with the appropriate 
 properties and coordination geometry for heme binding interactions 
 ( table 1 ) . a preliminary 
 series of ar2 analogues , substituted at the meta - position 
 with aniline or heteroaromatic groups , were synthesized based on the 
 monoaryl scaffold 4 ( scheme 2 ( i ) ) . only primary 3- or 4-aminophenyl analogues 19a and 19d were found to cause a significant 
 red - shift ( max > 1 nm ) in the soret band 
 of cyp121 , which occurs at 416.5 nm in the water - ligated resting state . indole 18g , h , pyrazole 18i , and 3-pyridyl 18e substituents showed no 
 heme binding interactions ( max = 0 nm ) , and 
 only minor perturbation of the soret band was observed for the 4-pyridyl 
 isomer 18f ( max = 1 nm ) . the change in the 
 wavelength of the soret band ( max , nm ) of 
 cyp121 ( 5 m ) was calculated as the difference of that found 
 in the presence of analogues compared to 1% v / v dmso - d6 alone ( max = 416.5 nm ) . binding affinities 
 ( kd ) for selected compounds were calculated 
 from the titration of compounds ( 0.2 l aliquots of 10100 
 mm stocks ) into cyp121 ( 5 m ) . the binding affinities of the 3-aminophenyl 19a ( kd = 14 m ) and 4-aminophenyl 19d ( kd = 230 m ) substituted 
 analogues 
 were determined by optical titration according to the previously described 
 procedure . the 16-fold difference in affinity 
 between the two regioisomers clearly indicated that the 3-aminophenyl 
 substituent provided a better geometry for heme ligation , and consequently 
 this functional group was incorporated into all subsequent analogues 
 in the ar2 series . all subsequent ar2 analogues containing a 
 3-aminophenyl substituent 
 ( 19b , c , 24a , 25a , b ) caused type ii shifts in the cyp121 optical spectrum , 
 allowing their binding affinity to be determined by optical titration 
 in addition to itc ( table 2 and figure 5 ) . the magnitude of the type ii shift decreased 
 with increasing steric bulk ortho to the 3-aminophenyl 
 substituent . these results supported the hypothesis that torsional 
 strain in the biphenyl system , or the steric bulk of the benzyl or 
 methyl hydroxy protecting groups , in analogues 19b and 19c , respectively , prevented the ligands approaching the cofactor 
 for heme coordination . binding affinity of 14d previously reported . binding affinity 
 ( kd , m ) values for all analogues 
 were measured by 
 itc . the kd values for the ar2 series 
 of heme binding compounds were also determined by uv  vis optical 
 titration as described previously . ligand 
 efficiency ( le ) was calculated from the gibbs free energy of binding 
 divided by number of heavy atoms ( g / ha ) . optical titration difference spectra ( top panel ) and concentration - dependent 
 changes in cyp121 absorbance ( bottom panel ) for representative heme 
 binding ligands ( a ) 24a , ( b ) 25b , and ( c ) 19a . absorption spectra of cyp121 ( 5 m ) were collected 
 between 250 and 800 nm after sequential additions of 0.2 l 
 aliquots of dmso - d6 stock solutions ( 0.1583 
 mm ) of ligands . difference spectra were obtained from the subtraction 
 of the ligand - free cyp121 spectra from those obtained over the course 
 of the titration . the maximum change in absorbance from each difference 
 spectrum was plotted against ligand concentration and data were fitted 
 using a modified version of the morrison equation for tight binding 
 ligands 24a and 25b(17 ) or a hyperbolic function for 19a . the only analogue in the ar1 or ar3 series of compounds to 
 perturb 
 the soret band was the 3(4)-imidazole - methylamine substituted compound 26h . the type ii shift observed was small ( max26h ( 100 m ) = + 1.5 nm ) compared to 
 that observed for the 3-aminophenyl substituted ar2 analogues ( max25a [ 100 m ] = + 5.0 nm ) , which was consistent 
 with results observed for heteroaromatic nitrogen substituents during 
 our initial selection of a heme binding functional group for ar2 analogues 
 ( table 1 ) . a 2.0  
 x - ray crystal structure of 26h in complex with cyp121 
 indicated that the compound must adopt multiple different binding 
 modes , as the 3(4)-imidazole substituent of the crystallized compound 
 resided in the ar3 pocket , distal to the heme cofactor ( figure 8b ) . these competing binding 
 modes might account for the weak affinity of 26h ( kd = 274 m ) compared to the structurally 
 similar 3-thiophenyl 26c or 2-furanyl 26d methylamine analogues , which did not perturb the optical spectrum 
 of cyp121 ( table 2 ) . owing to a lack of spectral perturbation , the binding affinity of 
 all other ar1 and ar3 analogues was assumed to result from binding 
 interactions made with residues in the distal active site pocket and 
 provided support that these analogues retained a similar binding mode 
 to lead 2 . the binding 
 affinity ( kd ) of analogues for cyp121 
 was determined by itc ( table 2 , figure 6a  c ) . comparison of the kds obtained for the 
 ar2 series by itc and optical titration enabled robust comparisons 
 to be made with the affinity of the non - heme binding ar1 and ar3 series 
 of analogues , as well as with previously reported type ii inhibitors 
 in the literature . in general , kd values obtained by optical 
 titration were 10100-fold lower than those measured in itc , 
 consistent with previous reports in the literature . variations in affinity data generated using different biophysical 
 techniques have been previously attributed to the kinetically significant 
 but spectrally silent , multistep binding interactions of p450s with 
 ligands , the potential presence of heterogeneous enzyme populations 
 in solution , requirement for conformational change , and/or enzyme 
 cooperativity . high resolution ( 1.62.2 
  ) x - ray crystal structures of cyp121 in complex with a number 
 of analogues were obtained , allowing inhibitor binding modes to be 
 confirmed and enabling robust comparison of the sar contributing to 
 binding affinity ( figure 7,8 ) . representative itc binding isotherms and 
 integrated enthalpy plots 
 for ( a ) fused ar1ar3 fragment 6 ( 500 m ) ; 
 ( b ) non - heme binding ar3 analogue 26a ( 1 mm ) ; ( c ) heme 
 binding analogue 25a ( 500 m ) . the isotherm of 
 each compound titrated into buffer alone was subtracted from that 
 containing cyp121 ( 50 m ) . the integrated enthalpy change for 
 each injection plotted as a function of molar ratio of ligand - to - cyp121 was fitted using a one - site equilibrium binding model to calculate 
 binding affinity ( kd ) . the binding stoichiometry 
 ( n ) was allowed to vary for analogues 6 , 26a , and 25a . changing the position of the original 
 4-hydroxy group to the 3-position 14b resulted in a moderate 
 reduction in binding affinity , while substitution with a 3-fluoro 
 substituent increased the kd of 14c 5-fold compared to 5 . these results suggest 
 that aromatic stacking interactions made by ar1 contribute to binding 
 affinity in this region . the change in -electron density from 
 the introduction of m - hydroxy or m - fluoro substituents would likely weaken both offset face - to - face 
 aromatic interactions with phe168 and edge - to - face interactions with 
 trp182 , in addition to disrupting hydrogen bonding interactions with 
 two conserved water molecules shown to solvate the p - hydroxy substituent of fragments 1 , 5 , 
 and lead compound 2 ( figure 1 and 2d ) . disubstitution of ar1 with hydroxy groups in 
 the 2- and 4-positions , 14d , also lowered the binding 
 affinity ( kd = 180 m ) despite introducing 
 polar interactions with residues thr77 and ala167 as shown in the 
 x - ray crystal structure of 14d . intramolecular hydrogen bonding between the 2-hydroxy group and 
 the 1-nh - pyrazole heterocycle might account for this 
 result by favoring the 3-aminopyrazole tautomer and thus weaken bonding 
 interactions to gln385 . no overall change in the conformation of the 
 ligands was observed due to changes made to ar1 substituents , consistent 
 with our initial docking results . the 
 sensitivity of ar1 to even minor structural changes confirmed our 
 previous predictions from retrofragmentation and ge analysis that 
 binding interactions in this region represented a key affinity hotspot 
 for cyp121 that might be of use during later stages of compound development . incorporation of a 
 3-aminophenyl substituent 
 onto the ar2 ring of the monoaryl scaffold 4 or the biaryl 
 scaffold 5 resulted in a more than 100-fold improvement 
 in binding affinity . analogue 19a had an equivalent kd of 14 m ( le   =   0.35 ) 
 by optical titration to that of lead compound 2 and was 
 made significantly more le by removal of the ar3 motif . the reincorporation 
 of the ar1 phenol ring to give compound 25b ( kd = 0.29 m ) resulted in a further 50-fold 
 improvement in binding affinity , as had been previously observed during 
 our lead - deconstruction analysis when the ar1 and ar2 fragments 3 and 4 were fused to give biaryl retrofragment 5 ( figure 3 ) . removal of the ar2 4-hydroxy group of 25b to relieve 
 torsional strain in the heme binding biphenyl - amine system improved 
 cyp121 affinity a further 20-fold to produce the low nanomolar , high 
 le compounds 24a and 25a . while the 4-hydroxy 
 analogue of 19a was not analyzed , the effect of ortho - substitution on weakening heme binding interactions 
 was made apparent through assessment of the 4-benzyloxy and 4-methoxy 
 substituted analogues 19b and 19c . while 
 both 19b and 19c caused type ii shifts in 
 the cyp121 soret band at high concentrations ( 1001000 m ) , 
 the bulky protecting groups clearly prevented ligands obtaining the 
 optimal geometry for heme ligation and a kd could not be obtained for these compounds by optical titration within 
 solubility limits . the relatively good binding affinity of 19b ( kd = 28 m ) determined by itc 
 suggested that this compound might instead adopt an alternative  heme - silent  
 binding mode in the distal active site . the improved le of 19a , 24a , and 25a  b compared to lead 2 ( le 
 = 0.24 ) was reflected in the good ge value of 0.42 calculated for 
 the 3-aminophenyl substituent based on the introduction of seven heavy 
 atoms to retrofragment 5 . comparison 
 with the low ge of 0.08 calculated previously for the ar3 phenol ( figure 3 ) , which had the 
 same number of heavy atoms , demonstrated the utility of our lead deconstruction - elaboration 
 approach in allowing the identification of maximally efficient scaffolds 
 and in targeting binding hotspots . the 2.1  crystal structure 
 of 25b ( figure 7a ) , the structurally most similar compound 
 to retrofragment 5 , in complex with cyp121 revealed that 
 the analogue accurately 
 replicated the conserved binding mode of the lead 2 ( figure 1 ) . all intermolecular 
 interactions that had been identified as binding hotspots , such as 
 aromatic stacking of ar1 with phe168 and trp182 , interaction of the 
 ar2 4-hydroxy group with a water - bridged heme propionate network , 
 and hydrogen bonding between the aminopyrazole ring and gln385 were 
 satisfied . the 3-aminophenyl substituent was located 2.5  from 
 the heme - iron and at an angle of 36 , consistent with that required 
 for tetrahedral nh2fe(iii ) coordination . hydrogen bonding interactions were also identified 
 between the 3-amino group of 25b and ser237 , which is 
 thought to be the conserved alcohol residue of cyp121 required for 
 the catalysis of proton transfer to the heme iron and for organization 
 of the axial water ligand . x - ray crystal structures 
 of heme binding ar2 analogues 25b ( pdb 5ibj ) , 25a ( pdb 5ibe ) , 
 and 19a ( pdb 5ibf ) in complex with cyp121 . ( a ) 25b ( cyan ) 
 closely recapitulated the binding of lead 2 , 
 forming polar contacts ( yellow dashes ) with amino acids ( blue sticks ) 
 in binding hotspots as well as additional binding contacts to ser237 , 
 val228 , and the heme iron . ( b ) 25a ( orange ) was rotated 
 away from the i - helix ( yellow cartoon ) , placing the 5-aminopyrazole 
 ring in the ar3 pocket . polar contacts to the heme iron and ser237 
 are indicated ( yellow dashes ) . ( c ) two molecules of 19a bound per active site , each ligand fulfilling a subset of the interactions 
 identified as binding hotspots . inset : heme binding 19a recapitulated the binding mode of 25a while the non - heme 
 binding 19a ligand satisfied aromatic interactions in 
 the ar1 pocket , as well as the hydrogen bonding interactions of fragment 7 . ( d ) extracted structures of 25b , 25a , and 19a annotated to show metal coordination ( yellow 
 dashes ) distance , angle of approach to the porphyrin ring ( yellow 
 angle ) , and dihedral torsion in the biphenyl - aniline motif . removal 
 of the ar2 4-hydroxy group reduced the dihedral angle of the biphenyl 
 aniline system in 19a and 25a by 30 
 compared to 25b and allowed the analogues to approach 
 the plane of the porphyrin at a sharper angle ( 3133 ) . 
 the omit fo  fc electron density maps of ligands 19a , 25a , and 25b contoured to 3 the 1.6  crystal structure 
 of 25a confirmed 
 that removal of the ar2 4-hydroxy group resulted in a 30 reduction 
 in the dihedral angle of the biphenylamine motif ( figure 7b , d ) . as for analogue 25b , the 3-aminophenyl substituent of 25a bound 
 in close proximity ( 2.3  ) and at an appropriate orientation 
 ( 34 ) for heme coordination . however , reduced rotation in the 
 biphenyl system allowed a slightly sharper angle of approach to the 
 heme ring . close overlap between the aniline ring of 25a and the sterically unhindered aniline - containing fragments 8 and 9 suggested that aromatic stacking interactions 
 were optimized by the removal of the 4-hydroxy substituent from 25b . these improved interactions might account for the 9-fold 
 improvement in potency between the two analogues and illustrate the 
 utility of fragments as tools for elucidating preferred binding motifs 
 ( figure 4b , and figure s3 , supporting information ) . the combination 
 of improved heme interactions and removal of functional groups identified 
 to interact with binding hotspots , such as the ar2 4-hydroxy group , 
 resulted in a change in orientation of 25a compared to 
 the conserved position of 25b and lead 2 . analogue 25a was tilted sideways to place the exocyclic 
 5-amino group of the pyrazole ring into the pocket normally occupied 
 by ar3 , resulting in a loss of interactions with gln385 . the fact 
 that 24a retained good affinity despite this loss of 
 interactions with gln385 was consistent with observations made about 
 the binding of retrofragments during lead deconstruction ( vide supra ) . the equipotent ( kd = 33 nm ) 4-methoxy 
 protected precursor 24a was found to bind in an identical 
 orientation to 25a , demonstrating some tolerance of the 
 ar1 group to substituent variation ( figure s4 , supporting information ) . two molecules of analogue 19a , which can be considered 
 a fragment of 25a ( lacking only the ar1 ring ) , were found 
 to bind within each active site of the cyp121 crystallographic dimer 
 ( figure 7c ) . the orientation 
 of the two overlapping molecules of 19a illustrated both 
 the importance of the ar1 region as a binding hotspot and also demonstrated 
 optimization strategies which might further improve selectivity . one 
 molecule of 19a ligated the heme iron as observed for 25a , with a coordination distance of 2.4  and a 32 
 angle of approach to the heme ring . the second molecule of 19a satisfied the requirements of the remaining binding hotspots , 
 including hydrogen - bonding interactions with gln385 and aromatic stacking 
 interactions in the ar1 site . extension of the 3-aminobiphenyl system 
 into the ar1 pocket demonstrated a route to achieving the additional 
 hydrogen bonding interactions with the f / g - helix residues thr77 , ala178 , 
 and asp185 that had earlier been suggested by the identification of 
 hit fragment 7 ( figure 7c , inset and figure 2a ) . the binding affinity 
 of ar3 analogues 
 was largely unaffected by the structural changes made to the 5-aminopyrazole 
 substituent . analogues substituted with a range of aromatic or heteroaromatic 
 groups maintained low micromolar binding affinities ( kd < 100 m ) , and a number of compounds ( 26b  d ) had comparable potency to lead 2 ( kd = 15 m ) ( table 2 ) . efforts to grow 
 further out into the active site pocket occupied by ar3 by the addition 
 of a methylene linker resulted in a small improvement in binding affinity 
 with a kd of 6.3 m determined for 
 the 4-hydroxybenzyl analogue 26a , making it the most 
 potent non - heme binding cyp121 ligand described to date . the poorly 
 defined electron density of the 4-hydroxybenzyl ring in the x - ray 
 crystal structure of 26a suggested that it was conformationally 
 flexible and only interactions with active site solvent molecules 
 were observed ( figure 8a ) . all ar3 analogues containing methylamine - linked 
 aromatic substituents were more potent than their carbonyl - linked 
 amido - pyrazole counterparts 29a  c . for example , comparison of 4-hydroxy - benzyl 26a and 
 benzamide 29b analogues or 4-pyridinyl - methylamine 26 g and isonicotinamide 29c analogues revealed 
 a 2.516-fold loss in affinity , respectively , for the carbonyl - linked 
 analogues . increased conformational rigidity enforced by the 5-amido 
 group or changes to the hydrogen bond donor  acceptor interactions 
 of the 5-amino - nh with gln385 a 
 preference for 4-hydroxy substituted aromatics was observed in both 
 the methylamine ( 26 ) and amide - linked ( 29 ) analogues , which was unsurprising considering the structure of 
 the natural cyy cyp121 substrate . the addition 
 of bromine ortho to the 4-hydroxy group in analogue 26b had little effect on binding affinity and indicated that 
 larger substituents could be tolerated in this region of the active 
 site . indole 26e and indazole 26f bioisosteres 
 of the 4-hydroxyphenyl group resulted in a 34-fold increase 
 in kd , possibly indicating that the greater 
 acidity or hydrogen bonding ability of the hydroxyl group was favorable 
 for binding . further supporting this assumption , analogues containing 
 basic aromatic nitrogens , such as the pyridinyl 26 g or 
 3(4)-imidazole 26h methylamine - linked analogues , significantly 
 weakened cyp121 binding affinity . x - ray crystal structures of ar3 analogues 
 ( a ) 26a ( pdb 5ibi ) and ( b ) 26h ( pdb 5ibh ) 
 in complex with cyp121 . both compounds closely recapitulated the binding 
 mode of lead 2 . ar3 substituents remained flexible , interacting 
 only with active site water molecules for 26a or forming 
 novel interactions with active site residues ala75 and asn74 for 26h . the omit fo fc electron density maps of ligands 26a and 26h contoured to 3 have been provided in 
 supporting information figure s5 . figures 
 prepared using pymol v1.7.4 ( schrdinger , llc ) . as previously highlighted , a 2.0  x - ray crystal 
 structure 
 of 26h in complex with cyp121 was obtained that showed 
 the imidazole ring binding distal to the heme cofactor in the ar3 
 pocket ( figure 8b ) . hydrogen bonding interactions with asn74 and ala75 had not been previously 
 observed in other cyp121ligand structures and could provide 
 a new route for compound optimization . despite these novel interactions 
 the binding affinity of 26h was weak ( kd = 274 m ) , which could have been the result of 
 the mixed binding modes suggested by uv  vis spectral analysis 
 ( vide supra ) . the consistency of the binding mode of 26a and 26h with lead 2 demonstrated in x - ray 
 crystallography provided confidence when interpreting the sar of the 
 remainder of the ar3 analogues . the binding affinity of thiophenyl26c or furanyl26d methylamine - linked 
 analogues was comparable to that of lead 2 and tolerance 
 to these smaller , hydrophobic heterocycles might enable the pharmacokinetic 
 properties of subsequent analogues to be modulated during later phases 
 of drug development . the insensitivity of the ar3 pocket to structural 
 modifications could indicate a functional adaption of the enzyme to 
 enable the accommodation of different substrates . recent evidence 
 that cyp121 binds a number of other cyclic dipeptides , including cyf 
 and cyw , supports this proposal . the relative affinity 
 of lead compounds , their binding stoichiometry to cyp121 and their 
 effect on the oligomerization state of the enzyme were investigated 
 using native mass spectrometry . . however , some form higher homomeric 
 oligomerization states , heterodimers with their cognate reductase 
 proteins , or are expressed as fusion products covalently linked to 
 a reductase domain . we have previously reported that 
 cyp121 exists predominantly as a dimer in solution , with a small percentage 
 of monomeric protein present . dimeric 
 cyp121 forms complexes with two ligand molecules simultaneously and 
 the dimer interface does not appear to occlude the substrate entry 
 channel . the stability of the dimer is reduced by the binding of azole 
 antifungal compounds . in contrast , other small molecule cyp121 inhibitors 
 and the cyp121 substrate ( cyy ) form stable complexes with the protein 
 and do not affect the oligomerization state . native mass spectra were collected for cyp121 with the most 
 potent ar3 analogue 26a , ar2 analogues 19a , 24a , 25a , and 25b and the 
 biaryl retrofragment 6 ( figure 9a  c ) . the proportion of ligand - bound cyp121 increased in a concentration - dependent 
 manner as the ratio of ligand - to - protein was increased from sub- ( 0.5:1 ) 
 to superstoichiometric ( 25:1 ) values . the relative occupancy of monomeric 
 and dimeric cyp121 and binding stoichiometry observed at three different 
 ligand concentrations allowed analogues to be ranked in terms of their 
 relative binding affinities ( table s1 , 
 supporting information ) . the rank order of affinities correlated well 
 with kd values calculated previously by 
 itc and uv  vis optical titration ( table 2 ) . both the cyp121 monomer and dimer were 
 entirely in the bound state at stoichiometric ligand - to - protein concentrations 
 of ar2 analogues 25a and 25b , consistent 
 with the low nanomolar affinities of these compounds . in contrast , 
 complete occupancy of the cyp121 dimer by the more weakly binding 
 analogues 19a and 26a was not achieved until 
 ligands were present in 25-fold excess ligand - to - protein concentrations . 
 despite the comparable kd of retrofragment 6 to ar2 analogue 19a , the cyp121 dimer remained this apparent difference in binding strength between 6 and 19a observed by native mass spectrometry might 
 reflect the different binding mode of the two analogues . heme binding 
 interactions made by 19a would likely be strengthened 
 in the gas phase of the mass spectrometer , while the closer proximity 
 of 6 to the entrance of the active site could allow the 
 ligand to be displaced more easily during ionization . the double - occupancy 
 of the cyp121 monomer observed for analogue 19a in x - ray 
 crystallography was not detected within the concentration ranges tested 
 in native mass spectrometry experiments ( 2.5218 m ligand ) . representative 
 native mass spectra of cyp121 and selected compounds 26a , 25a , and 19a demonstrated that 
 compounds formed stable complexes with both monomeric ( m / z 52506750 ) and dimeric ( m / z 52506750 ) cyp121 . the number of bound 
 ligands ( orange spheres ) increased in a concentration - dependent manner 
 and was proportionate to the binding affinity ( kd ) of the compounds . spectra were collected for cyp121 ( 8.7 
 or 5 m ) at three different ligand - to - protein ratios ( 0.5:1 , 
 1:1 , and 25:1 ) and in the presence of dmso - d6 ( 2.2% or 5% v / v ) alone . the concentration of protein or dmso - d6 did not affect the quality of the spectra 
 or ligand binding interactions , but higher concentrations of dmso - d6 reduced the charge state of the protein . ligand 
 binding stoichiometry was calculated from the difference in mass of 
 ligand - bound and unbound ( orange dashed lines ) protein peaks divided 
 by the molecular weight of ligands ( table s1 , supporting information ) . no significant change in the oligomerization state of cyp121 
 was 
 observed for any of the analogues compared to that previously noted 
 with the azole antifungals ( table s2 , supporting 
 information ) . the different effects of 
 ar2 analogues and azole antifungal compounds on the oligomerization 
 state might reflect the nature of the metal binding functional group 
 in each series . greater conformational reorganization of the cyp121 
 active site might be expected in order to accommodate axial coordination 
 of an sp - hybridized imidazole ring ( 75 ) , while the primary aniline present in the current 
 analogues approaches the porphyrin at a sharper angle ( 3136 , figure 7d ) . selectivity 
 screening against off - target microbial p450s is far less common during 
 pharmaceutical development than screening against mammalian p450s 
 and consequently the functions and promiscuity of these enzymes remain 
 largely uncharacterized . all of the mtb p450s that 
 have been studied to date bind with varying affinity to compounds 
 from the azole series of antifungal drugs , but there have been few 
 reports detailing their interactions with non - imidazole / triazole ligands . compounds from our development of cyp121 inhibitors were screened 
 against a panel of mtb p450s using uv  vis 
 spectroscopy ( table 3 ) to both assess their selectivity and also to aid the development 
 of chemical tools which could be used to help characterize these p450 
 systems in mtb . the 
 binding mode of compounds 
 is indicated as type ii heme binding ( ii , orange ) , binding to the 
 distal pocket ( n , green ) , or nonbinding ( x , unshaded ) . binding was 
 determined using a single shot uv  vis assay in which analogues 
 ( 100 m or 1 mm ) were added to a solution of each p450 enzyme 
 ( 2.55 m ) and the absorbance spectrum was collected 
 between 800 and 250 nm . compounds were prepared as dmso - d6 stocks and added to give a maximum concentration of 
 1% v / v dmso - d6 . the change in the maximum 
 wavelength of the soret band ( max , nm ) compared 
 to that measured for each p450 in buffer containing 1% v / v dmso - d6 was used to assess the binding mode and the 
 relative strength of binding . compounds that did not change the soret 
 max were tested in competition with known type ii 
 inhibitors ( 50 m ) , with compounds that decreased the max compared to that observed in the presence of the type ii 
 inhibitor alone being designated as  non - heme binding the binding affinity of analogue 25a was calculated by direct or competitive optical titration , as described 
 previously , depending on the mode of binding to each p450 respectively . * kd calculated by itc for analogue 25b . a qualitative assessment of the 
 binding promiscuity of the cyp121 
 ligands developed in this study was obtained by measuring the change 
 in the absorbance maximum ( max , nm ) of the 
 characteristic soret band of each p450 isoform when incubated with 
 the ligands ( 100 m to 1 mm ) compared to a dmso ( 1% v / v ) control . binding interactions made by the compounds with the distal part of 
 the p450 active site , which do not directly perturb the heme environment 
 or max of the soret band , were characterized by 
 measuring the decrease in the max caused 
 by known type ii ligands when mtb p450s were coincubated 
 with mixtures of the type ii ligand and the novel cyp121 inhibitors . the binding affinity of the most potent cyp121 compound 25a has been provided to give a quantitative assessment of ligand selectivity 
 across the different p450 isoforms . experiments were performed as 
 direct titrations for mtb p450s cyp121 , cyp126 , and 
 cyp142 , or in competition with a known type ii inhibitor where 25a exhibited a binding mode that did not influence the max of the heme absorbance spectrum , as in the case of mtb p450 isoforms cyp124 and cyp125 . owing to the weak binding 
 affinity of 25a for the mtb p450 cyp144 , 
 a kd was obtained instead for the related 
 compound 25b by itc . subsequent to commencing work 
 on these compounds , the original 
 4-(1h-1,2,4-triazol-1-yl)phenol fragment hit 1 for cyp121 had been identified to bind to all of the p450s 
 reported here in ligand - observed nmr experiments ( unpublished data ) . the biaryl retrofragment 5 , which consisted of the fused 
 ar1 and ar2 motifs of lead 2 and could be seen to represent 
 a  merged  analogue of hit fragment 1 , 
 was less promiscuous , only binding to five of the seven p450s screened . elaboration of 5 to include an ar3 substituent , as seen 
 in the 4-hydroxybenzyl substituted analogue 26a , further 
 enhanced cyp121 selectivity . in contrast , the incorporation of a heme 
 binding 3-aminophenyl substituent onto the scaffold of 5 , as in 25a and 25b , reduced the selectivity 
 for inhibitors for cyp121 , as might be expected from the introduction 
 of a binding group targeting a conserved active site feature . while 
 type ii spectral changes were identified for 4 of the 7 p450s studied , 25a and 25b bound in positions that did not perturb 
 the p450 heme spectrum in 3 of the 7 isoforms tested , illustrating 
 the preference of different p450s for certain metal - binding pharmacophores 
 and the impact of protein microenvironment on metal chelation . the kd of 25a varied between 
 0.44 and 27 m across the different p450 isoforms , which gave 
 a 301800-fold selectivity ratio ( sr = off - target binding affinity / on - target 
 binding affinity ) for cyp121 . the low 
 solubility of ar2 analogue 19a limited testing to the 
 lower concentration of 100 m which , in combination with the 
 small size of the compound , possibly accounted for the apparently 
 greater selectivity observed in the single - shot uv  vis assay . these results demonstrated how optimizing ligand affinity by targeting 
 binding interactions made in the distal pocket , as through the introduction 
 of ar1 or ar3 motifs , can enhance drug selectivity between p450s . drug  drug interactions 
 resulting from the inhibition of human p450s , or toxicities resulting 
 from the metabolic activation of drugs by human p450s , are leading 
 causes of attrition during pharmaceutical development . thus , understanding 
 the sar influencing p450 promiscuity and isoform selectivity is a 
 topic of significant interest . the five human p450s cyp1a ( cyp1a1 
 and cyp1a2 isoforms ) , cyp2c9 , cyp2c19 , cyp2d6 , and cyp3a4 are responsible 
 for the phase i metabolism of more than 80% of marketed drugs . compounds 19a , 25a , and 26a were screened for inhibitory activity against 
 these five human p450s and were found to have good selectivity profiles , 
 showing weak or no inhibition ( ic50 > 10 m ) against 
 all five human p450 isoforms ( table 4 ) . the exception was compound 19a , which was a moderately potent ( 1 m < ic50 < 10 m ) inhibitor of cyp1a ( ic50 = 
 1.22 m ) , an isoform which has a documented preference for aromatic 
 and heterocyclic amine substrates . the 
 reincorporation of ar1 into analogue 19a to give potent 
 analogue 25a resulted in a 20-fold increase in ic50 against cyp1a ( ic5025a = 21.4 m ) . 
 when the corresponding improvement in cyp121 binding affinity for 
 the two compounds was also taken into account , this amounted to an 
 approximate 15000-fold better apparent selectivity ratio ( app sr = 
 off - target potency / on - target potency ) . the 4-hydroxybenzyl substituted ar3 analogue 26a did 
 not inhibit any of the human p450s by 50% within the tested concentration 
 range . taken together , the improved selectivity from reintroducing 
 ar1 to the heme binding analogue 19a to give 25a , and the high selectivity of the non - heme binding analogue 26a , supported our hypothesis that targeting variable residues 
 and hotspots in the distal pocket of the cyp121 active site could 
 be used to achieve p450 isoform selectivity . aromatic amines , such 
 as those present in the potent cyp121 ligands 25a , 24a , 25b , and 19a , are commonly 
 identified as potential toxicophores . however , considering 13% of 
 marketed drugs contain aniline functional groups , it is premature 
 to exclude all compounds with this functional group from drug - like 
 chemical space . the toxicity of aniline 
 structures requires metabolic activation , which is primarily mediated 
 by human p450s . the good selectivity 
 of cyp121 inhibitors , particularly compound 25a , demonstrated 
 here against human p450s , is promising for the development of this 
 series of cyp121 inhibitors , however further assessment against other 
 p450 , monooxygenase , and peroxidase enzymes is warranted . the selectivity 
 of compounds derived from lead 2 against human p450s 
 was good compared to that of other classes of p450 inhibitors that 
 have been reported recently and suggests that the 
 careful optimization of metal binding functional groups and targeting 
 active site residues are methods which could reduce p450 liability 
 during drug development . the concentration of analogues 19a , 25a , and 26a required to inhibit 
 the activity of human p450s by 50% ( ic50 , m ) was 
 calculated from a seven - point dose  response curve . compounds 
 that did not achieve 50% inhibition over the tested concentration 
 range 025 m were considered non - inhibitors . , 
 tolbutamide ; cyp2c19 , mephenytoin ; cyp2d6 , dextromethorphan ; cyp3a4 , 
 midazolam ) was detected by lc - ms / ms or fluorescence for cyp1a . reported 
 cyp1a activity represents the combined activities of both the cyp1a1 
 and cyp1a2 isoforms . the apparent selectivity ratio ( app sr = off - target 
 potency ( ic50)/on - target potency ( kd ) ) was calculated as a ratio of the ic50 ( m ) 
 of each compound for cyp1a versus their respective cyp121 kd ( m ) . the 
 ic50 values of known inhibitors were determined as positive 
 controls and are listed in table s3 of 
 the supporting information . the antimycobacterial activity of 
 the most potent analogues 19a , 24a , 25a , 25b , 26a , and biaryl retrofragment 5 was assessed in vitro against mtb h37rv . no significant inhibition of bacterial growth was detected for any 
 of the lead compounds ( mic90  50 m ) over 
 the tested concentration range ( 0100 m ) ( table s4 , supporting information ) . achieving 
 adequate intracellular drug exposure is a particular challenge for 
 tb therapeutics because of the unique cell - wall structure of the bacterium 
 and the expression of numerous multisubstrate efflux transporters . efforts to determine whether low membrane permeability or efflux 
 might account for the poor cellular activity of the potent cyp121 
 ligands described here are currently in progress . using a fragment - based approach , a novel series of potent and isoform 
 selective inhibitors of the essential mtb enzyme 
 cyp121 have been developed . an initial fragment screen and synthetic 
 optimization strategy yielded the previously reported non - heme binding 
 lead 2 , which had micromolar affinity for cyp121 but 
 low le . the deconstruction of lead compound 2 and synthesis 
 of component retrofragments 36 allowed 
 the ge of individual structural motifs ar1 , ar2 , and ar3 to be assessed . 
 analysis of the binding mode of retrofragments enabled the identification 
 of activity hotspots and the selection of more le scaffolds for synthetic 
 optimization . the rational incorporation of a metal - binding functional 
 group onto ar2 , guided by x - ray crystallography and uv  vis 
 spectroscopy , was used to achieve a 1000-fold improvement in binding 
 affinity over lead compound 2 , while the incorporation 
 of structural groups that projected into the ar1 or ar3 regions of 
 the distal active site was shown to modulate the isoform selectivity 
 of inhibitors against human p450s . extensive characterization of analogues 
 by x - ray crystallography , uv  vis spectroscopy , and native mass 
 spectrometry validated our design strategy , demonstrating that analogues 
 maintained binding interactions in identified activity hotspots and 
 formed stable complexes with the native cyp121 dimer . analogues with 
 low nanomolar potency ( kd = 15 nm ) for 
 cyp121 are reported , and these molecules have good selectivity against 
 human drug metabolizing p450s and a 30-fold selectivity ratio for 
 cyp121 over other mtb p450 isoforms . analysis of 
 selectivity data from this series provides insight into factors governing 
 the selectivity of p450s , including the effect of the protein microenvironment 
 and preference for specific metal - binding pharmacophores , which has relevance more broadly for drug development . further structural 
 optimization to improve the cellular activity of these analogues is 
 ongoing with the aim of developing novel classes of anti - tb agents . ( poole , uk ) or alfa aesar ( heysham , uk ) 
 unless otherwise specified . unless otherwise stated , reactions were 
 conducted under positive pressure of a dry nitrogen atmosphere . temperatures 
 of 0 c , 10 c , and 78 c were obtained 
 by cooling the reaction vessel in a bath of ice , salt , and ice or 
 acetone and dry ice ( co2(s ) ) , respectively . thf was freshly 
 distilled over calcium hydride and lithium aluminum hydride , using 
 triphenylmethane as an indicator . other anhydrous solvents were purchased 
 directly from commercial sources and used without further purification . analytical thin layer chromatography ( tlc ) was performed using 
 merck glass - backed silica ( kieselgel 60 f254 0.25 mm ) plates . compounds 
 were visualized using short wave ( 254 nm ) or long wave ( 365 nm ) ultraviolet 
 light or potassium permanganate stain . retention factors ( rf ) are quoted with respect to the solvent system 
 used to develop the plate . flash column chromatography was performed 
 using an isolera spektra one / four purification system and the appropriately 
 sized biotage snap column containing kp - silica gel ( 50 m ) . infrared absorption spectra were recorded 
 on a bruker alpha ft - ir 
 or spectrum one ft - ir ( perkinelmer ) spectrometer by attenuated total 
 reflectance ( atr ) using a diamond crystal . data are reported as vibrational 
 frequency ( cm ) and intensity ( strong , medium , 
 weak , or broad ) for the assigned functional group . nuclear magnetic 
 resonance ( nmr ) spectra were recorded at 300 k 
 unless otherwise stated , using either a bruker 400 mhz avance iii 
 hd smart probe , 400 mhz qnp cryoprobe , or 500 mhz dch cryoprobe spectrometer . spectra acquired on the 500 mhz dch cryoprobe have been converted 
 to analogue and back to reduce baseline roll . data are reported as chemical 
 shift in parts per million (  ppm ) relative to the residual 
 protonated solvent resonance peak . the relative integral , multiplicity , 
 coupling constants ( j hz ) , and assignment of peaks 
 has been provided where possible . assignment of h nmr 
 and c nmr spectra was assisted by dept , homonuclear ( cosy ) , 
 and heteronuclear ( edited h c nmr spectra were processed with line broadening ( lb ) of 
 112 hz for individual compounds to enable resolution of signals 
 from slowly relaxing quaternary pyrazole carbons c3 , c4 , and c5 and 
 resolution of closely overlapping aromatic signals . lb has been noted 
 for relevant spectra and c signals requiring lb for resolution 
 have been noted as broad ( br ) . c signals that were obscured 
 or were only identified through proton relaxation in 2d - nmr experiments 
 are also noted . liquid chromatography mass spectrometry ( lcms ) 
 was carried out 
 using an aquity uplc h - class system ( waters , manchester uk ) . samples 
 were run using a gradient of water ( 15% ) ( + 0.1% formic acid ) 
 in acetonitrile over a period of 4 min . high resolution mass spectra 
 ( hrms ) were recorded using a micromass quadrapole - time - of - flight ( q - tof ) 
 spectrophotometer or an orbitrap lcms spectrometer attached to a dionex 
 ultimate 3000 hplc . the mass to charge ratio ( m / z ) of the molecular ion and difference from calculated mass 
 (  ppm ) have been quoted . all final compounds had a purity 
 greater than 98% by lcms analysis , 
 except for compounds 14b , 18 g , 18f , 19c , and 26b , which were 9298% 
 pure by lcms analysis . methyl 4-methoxybenzoate ( 831 mg , 5 mmol ) and acetonitrile ( 1 ml ) 
 were added to a stirred suspension of nah ( 60% dispersion in mineral 
 oil , 490 mg , 12.25 mmol ) in anhydrous thf ( 25 ml ) . the reaction was 
 stirred at 60 c for 40 h , and then the volatiles were removed 
 under reduced pressure . the crude residue was diluted with h2o ( 15 ml ) and acidified with 1 m hcl , and then the product was extracted 
 into dcm ( 3  20 ml ) . the combined organic fractions were washed 
 with brine ( 5 ml ) , dried over anhydrous na2so4 , and the solvent removed under reduced pressure . the crude product 
 was purified by flash chromatography ( 40100% v / v dcm in petroleum 
 ether ) to yield 3-(4-methoxyphenyl)-3-oxopropanenitrile as a white 
 solid ( 578 mg , 3.30 mmol , 66% ) , rf 0.30 
 ( 1:2 v / v etoac in petroleum ether ) . lcms ( esi ) m / z 174.1 [ m  h ] , retention 
 time 1.68 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c10h10no2 [ m + h ] = 176.0712 ) , obsd 176.0703 (  ppm = 5.1 ) . h nmr 
 ( 500 mhz , cdcl3 )  7.90 ( d , j = 
 9.0 hz , 2h ) , 6.98 ( d , j = 9.0 hz , 2h ) , 4.02 ( s , 2h ) , 
 3.90 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  
 185.5 , 164.9 , 131.1 , 127.4 , 114.5 , 114.2 , 55.8 , 29.2 ppm . n ) , 1685 ( s , c = o ) , 1623 
 ( w , c = c ) , 1596 ( s ) , 1514 ( s ) , 1465 , 1450 ( s , ch3 ) , 1439 ( m , c = c ) , 1326 ( s , ch3 ) , 1268 ( s , c  o , 
 ch2 ) , 1224 , 1172 ( s , c  o ) , 1011 ( s , c  o , 
 ch2 ) , 930 , 828 , 814 ( s , c  h ) cm . concentrated hcl ( 37% v / v , 2.4 ml ) was added slowly to a stirred 
 solution of 3-(4-methoxyphenyl)-3-oxopropanenitrile ( 552 mg , 3.15 
 mmol ) in absolute etoh ( 20 ml ) . the reaction was heated to reflux , 
 and then hydrazine hydrate ( 750 l , 15.5 mmol ) was added dropwise 
 over 10 min . the reaction was heated under reflux for 5 h and then 
 concentrated under reduced pressure ( 5 ml ) , diluted with h2o ( 10 ml ) , and brought to ph 8.0 using saturated nahco3 . the product was extracted into etoac ( 3  20 ml ) , and the 
 combined organic fractions were washed with brine ( 5 ml ) , dried over 
 anhydrous na2so4 , and the solvent removed under 
 reduced pressure . the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 3-(4-methoxyphenyl)-1h - pyrazol-5-amine as a white solid ( 512 mg , 2.71 mmol , 86% ) , rf 0.18 ( 5% v / v meoh in dcm ) . lcms ( esi+ ) m / z 190.2 [ m + h ] , retention 
 time 1.27 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c10h12n3o [ m + h ] = 190.0975 ) , obsd 190.0970 (  ppm = 2.4 ) . h nmr 
 ( 500 mhz , meod )  7.50 ( br , s , 1h ) , 7.38 ( d , j = 8.8 hz , 2h ) , 6.93 ( d , j = 8.8 hz , 2h ) , 3.79 ( s , 
 3h ) ppm . c nmr ( 125 mhz , meod )  159.4 , 152.7 , 
 128.9 ( 2  c ) , 127.0 , 115.2 , 109.6 , 55.7 ppm . ir ( thin film ) vmax 3397 , 3148 ( m , br , n ( m , c  h ) , 1615 ( m , n  h ) , 1505 , 1479 , 1462 ( s , n h , 
 c  h , c = c ) , 1439 ( m , c = c ) , 1284 ( m , c  n , 
 c  o ) , 1244 ( s , c c  h ) , 
 1028 ( s , c  o ) , 831 , 817 , 769 ( s , c  h ) , 710 ( m , c  h ) 
 cm .   step 3 : synthesis of 4-(5-amino-1h - pyrazol-3-yl)phenol 
 ( 3 ) . a solution of 1 m bbr3 in dcm 
 ( 12.5 ml , 12.5 mmol ) was added dropwise over a period of 10 min to 
 a stirred solution of 3-(4-methoxyphenyl)-1h - pyrazol-5-amine 
 ( 473 mg , 2.5 mmol ) in anhydrous dcm ( 22 ml ) at 0 c . the reaction 
 was stirred at 0 c and allowed to come to room temperature over 
 14 h. when complete , the reaction was quenched with saturated nahco3 and extracted into etoac ( 3  30 ml ) . the organic phase 
 was combined , washed with brine ( 10 ml ) , dried over anhydrous na2so4 , and the solvent removed under reduced pressure 
 to yield 4-(5-amino-1h - pyrazol-3-yl)phenol 3 as an off - white solid ( 342 mg , 1.95 mmol , 75% ) , rf 0.35 ( 10% v / v meoh in etoac ) . lcms ( esi+ ) m / z 176.2 [ m + h ] , retention 
 time 0.81 min ( 100% ) . hrms ( + esi ) : m / z ( calcd c9h10n3o [ m + h ] = 176.0824 ) , obsd 176.0823 (  ppm = 0.6 ) . h nmr 
 ( 500 mhz , dmso - d6 )  9.49 ( s , 1h ) , 
 7.44 ( d , j = 9.0 hz , 2h ) , 6.76 ( d , j = 8.5 hz , 2h ) , 5.63 ( s , 1h ) ppm . c nmr ( 125 mhz , dmso - d6 )  156.9 , 153.6 , 145.2 , 126.1 , 122.7 , 
 115.4 , 86.9 ppm . h ) , 1615 ( m , n  h , c = c ) , 
 1519 ( s , n n , c  o ) , 1179 ( m , c  o ) , 1111 , 
 1014 ( m , c  h ) , 834 ( s , c  h ) , 765 ( s , c  h ) cm step 1 : synthesis of methyl 3-(4-methoxyphenyl)-3-oxopropanoate . dimethyl carbonate ( 1.26 ml , 15 mmol ) was added to a stirred suspension 
 of nah ( 60% dispersion in mineral oil , 600 mg , 15 mmol ) in anhydrous 
 toluene ( 2 ml ) . the reaction was brought to reflux , and a solution 
 of 4-methoxyacetophenone ( 0.90 g , 6 mmol ) in anhydrous toluene ( 3 
 ml ) was added dropwise . the reaction was heated at reflux for a further 
 15 min and then allowed to cool to room temperature . the reaction 
 was quenched with a solution of acoh ( 0.5 ml ) in water ( 2 ml ) . the 
 organic layer was separated , and the aqueous fraction was extracted 
 with et2o ( 2  5 ml ) . the organics were combined , 
 washed with a solution of saturated k2co3 , dried 
 over anhydrous na2so4 , and the solvent removed 
 under reduced pressure . the crude product was purified by flash chromatography 
 ( 1050% v / v etoac in petroleum ether ) to yield methyl 3-(4-methoxyphenyl)-3-oxopropanoate 
 as a yellow oil ( 1.05 g ) . the product was 90% pure by h nmr integration and was taken forward without further purification ; rf 0.18 ( 20% v / v etoac in petroleum ether ) . lcms 
 ( esi+ ) m / z 207.2 [ m  h ] , retention time 1.75 min ( 96% ) . hrms ( esi+ ) : m / z ( calcd c11h13o4 [ m + h ] = 209.0808 ) , obsd 209.0803 (  
 ppm = 2.4 ) . h nmr ( 500 mhz , cdcl3 )  
 7.92 ( d , j = 9.0 hz , 2h ) , 6.94 ( d , j = 9.0 hz , 2h ) , 3.96 ( s , 2h ) , 3.87 ( s , 3h ) , 3.74 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  190.9 , 168.3 , 164.2 , 
 130.0 , 129.2 , 114.1 , 55.7 , 52.6 , 45.7 ppm . ir ( thin film ) vmax 2954 , 2842 ( w , c  h ) , 1739 ( s , c = o ) , 
 1675 ( s , c = o ) , 1599 ( s , c = c ) , 1575 , 1511 ( m , c = c ) , 
 1437 , 1421 ( m , ch2 , ch3 ) , 1323 ( m , c  o , 
 c  h ) , 1258 ( s , c  o ) , 1215 ( m ) , 1147 , 1170 ( s , c  o , 
 c  h ) , 1025 ( m , c  o ) , 986 ( m ) , 840 ( m , c  h ) cm . a solution of 4-methoxyaniline ( 406 mg , 3.3 mmol ) in m - xylene ( 6 ml ) was added to a stirred solution of methyl 3-(4-methoxyphenyl)-3-oxopropanoate 
 ( 623 mg , 3 mmol ) in m - xylene ( 6 ml ) , and the reaction 
 was heated to 150 c for 20 h. the product precipitated on cooling 
 to room temperature and was purified by trituration at 0 c with 
 ice - cold etoac : hexane ( 1:5 v / v ) . the precipitate was collected by 
 vacuum filtration , washed with 1:5 etoac : hexane followed by 1:3 etoac : hexane , 
 and dried under reduced pressure to give n,3-bis(4-methoxyphenyl)-3-oxopropanamide 
 as a brown powder ( 567 mg , 1.9 mmol , 63% ) , rf 0.06 ( 1:2 etoac : petroleum ether ) . lcms ( esi+ ) m / z 322.0 [ m + na ] , retention time 1.87 
 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c17h18no4 [ m + h ] = 300.1236 ) , 
 obsd 300.1251 (  ppm = 5.0 ) . h nmr ( 500 mhz , cdcl3 )  9.26 ( s , 1h ) , 8.02 ( d , j = 8.9 
 hz , 2h ) , 7.48 ( d , j = 9.0 hz , 2h ) , 6.97 ( d , j = 8.9 hz , 2h ) , 6.86 ( d , j = 9.0 hz , 2h ) , 
 4.04 ( s , 2h ) , 3.89 ( s , 3h ) , 3.79 ( s , 3h ) ppm . c nmr ( 125 
 mhz , cdcl3 )  195.1 , 164.7 , 164.0 , 156.6 , 131.2 , 
 130.9 , 129.2 , 122.0 , 114.3 , 114.2 , 55.8 , 55.6 , 45.1 ppm . h ) , 3144 , 
 2963 , 2840 ( w , c  h ) , 1685 ( m , c = o ) , 1598 ( s , c = c ) , 
 1556 ( m , c = c ) , 1509 ( s , n h ) , 1465 , 1419 ( w , ch2 , ch3 ) , 1323 , 1263 , 1246 , 1217 , 1170 ( m , c = c , 
 c  o , c n ) , 1028 , 1013 ( c  o ) , 826 ( s , c  h ) 
 cm . lawesson s reagent ( 419 mg , 1.04 mmol ) was added to a stirred 
 solution of n,3-bis(4-methoxyphenyl)-3-oxopropanamide 
 ( 120 mg , 0.4 mmol ) in dry thf ( 20 ml ) . the reaction was heated to 
 reflux for 4 h and then concentrated under reduced pressure to give 
 a red oil . the sulphonamide intermediate was redissolved in absolute 
 etoh ( 5 ml ) , and glacial acoh ( 69 l , 1.3 mmol ) was added . the 
 flask was evacuated , flushed with n2 gas , and then hydrazine 
 monohydrate ( 66 l , 1.3 mmol ) added . the reaction was heated 
 to reflux for 3 h and then allowed to stir at room temperature for 
 24 h. when complete , the reaction was concentrated under reduced pressure . the crude orange oil was diluted with dcm ( 20 ml ) and made basic with 
 a satd nahco3 solution ( 5 ml ) . the phases were separated , 
 and the aqueous layer was extracted with dcm ( 2  2 ml ) . the 
 organic fractions were combined , dried over anhydrous na2so4 , and the crude product dried directly onto silica 
 gel . flash chromatography ( 2580% v / v etoac in petroleum ether ) 
 yielded an off - white solid that was further purified by trituration 
 with et2o to give n,3-bis(4-methoxyphenyl)-1h - pyrazol-5-amine as a white powder ( 41 mg , 0.14 mmol , 35% ) , rf 0.11 ( 2:1 v / v etoac in petroleum ether ) . lcms 
 ( esi+ ) m / z 296.3 [ m + h ] , retention time 1.95 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c17h18n3o2 [ m + h ] = 296.1399 ) , obsd 296.1393 (  
 ppm = 2.0 ) . h nmr ( 500 mhz , acetone - d6 )  11.42 ( s , 1h ) , 7.67 ( d , j = 
 8.7 hz , 2h ) , 7.35 ( m , 2h ) , 6.99 ( d , j = 8.9 hz , 2h ) , 
 6.81 ( d , j = 9.0 hz , 2h ) , 6.13 ( d , j = 1.2 hz , 1h ) , 3.83 ( s , 3h ) , 3.73 ( s , 3h ) ppm . c nmr 
 ( 125 mhz , acetone - d6 )  160.9 , 154.2 , 
 144.7 , 139.2 , 127.7 , 124.6 , 118.0 , 117.97 , 115.42 , 115.40 , 91.0 , 56.1 , 
 56.0 ppm . c peak at 144.7 identified from h  c - hmbc spectrum . ir ( thin film ) vmax 3402 ( m , n  h ) , 3270 ( br , w , n h ) , 2968 , 
 2840 ( w , c  h ) , 1612 , 1598 , 1587 ( m , c = c ) , 1554 , 1505 , 
 1464 , 1427 ( s , c  h , c = n ) , 1294 ( m , c n ) , 1239 
 ( s , c  o ) , 1182 ( s , c c  o ) , 
 959 ( m ) , 823 , 762 ( s , c  h ) cm . step 4 : 4-((3-(4-hydroxyphenyl)-1h - pyrazol-5-yl)amino)phenol 
 ( 6 ) . a 1 m solution of bbr3 in dcm 
 ( 1.4 ml , 1.4 mmol ) was added dropwise over a period of 15 min to a 
 stirred suspension of n-3-bis(4-methoxyphenyl)-1h - pyrazol-5-amine ( 104 mg , 0.35 mmol ) in dry dcm ( 7.5 ml ) 
 at 0 c . the reaction was allowed to come slowly to room temperature 
 and stirred for 8 h , then quenched with ice water ( 40 ml ) . etoac ( 20 
 ml ) was added , and the aqueous phase was made slightly basic with 
 satd nahco3 . the phases were separated , and the aqueous 
 fraction was extracted with etoac ( 3  15 ml ) . the organic fractions 
 were combined , dried over anhydrous na2so4 , 
 and the solvent removed under reduced pressure . the crude product 
 was purified by flash chromatography ( 510% v / v meoh in dcm , 
 followed by 5% meoh in etoac ) to yield 4-((3-(4-hydroxyphenyl)-1h - pyrazol-5-yl)amino)phenol ( 6 ) as an off - white 
 solid ( 43 mg , 0.16 mmol , 46% ) , rf 0.52 
 ( 10% v / v meoh in etoac ) . lcms ( esi+ ) m / z 268.2 [ m + h ] , retention time 1.37 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c15h14n3o2 [ m + h ] = 268.1081 ) , 
 obsd 296.1070 (  ppm = 4.0 ) . h nmr ( 500 mhz , meod ) 
  7.49 ( d , j = 8.5 hz , 2h ) , 7.05 ( d , j = 8.0 hz , 2h ) , 6.82 ( d , j = j = 8.7 hz , 2h ) , 6.70 ( d , j = 8.9 hz , 2h ) , 6.03 ( s , 
 1h ) ppm . c nmr ( 125 mhz , meod )  158.9 , 155.5 ( br ) , 
 152.0 , 145.5 ( br ) , 137.9 , 127.8 , 122.8 , 119.2 , 116.6 , 116.6 , 90.7 
 ( br ) ppm . ir ( thin film ) vmax 3417 ( s , 
 br , o h ) , 1607 , 1615 ( w , c = c ) , 
 1574 ( s , c = c , n  h ) , 1522 ( s , c = h ) , 
 1383 ( w , c  o ) , 1254 ( m , br , c  o , c n ) , 1107 ( w , 
 c  h ) , 830 ( s , c  h ) , 759 ( m , o  h ) cm . 2-(4-methoxyphenyl)acetonitrile 11 ( 2.32 
 ml , 17.1 mmol ) and methyl 4-methoxybenzoate 10a ( 2.58 
 g , 15.5 mmol ) were added to a stirred suspension of nah ( 60% in mineral 
 oil , 1.34 g , 33.6 mmol ) in anhydrous thf ( 30 ml ) . the reaction was 
 heated to 60 c for 20 h then cooled to 0 c and quenched 
 with h2o ( 10 ml ) . the suspension was concentrated under 
 reduced pressure then diluted with dichloromethane ( 50 ml ) and acidified 
 with 3 m hcl to ph 23 . the organic layer was separated off , 
 and the aqueous phase was extracted with dichloromethane ( 3  
 50 ml ) . the organic fractions were combined , washed with brine ( 10 
 ml ) , dried over anhydrous mgso4 , and the solvent removed 
 under reduced pressure to yield 2,3-bis(4-methoxyphenyl)-3-oxopropanenitrile 12a as a yellow gum ( 3.84 g , 13.7 mmol , 88% ) . the crude oxopropanenitriles 12a was used in subsequent reactions without further purification , 
 although other analogues were purified by flash chromatography eluting 
 with the solvent system specified . lcms ( + esi ) : m / z 280.2 [ m + na ] ; retention time 2.11 
 min ( 93% ) . hrms ( + esi ) : m / z ( calcd 
 c17h16no3 [ m + h ] = 282.1130 ) , 
 obsd 282.1135 (  ppm = 1.8 ) . h nmr ( 500 mhz , cdcl3 )  7.92 ( d , j = 9.0 hz , 2h ) , 7.34 
 ( d , j = 8.8 hz , 2h ) , 6.92 ( d , j = 
 7.8 hz , 2h ) , 6.90 ( d , j = 7.8 hz , 2h ) , 5.50 ( s , 1h ) , 
 3.85 ( s , 3h , ) , 3.78 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  187.6 , 164.5 , 160.1 , 131.8 , 129.5 , 126.5 , 122.7 , 
 117.2 , 115.1 , 114.3 , 55.7 , 55.5 , 45.8 ppm . ir ( thin film ) vmax 3008 , 2936 , 2839 ( w , c  h ) , 2248 ( w , 
 nitrile cn ) , 1677 ( m , c = o ) , 1597 , 1510 ( s , c = c ) , 1462 , 
 1422 ( w ) , 1305 ( m , c  o ) , 1254 , 1237 , 1173 ( s , c  o ) , 
 1027 ( m ) , 939 , 822 , 780 ( m ) cm . prepared as an orange oil ( 0.703 g , 2.50 
 mmol , 83% ) according to general procedure a. the crude product was 
 purified by flash chromatography ( 050% v / v etoac in petroleum 
 ether ) , rf 0.55 ( 50% v / v etoac in petroleum 
 ether ) . lcms ( esi ) m / z 294.2 
 [ m + h ] , retention time 2.13 min . h nmr ( 400 
 mhz , cdcl3 )  7.49 ( d , j = 7.6 hz , 
 1h ) , 7.45 ( app t , j = 2.4 , 1.6 hz , 1h ) , 7.34 ( m , 
 3h ) , 7.11 ( d , j = 8.4 , 2.0 hz , 1h ) , 6.90 ( d , j = 8.8 hz , 2h ) , 5.53 ( s , 1h ) , 3.82 ( s , 3h ) , 3.78 ( s , 3h ) 
 ppm . c nmr ( 100 mhz , cdcl3 )  189.1 , 
 160.2 , 160.1 , 135.1 , 130.0 , 129.6 , 122.3 , 121.8 , 121.0 , 116.9 , 115.2 , 
 113.7 , 55.6 , 55.5 , 46.2 ppm . prepared as an orange solid ( 229 mg , 0.852 
 mmol , 
 85% ) according to general procedure a. the crude product was purified 
 by flash chromatography ( 1050% v / v etoac in petroleum ether ) . lcms ( esi+ ) m / z 270.2 [ m + h ] , retention time 2.08 min ( 89% ) . hrms ( esi+ ) m / z ( calcd c16h13fno2 [ m + h ] = 270.0930 ) , obsd 270.0933 (  ppm = 1.1 ) . h nmr ( 500 mhz , cdcl3 )  7.71 ( d , j = 8.0 hz , 1h ) , 7.61 ( d , j = 9.0 hz , 1h ) , 
 7.43 ( m , 1h ) , 7.33 ( d , j = 8.8 hz , 2h ) , 7.29 ( m , 
 1h ) , 6.91 ( d , j = 8.9 hz , 2h ) , 5.49 ( s , 1h ) , 3.79 
 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  
 188.1 , 162.8 ( d , jc f = 248 hz ) , 
 160.4 , 135.6 ( jc  f = 6.4 hz ) , 
 130.8 ( d , jc f = 10 hz ) , 121.5 , 116.5 , 
 116.0 ( d , jc ir ( thin 
 film ) vmax 3076 , 2959 , 2936 , 2840 ( w , 
 c  h ) , 2248 , 2207 ( w , nitrile cn ) , 1694 ( m , c = o ) , 1608 , 
 1588 ( m , c = c ) , 1510 ( s ) , 1439 , 1304 ( m ) , 1252 ( s ) , 1149 , 1030 ( m , c  o ) , 885 ( w ) , 834 , 803 , 788 ( m ) cm . prepared as a yellow semisolid ( 2.18 g ) according 
 to general procedure a and used in subsequent reactions without further 
 purification . the crude product contained 15% unreacted starting material 
 by h nmr integration ; rf 0.18 
 ( 50% v / v etoac in petroleum ether ) . lcms ( esi ) m / z 328.1 [ m  h ] , retention 
 time 2.23 min ( 59% ) . h nmr ( 400 mhz , cdcl3 ) 
  7.94 ( d , j = 8.9 hz , 2h ) , 7.62 ( s , 1h ) , 7.49 
 ( d , j = 8.0 hz , 1h ) , 7.8 ( d , j = 
 7.8 hz , 1h ) , 7.27 ( m , 1h ) , 6.95 ( d , j = 8.9 hz , 2h ) , 
 5.53 ( s , 1h ) , 3.89 ( s , 3h ) ppm . c nmr ( 100 mhz , cdcl3 )  186.7 , 164.9 , 132.9 , 132.4 , 131.9 , 131.3 , 127.0 , 
 126.3 , 123.6 , 121.7 , 116.4 , 114.6 , 55.8 , 45.7 ppm . ir ( thin film ) vmax 2937 , 2842 ( w , c  h ) , 2252 ( w , cn ) , 
 1679 ( m , c = o ) , 1597 ( s ) , 1572 , 1512 , 1424 , 1323 ( m , c = c , 
 c  h ) , 1264 , 1238 , 1172 ( s , c n , c  o ) , 1026 ( m ) , 
 941 , 833 , 775 , 692 ( m , c  h ) cm . prepared as an orange semisolid ( 0.845 
 g , 1.56 mmol , quant . ) according to general procedure a and either 
 used directly in subsequent reactions or further purified by flash 
 chromatography ( 010% v / v meoh in dcm ) . lcms ( esi+ ) : m / z 436.1 [ m + h ] , retention 
 time 2.44 min ( 94% ) . hrms ( esi+ ) : m / z ( calcd c23h19brno3 [ m + h ] = 436.0548 ) , obsd 436.0561 (  ppm = 3.0 ) . h nmr ( 500 mhz , cdcl3 )  7.92 ( d , j = 8.9 hz , 2h ) , 7.63 ( d , j = 2.4 hz , 1h ) , 7.44 ( d , j = 7.4 hz , 2h ) , 7.38 ( appt , j = 7.6 , 7.2 
 hz , 2h ) , 7.347.30 ( m , 2h ) , 6.93 ( m , 3h ) , 5.45 ( s , 1h ) , 5.13 
 ( s , 2h ) , 3.87 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  186.8 , 164.6 , 155.6 , 136.0 , 133.1 , 131.8 , 128.7 , 128.3 , 
 128.1 , 126.9 , 126.2 , 124.2 , 116.7 , 114.5 , 114.3 , 113.5 , 70.9 , 55.7 , 
 44.9 ppm . ir ( thin film ) vmax 3064 , 3033 , 
 2935 , 2840 ( w , c  h ) , 2249 ( w , nitrile c n ) , 1678 ( m , 
 c = o ) , 1597 ( s ) , 1572 , 1510 ( m , c = c ) , 1492 ( s ) , 1454 
 ( m ) , 1382 ( w , c n , c  o ) , 
 1052 , 1024 ( m , c  o ) , 808 , 735 ( m ) cm . concentrated hcl ( 37% 
 v / v , 6.25 ml ) was added slowly to a stirred solution of crude 2,3-bis(4-methoxyphenyl)-3-oxopropanenitrile 12a ( 4.16 g , 11.84 mmol , 1 equiv ) in absolute etoh 
 ( 63 ml ) . the reaction was then heated to reflux , and hydrazine hydrate 
 ( 3.6 ml , 74 mmol , 5 equiv ) was added dropwise over 10 min . after heating 
 at reflux until all starting material had been consumed ( typically 
 515 h ) , the reaction was cooled to room temperature and concentrated 
 under reduced pressure ( 20 ml ) . the reaction was diluted in water 
 ( 15 ml ) and brought to ph 8.0 using saturated nahco3 . the organic 
 fractions were combined and washed with brine ( 10 ml ) , dried over 
 anhydrous mgso4 , and the solvent removed under reduced 
 pressure . the crude product was purified by flash chromatography ( 40% 
 v / v etoac in petroleum ether , then 010% v / v meoh in etoac ) 
 to yield 3,4-bis(4-methoxyphenyl)-1h - pyrazol-5-amine 13a as a yellow solid ( 3.171 g , 10.7 mmol , 91% ) , rf 0.29 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) : m / z 296.1 [ m + h ] ; retention time 2.01 
 min ( 95% ) . hrms ( esi+ ) : m / z ( calcd 
 c17h18n3o2 [ m + h ] = 296.1399 ) , obsd 296.1392 (  ppm = 2.4 ) . h nmr ( 500 mhz , dmso - d6 )  11.77 
 ( s , br , 1h ) , 7.22 ( d , j = 8.8 hz , 2h ) , 7.10 ( d , j = 8.5 hz , 2h ) , 6.89 ( d , j = 8.8 hz , 2h ) , 
 6.86 ( d , j = 8.8 hz , 2h ) , 4.39 ( s , br , 2h ) , 3.75 
 ( s , 3h ) , 3.74 ( s , 3h ) ppm . c nmr ( 125 mhz , dmso - d6 )  158.6 , 157.4 , 152.6 , 139.4 , 130.5 , 
 128.3 , 125.9 , 123.8 , 114.0 , 113.8 , 103.0 , 55.6 , 55.0 ppm . ir ( solid ) vmax 3432 , 3349 ( w , n  h ) , 3210 ( w , br , h ) , 3932 , 
 2833 ( w , c  h ) , 1622 ( w , c = c ) , 1513 , 1489 ( m , n  h , 
 c  h ) , 1303 , 1284 ( w ) , 1241 ( s , c  o , c n ) , 1179 
 ( s , c  o ) , 1111 ( w ) , 1020 ( s , c  o ) , 964 ( w , c  h ) , 
 834 ( s , c  h ) , 798 , 735 ( m , c  h ) cm . prepared as an orange amorphous solid 
 ( 680 mg , 2.30 mmol , 92% ) according to general procedure b. the crude 
 material was carried forward without further purification . lcms ( esi+ ) m / z 296.2 [ m + h ] , retention 
 time 1.80 min ( 93% ) . hrms ( esi+ ) m / z ( calcd c17h18n3o2 [ m 
 + h ] = 296.1394 ) , obsd 296.1381 (  ppm = 4.4 ) . h nmr ( 500 mhz , meod )  7.197.12 ( m , 3h ) , 6.90 
 ( m , 3h ) , 6.85 ( dd , j = 2.6 , 1.5 hz , 1h ) , 6.80 ( ddd , j = 8.3 , 2.6 , 1.0 hz , 1h ) , 3.77 ( s , 3h ) , 3.62 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  161.0 , 160.0 , 153.1 ( br ) , 143.3 
 ( br ) , 133.4 ( br ) , 132.2 , 130.6 , 126.4 , 120.8 , 115.3 , 115.0 , 113.8 , 
 106.3 ( br ) , 55.7 , 55.5 ppm . ir ( thin film ) vmax 3422 , 
 3336 , 3182 ( m , br , n h ) , 2999 , 2938 , 2834 ( m , c  h ) , 
 1607 , 1578 , 1525 , 1488 , 1464 , 1285 ( m , c = c ) , 1243 ( s , c n ) , 
 1177 , 1040 , 1022 ( m , c  o ) , 834 , 788 , 749 , 694 ( m ) cm . prepared as an orange solid ( 142 mg , 0.50 
 mmol , 97% ) according to general procedure b. the crude product was 
 purified by flash chromatography ( 05% v / v meoh in dcm ) . lcms 
 ( esi+ ) m / z 284.2 [ m + h ] , retention time 1.82 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c16h15fn3o [ m 
 + h ] = 284.1199 ) , obsd 284.1188 (  ppm = 3.9 ) . h nmr ( 500 mhz , cdcl3 )  7.24 ( m , 1h ) , 7.19 
 ( d , j = 8.7 hz , 2h ) , 7.12 ( dt , j = 7.7 , 1.3 hz , 1h ) , 7.03 ( ddd , j = 9.8 , 2.6 , 1.6 
 hz , 1h ) , 6.97 ( app tdd , j = 8.4 , 2.6 , 1.0 hz , 1h ) , 
 6.91 ( d , j = 8.7 hz , 2h ) , 3.83 ( s , 3h ) ppm . f = 249 hz ) , 158.7 , 152.8 , 140.2 , 132.7 
 ( d , jc f = 3 hz ) , 115.2 ( d , jc  f = 21 hz ) , 114.6 , 114.3 ( d , jc ir ( thin film ) vmax 3310 , 3190 ( m , br , n h ) , 3000 , 2934 , 
 2836 ( m , c  h ) , 1613 ( m , c = c ) , 1579 , 1524 ( m , n  h ) , 
 1488 ( m , c = c ) , 1286 ( m ) , 1245 ( s , c n , c  o ) , 
 1176 , 1021 ( m ) , 864 , 835 ( m , c  h ) , 788 , 751 ( m , c  h ) , 
 690 , 605 ( w ) cm . prepared as a brown amorphous solid ( 1.86 
 g , 5.42 mmol , 77% over two reaction steps from 3-bromophenylacetonitrile ) . the crude product was purified by flash chromatography ( 50100% 
 v / v etoac in petroleum ether ) , rf 0.08 
 ( 50% v / v etoac in petroleum ether ) . lcms ( esi+ ) m / z 346.1 [ m ( br ) + h ] , 
 retention time 1.96 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c16h15brn3o [ m + h ] = 344.0393 ) , obsd 344.0385 (  
 ppm = 2.5 ) . h nmr ( 500 mhz , meod )  7.37 ( m , 2h ) , 
 7.247.19 ( m , 4h ) , 6.88 ( d , j = 7.2 hz , 2h ) , 
 3.78 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  161.3 , 
 152.6 ( br ) , 144.2 ( br ) , 137.1 , 133.4 , 131.4 , 130.2 ( 2c ) , 129.5 , 124.2 
 ( br ) , 123.6 , 115.1 , 104.6 ( br ) , 55.7 ppm . ir ( thin film ) vmax 3063 , 
 2936 , 2841 ( w , c  h ) , 2250 ( w , nitrile c n ) , 1677 ( m , 
 c = o ) , 1596 ( s ) , 1574 , 1510 , 1474 , 1423 ( m , c = c ) , 1322 
 ( m ) , 1264 , 1239 ( s , c  o ) , 1173 ( s ) , 1027 ( m , c  o ) , 941 
 ( m ) , 832 , 772 , 693 ( m ) cm . prepared as 
 a yellow foam ( 881 mg , 1.96 mmol , 95% ) according to general procedure 
 b. no further purification by flash chromatography was required . lcms 
 ( esi+ ) m / z 450.2 , 452.2 [ m + h ] , retention time 2.17 min ( 98% ) . hrms ( esi+ ) : m / z ( calcd c23h21brn3o2 [ m + h ] = 450.0817 ) , obsd 450.0811 (  
 ppm = 1.3 ) . h nmr ( 500 mhz , cdcl3 )  
 7.52 ( d , j = 2.1 hz , 1h ) , 7.49 ( d , j = 7.3 hz , 2h ) , 7.40 ( app t , j = 7.5 hz , 2h ) , 7.33 
 ( t , j = 7.3 hz , 1h ) , 7.24 ( d , j = 
 8.7 hz , 2h ) , 7.11 ( dd , j = 8.4 , 2.1 hz , 1h ) , 6.91 
 ( d , j = 8.4 hz , 1h ) , 6.84 ( d , j = 
 8.8 hz , 2h ) , 5.16 ( s , 2h ) , 3.80 ( s , 3h ) ppm . c nmr ( 125 
 mhz , cdcl3 )  159.9 , 153.9 , 152.8 , 141.4 , 136.6 , 
 134.3 , 129.9 , 128.8 , 128.7 , 128.1 , 127.2 , 126.9 , 122.3 , 114.4 , 114.2 , 
 112.9 , 104.1 , 71.0 , 55.4 ppm . h ) , 2933 , 2836 ( w , c  h ) , 1613 ( m , c = c ) , 
 1523 ( s , n n ) , 
 1178 , 1031 ( m , c  o ) , 834 , 737 , 697 ( m ) cm . 2-(3-bromophenyl)acetonitrile 15a ( 1.09 g , 5.5 mmol ) 
 was added to a freshly prepared solution of sodium metal ( 139 mg , 
 6.05 mmol ) in etoh ( 7.5 ml ) at 0 c . after the reaction was stirred 
 on ice for 5 min , ethyl formate ( 1.11 ml , 13.8 mmol ) was added and 
 the reaction was heated to reflux for 1524 h. when no starting 
 material remained , the reaction was cooled and concentrated under 
 reduced pressure . the residue was diluted with h2o ( 5 ml ) 
 and acidified to ph 4 using 1 m hcl . the aqueous phase was extracted 
 with et2o or etoac ( 3  10 ml ) , and the combined organic 
 fractions were washed with brine ( 5 ml ) , dried over anhydrous na2so4 , and the solvent evaporated to yield an off - white 
 solid ( 1.18 g , 5.26 mmol , 96% ) . the crude product was carried forward 
 into subsequent reaction steps as a mixture of enantiomers and some 
 acetonitrile starting material ; rf 0.08 
 ( 50% v / v etoac in petroleum ether ) . lcms ( esi ) m / z 222.0 [ m  h ] , retention 
 time 2.01 min ( 97% ) . hrms ( esi ) : m / z ( calcd c9h6brno [ m  h ] = 221.9560 ) , obsd 221.9556 (  ppm = 1.7 ) . h nmr ( 400 mhz , meod )  7.94 ( s , 1h ) , 7.90 ( s , 1h ) , 
 7.66 ( d , j = 7.8 hz , 1h ) , 7.59 ( m , 1h ) , 7.57 ( s , 
 1h ) , 7.54 ( m , 1h ) , 7.51 ( d , j = 7.9 hz , 1h ) , 7.447.33 
 ( m , 4h ) , 7.337.20 ( m , 3h ) , 4.76 ( d , j = 7.4 
 hz , 1h ) ppm . ir ( thin film ) vmax 3103 , 
 2977 ( m , c  h ) , 2725 ( w , aldehyde c  h ) , 2223 ( m , nitrile 
 c  n ) , 1644 ( m , c = o ) , 1594 , 1558 ( m , c = c ) , 1473 
 ( m ) , 1378 ( s , aldehyde c  h ) , 1253 ( s ) , 1194 , 1073 ( m ) , 874 
 ( w ) , 771 ( s ) , 680 ( m ) cm . a solution of bbr3 in dcm 
 ( 1 m , 6.64 ml ) was added dropwise to a cooled ( 78 c ) 
 solution of 2-(3-bromo-4-methoxyphenyl)acetonitrile ( 500 mg , 2.2 mmol ) 
 in dcm ( 2.2 ml ) . the reaction was allowed to warm to room temperature 
 and stirred for 14 h or until all starting material had been consumed . the organic fractions were combined , washed with water ( 10 ml ) and 
 brine ( 10 ml ) , and then dried over anhydrous mgso4 . the 
 solvent was evaporated under reduced pressure to yield 2-(3-bromo-4-hydroxyphenyl)acetonitrile 
 as white crystals ( 0.460 g , 2.17 mmol , 98% ) , rf 0.23 ( 1:2 etoac : hexane ) . lcms ( esi ) m / z 210.0 [ m  h ] ; retention 
 time 1.76 min ( 95% ) . h nmr ( 500 mhz , cdcl3 ) 
  7.45 ( d , j = 1.8 hz , 1h ) , 7.18 ( dd , j = 8.2 hz , 1.9 hz , 1h ) , 7.02 ( d , j = 8.4 
 hz , 1h ) , 5.57 ( s , 1h ) , 3.67 ( s , 2h ) ppm . c nmr ( 125 mhz , 
 cdcl3 )  152.4 , 131.5 , 129.0 , 123.4 , 117.6 , 116.8 , 
 110.7 , 22.6 ppm . h ) , 2924 , 2904 ( w , c  h ) , 2272 ( w , nitrile 
 cn ) , 1613 ( w ) , 1511 , 1423 , 1410 ( s , c = c ) , 1356 ( m , c n ) , 
 1279 ( s ) , 1219 ( m , c  o ) , 802 ( s ) , 658 ( m ) cm . potassium carbonate ( 1.94 g , 14.0 mmol ) , potassium iodide 
 ( 1.28 g , 7.70 mmol ) , and benzyl bromide ( 1.67 ml , 14.0 mmol ) were 
 added to a stirred solution of 2-(3-bromo-4-hydroxyphenyl)acetonitrile 
 ( 1.48 g , 7.00 mmol ) in acetone ( 65 ml ) . the suspension was heated 
 to reflux for 15 h , at which point all of the starting material had 
 been consumed . the reaction was concentrated in vacuo , and the resulting 
 residue was diluted with etoac ( 120 ml ) . the organic fraction was 
 washed with water ( 3  50 ml ) and brine ( 50 ml ) , then dried over 
 anhydrous mgso4 . the crude product was purified by flash 
 chromatography ( 030% v / v etoac in petroleum ether ) to yield 
 2-(4-(benzyloxy)-3-bromophenyl)acetonitrile 15b as a 
 pale - yellow oil ( 1.960 g , 6.49 mmol , 93% ) , rf 0.44 ( 1:2 etoac : hexane ) . lcms ( esi+ ) m / z 306 , 308 [ m + h , reduced nitrile ] ; retention 
 time 2.60 min ( 98% ) . hrms ( esi+ ) : m / z ( calcd c15h13brno [ m + h ] = 302.0181 ) , 
 obsd 302.0166 (  ppm = 5.0 ) . h nmr ( 400 mhz , cdcl3 )  7.53 ( d , j = 2.2 hz , 1h ) , 7.45 
 ( m , 2h ) , 7.39 ( app t , j = 7.4 hz , 6 hz , 2h ) , 7.34 
 ( t , j = 7.3 hz , 1h ) , 7.20 ( dd , j = 8.5 hz , 2.2 hz , 1h ) , 6.92 ( d , j = 8.4 hz , 1h ) , 
 5.18 ( s , 2h ) , 3.60 ( s , 2h ) ppm . c nmr ( 100 mhz , cdcl3 )  154.9 , 136.2 , 133.0 , 128.8 , 128.2 , 128.1 , 127.1 , 
 123.6 , 117.7 , 114.2 , 113.2 , 71.0 , 22.6 ppm . ir ( thin film ) vmax 3089 , 3064 , 2915 , 2869 ( w , c  h ) , 
 2253 ( w , nitrile cn ) , 1604 ( m ) , 1493 ( s ) , 1454 , 1413 , 1382 ( m , c n ) , 
 1282 , 1254 ( s ) , 1157 ( w ) , 1051 ( s , c  o ) , 1006 ( m ) , 806 ( m ) , 
 737 , 696 ( s , c  br ) cm . prepared as an orange residue ( 334 mg , 
 1.01 mmol , 51% ) 
 according to general procedure c. the crude product was purified by 
 flash chromatography ( 05% v / v meoh in dcm ) to give a mixture 
 of isomers ; rf 0.07 ( 1:2 v / v etoac in 
 petroleum ether ) . lcms ( esi ) m / z 330.0 [ m  h ] , retention time 2.28 min 
 ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c16h13brno2 [ m + h ] = 
 330.0130 ) , obsd 330.0140 (  ppm = 3.0 ) . h nmr ( 400 
 mhz , cdcl3 )  7.99 ( d , j = 2.2 hz , 
 1h ) , 7.54 ( m , 2h ) , 7.46 ( m , 5h ) , 7.437.36 ( m , 5h ) , 7.33 ( m , 
 2h ) , 7.21 ( dd , j = 8.6 , 2.4 hz , 1h ) , 6.94 ( d , j = 9.0 hz , 1h ) , 6.91 ( d , j = 8.4 hz , 1h ) , 
 5.17 ( m , 4h ) , 3.52 ( s , 1h ) , 3.45 ( d , j = 3.2 hz , 
 1h ) ppm . ir ( thin film ) vmax 3142 ( br ) , 
 3034 ( w , c  h ) , 2722 ( w , c  h aldehyde ) , 2216 ( w , nitrile , 
 c  n ) , 1644 ( m , c = o ) , 1599 ( w , c = c ) , 1494 ( s ) , 
 1379 ( w , c  h aldehyde ) , 1285 ( s ) , 1079 , 1003 , 808 ( m ) , 731 
 ( s ) , 694 ( s , c  br ) cm . prepared as a cream - colored solid ( 508 
 mg ) according to 
 general procedure c. the crude product was taken forward without further 
 purification ; rf 0.11 ( 1/3 v / v etoac in 
 petroleum ether ) . lcms ( esi ) m / z 253.1 [ m  h ] , retention time 1.96 min 
 ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c10h9brno2 [ m + h ] = 
 253.9817 ) , obsd 253.9829 (  ppm = 4.7 ) . h nmr ( 400 
 mhz , meod )  7.98 ( d , j = 2.2 hz , 1h ) , 7.77 
 ( s , 1h ) , 7.66 ( dd , j = 8.7 , 2.2 hz , 1h ) , 7.60 ( dd , j = 6.3 , 2.2 hz , 1h ) , 7.56 ( s , 1h ) , 7.49 ( s , 1h ) , 7.38 ( dt , j = 8.8 , 2.4 hz , 1h ) , 7.34 ( dt , j = 8.8 , 
 2.4 hz , 1h ) , 7.06 ( d , j = 8.8 hz , 3h ) , 4.73 ( d , j = 6.6 hz , 1h ) , 3.953.80 ( m , 10h ) ppm . ir ( solid ) vmax 3243 ( m , br ) , 3063 , 2968 , 2838 ( w , c  h ) , 
 2216 ( m , cn ) , 1845 ( w ) , 1654 ( m , c = o ) , 1602 ( m ) , 1499 ( s , c n , 
 c  h ) , 1438 , 1375 , 1349 ( w , c = c , n h ) , 1289 ( m ) , 
 1256 ( s , c  o ) , 1208 ( m ) , 1056 , 1013 ( s , c  o ) , 870 ( m ) , 
 803 ( s , c  h ) , 686 ( m ) cm . glacial acoh 
 ( 679 l , 12.9 mmol ) followed 
 by hydrazine hydrate ( 500 l , 10.3 mmol ) was added to a stirred 
 solution of the crude 2-(3-bromophenyl)-3-oxopropanenitrile 16a ( 1.15 g , 5.14 mmol ) in etoh ( 7 ml ) at room temperature . the reaction was heated to 85 c for 35 h , and then the 
 solvent was removed under reduced pressure . the residue was diluted 
 with water ( 10 ml ) and et2o or etoac ( 10 ml ) , and brought 
 to ph 8.0 with a satd nahco3 solution . the phases were 
 separated , and the aqueous phase was extracted with et2o or etoac ( 3  10 ml ) . the combined organic fractions were 
 washed with brine ( 5 ml ) , dried over anhydrous na2so4 , and the solvent removed under reduced pressure . the product 
 was purified by trituration with petroleum ether and dcm to yield 
 4-(3-bromophenyl)-1h - pyrazol-5-amine 17a ( 865 mg , 3.70 mmol , 67% over two reactions steps from 3-bromophenylacetonitrile ) 
 as a pink solid ; rf 0.09 ( 2:1 v / v etoac 
 in petroleum ether ) . lcms ( esi+ ) m / z 240.1 [ m ( br ) + h ] , retention time 1.61 
 min ( 100% ) . hrms ( esi+ ) m / z ( calcd 
 c9h9brn3 [ m + h ] = 237.9980 ) , 
 obsd 237.9984 (  ppm = 1.7 ) . h nmr ( 500 mhz , meod ) 
  7.67 ( s , 1h ) , 7.62 ( s , br , 1h ) , 7.46 ( d , j = 7.4 hz , 1h ) , 7.32 ( d , j = 7.8 hz , 1h ) , 7.26 ( app 
 t. , j = 7.8 hz , 1h ) ppm . c nmr ( 125 
 mhz , meod )  153.2 ( br ) , 139.6 ( br ) , 137.2 , 131.5 , 130.1 , 129.4 , 
 126.0 ( br ) , 123.8 , 108.4 ( br ) ppm . ir ( thin film ) vmax 3392 , 
 3227 ( m , br , n  h ) , 2900 ( w , c  h ) , 1600 , 1567 , 1496 ( m , 
 c = c , c n ) , 1409 ( w ) , 1079 ( w ) , 872 ( m ) , 779 ( s ) , 687 
 ( m ) cm . prepared as a cream solid ( 648 mg , 1.88 
 mmol , 94% ) according to general procedure d. the crude product was 
 used without further purification ; rf 0.23 
 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) : m / z 346.1 [ m + h ] , retention time 1.94 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c16h15n3obr [ m + h ] = 344.0398 ) , obsd 
 344.0387 (  ppm = 3.2 ) . h nmr ( 500 mhz , 
 meod )  7.69 ( d , j = 2.2 hz , 1h ) , 7.54 ( s , 
 1h ) , 7.49 ( d , j = 7.5 hz , 2h ) , 7.36 ( m , 3h ) , 7.30 
 ( t , j = 7.4 hz , 1h ) , 7.09 ( d , j = 
 8.5 hz , 1h ) , 5.17 ( s , 2h ) ppm . c nmr ( 125 mhz , meod ) 
  154.4 , 138.3 , 132.3 , 129.5 , 129.1 , 128.9 , 128.3 , 127.7 , 115.6 , 
 113.6 , 107.3 , 71.9 ppm . ir ( thin film ) vmax 3400 , 3318 ( m , n  h ) , 
 3165 ( m , br , c  h ) , 3061 , 3036 ( m , c  h ) , 2889 ( m , c  h ) , 
 1600 , 1565 , 1500 ( m , c = c ) , 1486 ( s , n h ) , 1396 , 1381 , 
 1367 ( w ) , 1292 , 1257 ( s , c n ) , 1047 ( s , c  o ) , 1013 , 
 872 ( m ) , 802 ( s ) , 726 , 704 , 641 ( m ) cm . prepared as a white solid ( 339 mg , 1.26 
 mmol , 66% over two reaction steps ) according to general procedure 
 d. the crude material was purified by flash chromatography ( 210% 
 v / v meoh in etoac ) , rf 0.05 ( 50% v / v etoac 
 in petroleum ether ) . lcms ( esi+ ) m / z 268.1 [ m + h ] , retention time 1.59 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c10h11brn3o [ m + h ] = 268.0080 ) , obsd 
 268.0071 (  ppm = 3.4 ) h nmr ( 500 mhz , acetone - d6 )  11.05 ( br , s ) , 7.72 ( d , j = 2.1 hz , 1h ) , 7.63 ( s , 1h ) , 7.49 ( dd , j = 8.5 , 
 2.2 hz , 1h ) , 7.07 ( d , j = 8.5 hz , 1h ) , 4.38 ( br , 
 s , 2h ) , 3.88 ( s , 3h ) ppm . c nmr ( 126 mhz , acetone - d6 )  153.6 , 150.0 ( br ) , 130.6 , 128.4 , 
 126.3 , 124.3 , 112.6 , 112.5 , 111.4 , 105.5 ( br ) , 55.7 ppm . ir ( solid ) vmax 3407 , 3333 , 
 3225 ( m , br , n  h ) , 2937 , 2841 ( w , c  h ) , 1619 ( w , c = c ) , 
 1574 ( m , n  h ) , 1516 ( m ) , 1482 ( s , c = c , c  h ) , 
 1383 ( w , ch3 ) , 1286 ( m , c n , 
 c  o ) , 1184 ( w ) , 1045 , 1015 ( m , c  o , c  h ) , 744 
 ( m , c  h ) , 680 ( s , c  br ) cm . 4-(3-bromophenyl)-1h - pyrazol-5-amine 17a ( 238 mg , 1 mmol ) , 3-nitrophenyl 
 boronic acid ( 150 mg , 1.5 mmol ) ( 12 equiv ) , na2co3 ( 318 mg , 3 mmol ) , and pd(pph3)4 ( 116 mg , 0.1 mmol ) were combined in a microwave reaction vessel 
 in a 1:1 v / v mixture of 1,4-dioxane : water ( 12 ml ) . the reaction was 
 heated at 120 c in a microwave reactor for 1.53 h. the 
 reaction was then allowed to cool to room temperature and diluted 
 with dcm ( 20 ml ) and water ( 5 ml ) . the phases were separated , and 
 the aqueous phase was extracted with dcm ( 3  3 ml ) . the organic 
 fractions were combined , washed with brine ( 3 ml ) , dried over anhydrous 
 mgso4 , and the solvent removed under reduced pressure . the crude product was purified by flash chromatography ( 05% 
 v / v meoh in dcm ) to yield 4-(3-nitro-[1,1-biphenyl]-3-yl)-1h - pyrazol-5-amine 18a as a yellow amorphous 
 solid ( 152 mg , 0.50 mmol , 54% ) , rf 0.28 
 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 281.2 [ m + h ] , retention time 1.82 min ( 100% ) . hrms 
 ( esi+ ) m / z ( calcd c15h13n4o2 [ m + h ] = 281.1033 ) , 
 obsd 281.1023 (  ppm = 3.6 ) . h nmr ( 500 mhz , cdcl3 )  8.46 ( app t , j = 2.0 , 2.0 hz , 1h ) , 
 8.21 ( ddd , j = 8.3 , 2.0 , 2.0 hz , 1h ) , 7.93 ( ddd , j = 7.3 , 1.5 , 1.0 hz , 1h ) , 7.70 ( s , 1h ) , 7.62 ( app t , j = 8.0 , 7.5 hz , 1h ) , 7.59 ( s , 1h ) , 7.537.48 ( m , 
 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  151.2 
 ( br ) , 148.8 , 142.8 , 139.6 , 134.1 , 133.2 , 129.9 , 129.9 , 128.8 ( br ) , 
 126.9 , 125.7 , 125.1 , 122.3 , 122.1 , 108.5 ( br ) ppm . h ) , 2952 ( m , c  h ) , 1606 ( m , 
 n  h ) , 1524 ( s , no2 ) , 1470 , 1438 ( w , c = c ) , 
 1345 ( s , no2 ) , 1290 , 1265 ( w , c 1026 ( w , c  h ) , 793 , 738 , 685 ( m , c  h ) cm . prepared as 
 a yellow  brown foam ( 76 mg , 0.20 mmol , 70% ) according to general 
 procedure e. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) . lcms ( esi+ ) : m / z 387.0 [ m + h ] , retention time 2.07 min ( 94% ) . hrms ( esi+ ) : m / z ( calcd c22h19n4o3 [ m + h ] = 387.1457 ) , 
 obsd 387.1456 (  ppm = 0.3 ) . h nmr ( 500 mhz , meod ) 
  8.51 ( app t , j = 2.0 , 2.0 hz , 1h ) , 8.18 ( ddd , j = 8.3 , 2.0 , 1.0 hz , 1h ) , 7.97 ( ddd , j = 8.0 , 1.5 , 1.0 hz , 1h ) , 7.63 ( app t , j = 8.0 , 
 7.0 hz , 1h , 16-ch ) , 7.527.48 ( m , 2h ) , 7.35 ( m , 2h ) , 7.307.26 
 ( m , 2h ) , 7.24 ( d , j = 8.4 hz , 1h ) , 5.15 ( s , 2h ) ppm . c nmr ( 125 mhz , 
 meod )  155.0 , 152.8 , 149.3 , 141.6 , 138.4 , 136.9 , 130.3 , 130.2 , 
 129.9 , 129.5 ( 2c ) , 129.1 , 128.48 , 128.4 , 128.3 , 125.6 , 122.6 , 115.1 , 
 71.8 ppm . ir ( thin film vmax 3415 , 3325 , 
 3193 ( br , m , n h ) , 3033 , 2941 , 2880 ( w , c  h ) , 1613 , 
 1578 ( w , c = c ) , 1525 ( s , no2 ) , 1525 ( m ) , 1380 ( s , 
 no2 ) , 1287 , 1260 ( m , c n , c  o ) , 1055 , 1021 
 ( w , c  o ) , 890 ( w ) , 811 , 735 , 688 ( m ) cm . prepared as 
 a yellow amorphous solid ( 165 mg , 0.53 mmol , 53% ) according to general 
 procedure e , except that the reactions was carried out at 120 c 
 using conventional heating over 14 h. the crude product was purified 
 by flash chromatography ( 05% v / v meoh in dcm ) , rf 0.21 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 311.2 [ m + h ] , retention time 1.83 
 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c16h15n4o3 [ m + h ] = 311.1144 ) , obsd 311.1139 (  ppm = 1.6 ) . h nmr ( 500 mhz , meod )  8.41 ( d , j = 2.2 hz , 
 1h ) , 8.17 ( d , j = 8.2 hz , 1h ) , 7.93 ( d , j = 7.9 hz , 1h ) , 7.687.55 ( m , 2h ) , 7.51 ( d , j = 8.6 hz , 1h ) , 7.47 ( s , 1h ) , 7.14 ( d , j = 8.5 hz , 
 1h ) , 3.84 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  
 156.0 , 152.8 , 149.4 , 141.7 , 136.9 , 130.2 , 129.9 , 129.7 , 129.2 , 128.8 , 
 127.7 , 125.3 , 122.6 , 113.2 , 109.2 , 56.2 ppm . c nmr processed 
 with lb = 3.0 hz . ir ( thin film ) vmax 3421 , 
 3182 ( m , br , n h ) , 2952 ( m , c  h ) , 1613 , 1577 ( w , n h , 
 c = c ) , 1525 ( s , no2 ) , 1348 ( s , no2 ) , 1260 
 ( m , c  o , c n ) , 1181 ( w , c  h ) , 1022 ( m , c  o ) , 
 814 , 747 , 688 ( m , c  h ) cm . prepared according 
 to 
 general procedure e and crudely purified by elution through a short 
 silica plug ( 05% v / v meoh in dcm ) to remove inorganic byproducts . the product mixture was then carried forward with further purification 
 and characterization performed after reduction of the aromatic nitro 
 group . lcms ( esi+ ) : m / z 281.4 [ m 
 + h ] , retention time 3.89 min ( 52% ) . prepared according to general procedure 
 e. the crude product was purified by flash chromatography ( 05% 
 v / v meoh in dcm ) to yield 18e ( 0.030 g , 0.13 mmol , 61% ) . lcms ( esi+ ) : m / z 237.3 [ m + h ] , retention time 1.12 min ( 100% ) . h nmr ( 400 mhz , 
 cdcl3 ) :  8.84 ( dd , j = 2.4 , 0.8 
 hz , 1h ) , 8.58 ( dd , j = 4.9 , 1.4 hz , 1h ) , 7.86 ( ddd , j = 7.9 , 2.3 , 1.7 hz , 1h ) , 7.64 ( m , 1h ) , 7.54 ( s , 1h ) , 7.46 
 ( m , 2h ) , 7.42 ( m , 1h ) , 7.35 ( ddd , j = 8.0 , 4.8 , 0.8 
 hz , 1h ) , 5.65 ( br s , 3h ) ppm . c nmr ( 100 mhz , cdcl3 ) :  150.8 , 148.5 , 148.2 , 138.5 , 136.6 , 134.6 , 134.0 , 
 129.7 , 129.1 , 126.4 , 125.5 , 124.8 , 123.7 , 108.2 ppm . h ) , 
 2932 ( w , br , c  h ) , 1608 ( m ) , 1506 ( w ) , 1463 ( m ) , 1400 , 1380 
 ( w ) , 1188 , 1123 , 1059 , 1044 , 1027 ( w ) , 843 ( m ) , 774 , 708 , 695 ( s , 
 c  h ) cm . prepared according to general procedure 
 e. the crude product was purified by flash chromatography ( 05% 
 v / v meoh in dcm ) to yield 18f ( 0.014 g , 0.060 mmol , 29% ) . lcms ( esi+ ) : m / z 237.2 [ m + h ] ( calcd for c14h12n4 , 237.1 ) , 
 retention time 1.04 min ( 96% ) . h nmr ( 500 mhz , meod ) : 
  8.58 ( d , j = 1.6 hz , 1h ) , 8.57 ( d , j = 1.6 hz , 1h ) , 7.87 ( t , j = 1.6 hz , 1h ) , 
 7.77 ( d , j = 1.6 hz , 1h ) , 7.76 ( d , j = 1.6 hz , 1h ) , 7.70 ( s , 1h ) , 7.567.61 ( m , 2h ) , 7.51 ( t , j = 7.6 hz , 1h ) ppm . c nmr ( 125 mhz , meod ) 
  151.3 , 151.2 , 150.3 , 139.3 , 135.9 , 132.0 , 128.5 , 126.0 , 125.3 , 
 123.4 , 108.2 ppm . one unresolved quaternary c. ir ( thin 
 film ) vmax 3458 , 3159 ( m , br , n h ) , 
 291 ( m , c  h ) , 1618 ( m , c = c ) , 1594 ( s , n h , c  h ) ) , 
 1561 ( m ) , 1508 , 1402 ( s , c n ) , 1334 , 1223 , 1054 , 1023 ( m ) , 
 789 , 701 ( s , c  h ) cm . prepared according to 
 general procedure e. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 18 g ( 0.029 g , 
 0.11 mmol , 50% ) . lcms ( esi+ ) : m / z 275.3 [ m + h ] , retention time 1.74 min ( 94% ) . hrms ( esi+ ) : m / z ( calcd for c17h15n4 [ m + h ] = 275.1297 ) , obsd 275.1297 (  
 ppm = 0 ) . h nmr ( 500 mhz , meod ) :  7.82 ( app s , 
 1h ) , 7.75 ( app t , 1h ) , 7.65 ( s , 1h ) , 7.377.49 ( m , 5h ) , 7.25 
 ( d , j = 3.1 hz , 1h ) , 6.49 ( d , j = 
 3.2 hz , 1h ) ppm . c nmr ( 125 mhz , meod )  151.2 , 
 144.8 , 137.3 , 134.8 , 133.7 , 131.5 , 130.1 , 130.0 , 126.4 , 126.3 , 125.6 , 
 125.3 , 122.0 , 119.7 , 112.4 , 109.2 , 102.8 ppm . ir ( thin film ) vmax 3405 , 3181 ( m , br , n h ) , 2936 ( m , 
 c  h ) , 1602 ( s ) , 1504 , 1462 , 1415 , 1307 ( m , c = c , n h ) , 
 1016 ( s ) , 879 ( m ) , 787 , 763 , 728 , 667 ( s , c  h ) cm . prepared according to 
 general procedure e. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 18h ( 0.026 g , 
 0.095 mmol , 45% ) . lcms ( esi+ ) : m / z 275.3 [ m + h ] , retention time 1.82 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c17h14n4 [ m + h ] = 275.1297 ) , obsd 
 275.1288 (  ppm = 3.3 ) . h nmr ( 500 mhz , meod ) :  
 7.76 ( app t , j = 1.8 hz , 1h ) , 7.657.66 ( m , 
 2h ) , 7.60 ( dd , j = 8.3 , 0.6 hz , 1h ) , 7.497.33 
 ( dt , j = 7.0 , 1.8 hz ; 1h ) , 7.44 ( m , 2h ) , 7.33 ( dd , j = 8.2 , 1.6 hz , 1h ) , 7.25 ( d , j = 3.2 
 hz , 1h ) , 6.45 ( dd , j = 3.2 , 0.9 hz , 1h ) ppm . c nmr ( 125 mhz , meod ) :  151.1 , 144.5 , 138.3 , 135.9 , 
 134.8 , 131.6 , 130.2 , 128.9 , 126.4 , 126.3 , 125.7 , 125.6 , 121.4 , 119.8 , 
 110.6 , 109.2 , 102.2 ppm . h ) , 1602 ( s , n  h , 
 c = c ) , 1567 , 1455 ( s , c = c ) , 1350 , 1310 ( m , c n ) , 
 1227 , 1198 ( w ) , 1096 , 1055 , 1015 ( m , c n ) , 863 ( m ) , 785 , 722 
 ( s , c  h ) cm . prepared according to 
 general procedure e. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 18i ( 0.012 g , 
 0.05 mmol , 37% ) . lcms ( esi+ ) : m / z 226.2 [ m + h ] , retention time 1.30 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd for c12h12n5 [ m + h ] = 226.1093 ) , obsd 
 226.1104 (  ppm = 4.9 ) . h nmr ( 400 mhz , meod ) :  
 7.99 ( s , 2h ) , 7.69 ( s , 1h ) , 7.65 ( s , 1h ) , 7.42 ( dt , j = 7.0 , 1.9 hz , 1h ) , 7.397.30 ( m , 2h ) ppm . c 
 nmr ( 100 mhz , meod ) :  151.1 , 135.1 , 134.5 , 131.6 , 130.3 , 125.5 , 
 124.6 , 123.9 , 123.7 , 108.9 ppm . h ) , 
 2917 ( w , c  h ) , 2547 , 2358 , 2247 ( m , br ) , 1607 , 1588 ( m , n  h , 
 c = c ) , 1503 ( s ) , 1437 , 1373 , 1299 ( w , c n ) , 1034 , 903 
 ( m ) , 860 , 787 , 694 , 667 ( s , c  h ) cm . prepared according 
 to general procedure e. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) and then triturated with petroleum ether 
 and dcm to yield 23a as a cream - colored solid ( 434 mg , 
 1.12 mmol , 37% ) . lcms ( esi+ ) m / z 387.3 [ m + h ] , retention time 2.08 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c22h19n4o3 [ m + h ] = 387.1452 ) , 
 obsd 387.1443 (  ppm = 2.2 ) . h nmr ( 500 mhz , cdcl3 )  8.30 ( app t , j = 2.0 , 2.0 hz , 1h ) , 
 8.17 ( ddd , j = 8.2 , 1.3 , 1.0 hz , 1h ) , 7.81 ( ddd , j = 7.7 , 1.6 , 1.1 hz , 1h ) , 7.57 ( app t , j = 8.0 , 8.0 hz , 1h ) , 7.49 ( m , 3h ) , 7.38 ( app dt , j = 6.9 , 2.0 , 1.7 hz , 1h ) , 7.30 ( d , j = 8.9 hz , 2h ) , 
 6.89 ( d , j = 8.9 hz , 2h ) , 3.82 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  160.0 , 153.0 , 148.7 , 
 142.6 , 141.4 , 139.2 , 133.9 , 133.0 , 129.7 , 129.3 , 128.9 , 128.2 , 125.2 , 
 122.3 , 122.1 , 122.0 , 114.4 , 105.3 , 55.3 ppm . h ) , 3056 , 2970 , 2835 ( w , c  h ) , 1605 ( m , n  h ) , 
 1524 , 1506 ( s , no2 ) , 1436 ( m , c = c ) , 1347 ( s , no2 , c n , c  o ) , 1176 ( s ) , 
 1119 ( m ) , 1029 ( m , c  o ) , 835 , 795 ( m c  h ) , 735 , 721 
 ( s , c  h ) cm . prepared according 
 to general procedure e. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 23b as a brown 
 amorphous solid ( 459 mg , 0.93 mmol , 46% ) . m / z 493.3 [ m + h ] , retention time 2.28 
 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c29h25n4o4 [ m + h ] = 493.1876 ) , obsd 493.1890 (  ppm = 2.8 ) . h nmr ( 500 mhz , cdcl3 )  8.29 ( app t , j = 2.0 , 1.9 hz , 1h ) , 8.05 ( ddd , j = 8.3 , 2.3 , 1.0 
 hz , 1h ) , 7.70 ( dt , j = 7.8 , 1.5 , 1.2 hz , 1h ) , 7.41 
 ( app t , j = 8.0 , 8.0 hz , 1h ) , 7.35 ( m , 4h ) , 7.337.29 
 ( m , 4h ) , 7.28 ( m , 1h ) , 7.23 ( m , 1h ) , 7.21 ( m , 3h ) , 7.19 ( s , 1h ) , 7.00 
 ( d , j = 8.5 hz , 1h ) , 6.81 ( d , j = 
 8.8 hz , 1h ) , 5.13 ( s , 2h ) , 3.83 ( s , 3h ) ppm . c nmr ( 125 
 hz , cdcl3 )  159.9 , 154.2 , 153.2 , 148.0 , 141.1 , 139.7 , 
 136.5 , 135.6 , 131.8 , 130.7 , 128.9 , 128.8 , 128.7 , 128.6 , 128.0 , 127.1 , 
 126.0 , 124.7 , 122.4 , 121.8 , 114.3 , 113.5 , 104.9 , 70.6 , 55.3 ppm . ir ( thin film ) vmax 3422 , 3337 , 3202 ( w , br , 2853 ( w , c  h ) , 1614 ( m ) , 1523 ( s , no2 ) , 1348 ( s , 
 no2 ) , 1250 ( s ) , 1178 , 1028 ( m , c  o ) , 835 , 734 , 690 , 
 629 ( m ) cm . tin(ii ) chloride 
 dihydrate 
 ( 564 mg , 2.5 mmol ) was added to a solution of 4-(3-nitro-[1,1-biphenyl]-3-yl)-1h - pyrazol-5-amine 18a ( 140 mg , 0.5 mmol ) in 
 absolute etoh ( 50 ml ) . the reaction was heated to 85 c for 1.516 
 h until all starting material had been consumed . the reaction was 
 then concentrated under reduced pressure ( 10 ml ) and then 
 diluted with etoac ( 50 ml ) , brine ( 50 ml ) , and saturated sodium potassium 
 tartrate solution ( 40 ml ) . the product was extracted into etoac ( 2 
  20 ml ) , the combined organic fractions were dried over anhydrous 
 na2so4 , and the solvent removed under reduced 
 pressure . the crude material was purified by flash chromatography 
 ( 010% v / v meoh in dcm ) to give 4-(3-amino-[1,1-biphenyl]-3-yl)-1h - pyrazol-5-amine 19a as a yellow amorphous 
 solid ( 63 mg , 0.25 mmol , 52% ) , rf 021 
 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 251.2 [ m + h ] , retention time 1.21 min ( 100% ) . hrms 
 ( esi+ ) m / z ( calcd c15h15n4 [ m + h ] = 251.1291 ) , obsd 
 251.1284 (  ppm = 2.9 ) . h nmr ( 500 mhz , meod3 )  7.68 ( d , j = 1.5 hz , 1h ) , 7.63 
 ( s , 1h ) , 7.42 ( m , 3h ) , 7.17 ( app t , j = 8.0 , 7.5 
 hz , 1h ) , 7.02 ( app t , j = 2.0 , 2.0 hz , 1h ) , 6.98 
 ( appdt , j = 7.5 , 1.5 , 1.0 hz , 1h ) , 6.72 ( ddd , j = 8.0 , 2.3 , 1.0 hz , 1h ) ppm . c nmr ( 125 mhz , 
 meod )  151.3 ( br ) , 149.0 , 143.5 , 143.4 , 135.0 , 131.5 ( br ) , 
 130.5 , 130.1 , 126.3 , 126.1 , 125.3 , 118.2 , 115.7 , 115.2 , 108.9 ppm . ir ( thin film ) vmax 3341 , 3206 ( m , br , nh2 ) , 2972 , 2940 ( w , 
 c  h ) , 1602 ( s , n n , c  h ) , 1165 , 1052 
 ( w , c  h ) , 782 , 700 ( m , c  h ) cm . prepared according 
 to general procedure f. the crude product was purified by flash chromatography 
 ( 010% v / v meoh in dcm ) to yield 19b as a cream - colored 
 solid ( 99 mg , 0.28 mmol , 58% ) , rf 0.33 
 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) : m / z 357.2 [ m + h ] , retention time 1.64 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c22h21n4o [ m + h ] = 357.1715 ) , obsd 
 357.1724 (  ppm = 2.5 ) . h nmr ( 500 mhz , acetone - d6 )  10.99 ( br , s , 2h ) , 7.62 ( s , 1h ) , 
 7.47 ( d , j = 2.4 hz , 1h ) , 7.457.39 ( m , 3h ) , 
 7.33 ( app t , j = 7.6 hz , 2h ) , 7.27 ( t , j = 7.3 hz , 1h ) , 7.12 ( d , j = 8.4 hz , 1h ) , 7.08 ( app 
 t , j = 7.8 hz , 1h ) , 6.95 ( app t , j = 2.0 hz , 1h ) , 6.86 ( d , j = 7.6 hz , 1h ) , 6.63 ( dd , j = 7.7 , 2.2 hz , 1h ) , 5.12 ( s , 2h ) , 4.58 ( br , s , 2h ) , 4.32 
 ( br , s , 2h ) ppm . c nmr ( 125 mhz , d6-acetone )  154.7 , 151.3 ( br ) , 149.2 , 140.8 , 139.1 , 
 133.5 , 129.8 , 129.6 , 129.5 , 128.7 , 128.6 , 128.3 , 127.2 , 125.4 ( br ) , 
 119.6 , 117.1 , 115.2 , 114.2 , 108.0 ( br ) , 71.5 ppm . ir ( thin film ) vmax 3334 , 3196 ( m , br , n ( m , c  h ) , 1602 , 1584 ( s , c = c ) , 1498 , 1481 , 1452 ( m , 
 c = c ) , 1379 ( s , c n ) , 1145 ( w ) , 
 1021 ( m , c  o ) , 871 ( w ) , 787 , 734 ( m ) , 696 , 655 ( s ) cm prepared according 
 to general procedure f. the crude product was purified by flash chromatography 
 ( 010% v / v meoh in dcm ) to yield 19b as a white 
 amorphous solid ( 103 mg , 0.38 mmol , 77% ) , rf 0.34 ( 10% v / v meoh in etoac ) . lcms ( esi+ ) m / z 282.3 [ m + h ] , retention time 1.30 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c16h17n4o [ m + h ] = 281.1397 ) , obsd 
 281.1388 (  ppm = 3.3 ) . h nmr ( 500 mhz , meod )  
 7.53 ( s , 1h ) , 7.39 ( dd , j = 8.4 , 2.3 hz , 1h ) , 7.36 
 ( d , j = 2.4 hz , 1h ) , 7.12 ( app t , j = 7.8 hz , 1h ) , 7.06 ( d , j = 8.5 hz , 1h ) , 6.91 ( app 
 t , j = 2.0 hz , 1h ) , 6.86 ( d appt , j = 7.6 , 1.3 hz , 1h ) , 6.69 ( ddd , j = 7.9 , 2.4 , 1.0 
 hz , 1h ) , 3.78 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  
 156.2 , 152.7 , 148.1 , 140.8 , 132.9 , 130.1 , 129.5 , 128.6 ( br ) , 127.7 , 
 127.2 , 121.0 , 118.1 , 115.4 , 113.2 , 109.4 , 56.2 ppm . ir ( thin film ) vmax 3340 , 3210 ( m , br , n h ) , 2953 ( m , 
 c  h ) , 1603 , 1584 ( s , n h ) , 1500 ( m , n  h , c = c ) , 
 1482 ( s , n  h ) , 1454 ( m , c = c ) , 1282 ( m , c n , c  o ) , 1380 ( w , ch3 ) , 1055 , 
 1021 ( m , c  o ) , 873 ( w , c  h ) , 814 , 798 , 700 ( m , c  h ) 
 cm . prepared according 
 to 
 general procedure f. the crude product was purified by flash chromatography 
 ( 08% v / v meoh in dcm ) to yield 19d ( 0.005 g , 
 0.020 mmol , 10% , over two steps ) . lcms ( esi+ ) : m / z 251.2 [ m + h ] ; retention time 1.30 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c15h15n4 [ m + h ] = 251.1297 ) , obsd 
 251.1300 (  ppm = 1.2 ) . h nmr ( 500 mhz , meod ) :  
 7.667.59 ( m , 2h ) , 7.42 ( d , j 8.6 hz , 1h ) , 
 7.397.32 ( m , 3h ) , 6.79 ( d , j = 8.5 hz , 2h ) 
 ppm . c nmr ( 125 mhz , meod ) :  148.4 , 143.2 , 134.9 , 
 132.2 , 130.1 , 128.7 , 125.31 , 125.28 , 124.6 , 116.8 , 109.1 ( br ) ppm . prepared according 
 to general procedure f. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 24a as a brown 
 amorphous solid ( 65 mg , 0.18 mmol , 22% ) . impure fractions ( 157 mg ) , 
 which contained approximately 90% product , were also retained for 
 use in subsequent synthetic steps , with easier chromatographic purification 
 achieved after deprotection of the aryl methyl ether ; rf 0.32 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 357.2 [ m + h ] , retention time 1.60 
 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c22h21n4o [ m + h ] = 357.1710 ) , 
 obsd 357.1698 (  ppm = 3.5 ) . h nmr ( 500 mhz , meod ) 
  7.43 ( m , 2h ) , 7.37 ( dd , j = 7.8 , 7.5 hz , 
 1h ) , 7.29 ( d , j = 8.8 hz , 2h ) , 7.20 ( d , j = 7.6 hz , 1h ) , 7.10 ( app t , j = 7.8 hz , 1h ) , 6.87 
 ( m , 3h ) , 6.81 ( d , j = 7.4 hz , 1h ) , 6.68 ( ddd , j = 7.9 , 2.3 , 1.0 hz , 1h ) , 3.77 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  161.2 , 154.4 ( br ) , 149.0 , 143.3 , 143.2 , 
 142.1 ( br ) , 134.8 , 130.5 , 130.2 , 130.1 , 129.4 , 129.3 , 126.0 , 123.8 
 ( br ) , 118.1 , 115.7 , 115.1 ( 2c ) , 106.7 ( br ) , 55.8 ppm . h ) , 
 2833 ( w , c  h ) , 1601 ( s , n  h , c = c ) , 1520 ( s , c = c , 
 n  h ) , 1506 ( s , c = c , n h ) , 1436 ( m , c = c ) , 
 1290 ( w , c n ) , 1247 ( s , c  o ) , 1178 ( m , c  h ) , 
 1113 ( m , c  h ) , 1029 ( m , c  o ) , 972 ( m ) , 834 ( m , c  h ) , 
 783 ( m , c  h ) , 701 ( m , c  h ) cm . prepared according 
 to general procedure f. the crude product was purified by flash chromatography 
 ( 05% v / v meoh in dcm ) to yield 24b as a brown 
 amorphous solid ( 370 mg , 0.80 mmol , 65% ) , rf 0.16 ( 10% v / v meoh in dcm ) . m / z 463.3 [ m + h ] , retention time 1.95 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c29h27n4o2 [ m + h ] = 463.2134 ) , 
 obsd 463.2141 (  ppm = 1.5 ) . h nmr ( 500 mhz , meod ) 
  7.337.27 ( m , 6h ) , 7.23 ( app t , j = 
 7.1 hz , 1h ) , 7.13 ( m , 2h ) , 7.087.04 ( m , 2h ) , 6.896.82 
 ( m , 3h ) , 6.80 ( dt , j = 7.7 , 1.2 hz , 1h ) , 6.66 ( ddd , j = 7.9 , 2.3 , 1.0 hz , 1h ) , 5.03 ( s , 2h ) , 3.75 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  161.1 , 155.6 , 154.4 , 148.0 , 
 141.8 , 140.6 , 138.8 , 133.4 , 133.2 , 130.6 , 130.1 , 129.5 , 129.4 , 128.6 , 
 128.2 , 127.2 , 123.9 , 121.1 , 118.1 , 115.4 , 115.3 , 115.0 , 106.3 , 71.7 , 
 55.8 ppm . ir ( thin film ) vmax 3349 , 3211 ( br , w , n h ) , 2929 , 2833 
 ( br , w , c  h ) , 1603 ( s ) , 1585 , 1507 ( s , c = c , c  h ) , 
 1453 ( s ) , 1244 , 1177 ( s , c n , c  o ) , 1024 ( s , c  o ) , 
 833 , 735 , 696 ( s , c  h ) cm . 4-hydroxybenzaldehyde ( 83 mg , 0.68 mmol ) 
 and glacial acetic acid ( 72 l , 1.4 mmol ) were added to a stirred 
 solution of 4,4-(5-amino-1h - pyrazole-3,4-diyl)diphenol 5 ( 200 mg , 0.75 mmol ) in dmf ( 0.5 ml ) and dce ( 7 ml ) . the 
 reaction was stirred at room temperature for 2060 min then 
 cooled to 0 c , and na(oac)3bh ( 360 mg , 1.7 mmol ) 
 was added as a single portion . the reaction was allowed to warm to 
 room temperature and stirred for 20 h. for some substrates , an additional 
 equivalent of na(oac)3bh was then added and the reaction 
 was stirred for a further 7 h at room temperature until all aldehyde 
 had been consumed . the reaction was then diluted with etoac ( 50 ml ) 
 and water ( 10 ml ) and brought to ph 78 using saturated nahco3 solution . the phases were separated , and the aqueous fraction 
 was extracted with etoac ( 2  5 ml ) . the combined organic fractions 
 were dried over anhydrous na2so4 , and the solvent 
 was removed under reduced pressure . the crude material was purified 
 by flash chromatography ( 50100% etoac in petroleum ether ) 
 to yield 4,4-(5-((4-hydroxybenzyl)amino)-1h - pyrazole-3,4-diyl)diphenol 26a as a cream - colored powder 
 ( 184 mg , 0.49 mmol , 72% ) , rf 0.13 ( 10% 
 v / v meoh in dcm ) . lcms ( esi+ ) : m / z 374.2 [ m + h ] , retention time 1.25 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c22h20n3o3 [ m + h ] = 374.1505 ) , 
 obsd 374.1495 (  ppm = 2.7 ) . h nmr ( 500 mhz , meod ) 
  7.197.11 ( m , 4h ) , 7.01 ( d , j = 8.5 
 hz , 2h ) , 6.76 ( d , j = 8.5 hz , 2h ) , 6.70 ( m , 4h ) , 
 4.24 ( s , 2h ) ppm . c nmr ( 125 mhz , meod )  158.6 , 
 157.5 , 157.3 , 156.1 ( br ) , 142.3 ( br ) , 132.3 ( 2  ch ) , 129.9 , 
 125.3 , 123.1 ( br ) , 116.7 , 116.2 , 116.1 , 105.7 ( br ) , 49.9 ppm . c nmr processed 
 with lb = 19 hz . ir ( thin film ) vmax 3270 ( s , br , o  h , n h ) , 2940 ( w , c  h ) , 1612 , 
 1596 ( m , c = c ) , 1511 ( s , n  h ) , n ) , 1171 , 1102 , 1011 ( m , c  o ) , 
 834 ( s , c  h ) cm . prepared 
 according to general procedure g. the crude product was purified by 
 flash chromatography ( 50100% v / v etoac in petroleum ether ) 
 to yield 26b as a yellow solid ( 229 mg , 0.51 mmol , 74% ) , rf 0.11 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 454.2 [ m + h ] , retention 
 time 1.59 min ( 92% ) . hrms ( esi+ ) : m / z ( calcd c22h19brn3o3 [ m 
 + h ] = 452.0610 ) , obsd 452.0603 (  ppm = 1.5 ) . h nmr ( 500 mhz , meod )  7.41 ( d , j = 
 2.1 hz , 1h ) , 7.16 ( d , j = 8.7 hz , 2h ) , 7.10 ( dd , j = 1.9 , 8.4 hz , 1h ) , 7.03 ( d , j = 8.7 
 hz , 2h ) , 6.80 ( d , j = 8.4 hz , 1h ) , 6.78 ( d , j = 8.6 hz , 2h , 6.69 ( d , j = 8.7 hz , 2h ) , 
 4.24 ( s , 2h ) ppm . c nmr ( 125 mhz , meod )  158.6 , 
 157.3 , 155.5 , 154.1 , 142.6 , 134.5 , 133.2 , 132.3 , 129.9 , 128.9 , 125.2 , 
 123.1 , 117.0 , 116.7 , 116.2 , 110.6 , 105.8 , 48.3 ppm . h ) , 2928 ( m , c  h ) , 
 1651 , 1612 , 1590 ( m , c = c ) , 1527 , 1494 , 1432 ( s , n n ) , 1171 ( m ) , 
 1105 , 1010 ( m , c  o ) , 835 ( s , c  h ) , 666 , 527 ( m , c  br ) 
 cm . prepared 
 according to general procedure g. the crude product was purified by 
 flash chromatography ( 5090% v / v etoac in petroleum ether ) 
 to yield 26c as a white solid ( 156 mg , 0.43 mmol , 63% ) , rf 0.33 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 364.2 [ m + h ] , retention 
 time 1.71 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c20h18n3o2s [ m 
 + h ] = 364.1120 ) , obsd 364.1107 (  ppm = 3.6 ) . h nmr ( 500 mhz , dmso - d6dmso - d6 )  11.71 ( s , br , 1h , 1-nh ) , 9.50 ( s , 
 br , 1h ) , 9.30 ( s , br , 1h ) , 7.45 ( dd , j = 4.8 , 2.8 
 hz , 1h ) , 7.26 ( d , j = 2.0 hz , 1h ) , 7.10 ( m , 3h ) , 
 6.98 ( d , j = 8.4 hz , 2h ) , 6.72 ( d , j = 8.4 hz , 2h ) , 6.68 ( d , j = 8.4 hz , 2h ) , 4.49 ( s , 
 br , 1h ) , 4.27 ( d , j = 4.27 hz , 2h ) ppm . c nmr ( 125 mhz , dmso - d6 ) 157.0 , 
 155.6 , 154.1 , 142.7 , 139.1 , 130.7 , 128.3 , 128.0 , 125.5 , 123.9 , 121.3 , 
 115.6 , 115.2 , 103.5 , 42.8 ppm . ir ( thin film ) vmax 3306 ( s , br , 
 o  h , n h ) , 3101 , 2928 , 2855 ( w , c  h ) , 1613 , 1591 ( m , c = c ) , 1529 , 1512 ( s , c n , c  o ) , 1436 ( c = c),1375 
 ( m ) , 1262 , 1239 ( s , c n , c  o ) , 1172 ( m , c  o ) , 
 836 ( s , c  h ) cm . prepared 
 according to general procedure g , however no acoh was added to the 
 reaction and no nahco3 was used during reaction workup . the crude product was purified by flash chromatography ( 5090% 
 v / v etoac in petroleum ether ) to yield 26d as a yellow 
 amorphous solid ( 82 mg , 0.24 mmol , 38% ) , rf 0.54 ( 10% v / v meoh in etoac ) . lcms ( esi+ ) m / z 348.2 [ m + h ] , retention time 1.54 min ( 100% ) . 
 hrms ( esi+ ) : m / z ( calcd c20h18n3o3 [ m + h ] = 348.1348 ) , 
 obsd 348.1356 (  ppm = 2.3 ) . h nmr ( 500 mhz , meod ) 
  7.38 ( d , j = 1.8 hz , 1h ) , 7.16 ( d , j = 8.8 hz , 2h ) , 7.01 ( d , j = 8.6 hz , 2h ) , 
 6.78 ( d , j = 8.7 hz , 2h ) , 6.70 ( d , j = 8.8 hz , 2h ) , 6.30 ( m , 1h ) , 6.18 ( d , j = 3.2 hz , 
 1h ) , 4.33 ( s , 2h ) ppm . c nmr ( 125 mhz , meod )  
 158.6 , 157.3 , 155.6 ( br ) , 154.9 , 142.8 , 142.2 ( br ) , 132.3 , 129.9 , 
 125.1 , 122.9 ( br ) , 116.7 , 116.2 , 111.2 , 107.6 , 106.0 ( br ) , 42.5 ppm , c nmr processed with lb = 6 hz . h ) , 2935 , 2816 ( w , 
 c  h ) , 1614 ( m , c = c ) , 1529 , 1500 ( s , n h ) , 1434 
 ( m ) , 1378 , 1343 ( w ) , 1261 , 1238 ( s , c n , c  o ) , 1172 
 ( m , c  o ) , 1012 ( w ) , 836 ( s , c  h ) , 739 ( m , furan , c  h ) 
 cm . prepared according to general procedure g. the product was purified 
 by two sequential flash chromatography columns , eluting with 05% 
 v / v meoh in dcm , followed by 50100% v / v etoac in petroleum 
 ether , to yield 26e as a brown solid ( 52 mg , 0.13 mmol , 
 52% ) , rf 0.14 ( 10% v / v meoh in dcm ) . lcms 
 ( esi+ ) m / z 397.3 [ m + h ] , retention time 1.53 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c24h21n4o2 [ m + h ] = 397.1665 ) , obsd 397.1653 (  
 ppm = 3.0 ) . h nmr ( 500 mhz , meod )  7.50 ( d , j = 0.8 hz , 1h ) , 7.31 ( d , j = 8.3 hz , 1h ) , 
 7.19 ( d , j = 3.2 hz , 1h ) , 7.17 ( d , j = 8.7 hz , 2h ) , 7.09 ( dd , j = 8.4 , 1.7 hz , 1h ) , 
 7.01 ( d , j = 8.5 hz , 2h ) , 6.75 ( d , j = 8.6 hz , 2h ) , 6.70 ( d , j = 8.7 hz , 2h ) , 4.41 ( s , 
 2h ) ppm . c nmr ( 125 mhz , meod )  158.6 , 157.3 , 
 156.4 ( br ) , 142.1 ( br ) , 137.0 , 132.3 , 131.7 , 130.0 , 129.5 , 125.9 , 
 125.3 , 123.0 ( br ) , 122.6 , 120.4 , 116.7 , 116.2 , 112.1 , 105.7 ( br ) , 
 102.3 , 50.5 ppm . h ) , 
 2927 ( w , c  h ) , 1613 , 1594 ( m , c = c ) , 1529 , 1511 ( s ) 1451 , 
 1435 , 1349 ( m ) , 1262 , 1240 ( s , c  o , c n ) , 1172 ( m , c  o ) , 
 837 ( s , c  h ) , 731 ( m ) cm . glacial acoh 
 ( 1 ml ) was added to a suspension of 3,4-bis(4-methoxyphenyl)-1h - pyrazol-5-amine 13a ( 222 mg , 0.75 mmol ) and 
 pyridine-4-carbaldehyde ( 71 ul , 0.75 mmol ) in dry meoh the 
 reaction was stirred at room temperature for 3090 min , and 
 then nacnbh3 ( 47 mg , 0.75 mmol ) was added as a single portion . the reaction was stirred at room temperature for 1424 h and 
 then concentrated under reduced pressure . the crude product was redissolved 
 in etoac ( 40 ml ) and washed with water ( 10 ml ) that had been made 
 basic with saturated nahco3 solution . the aqueous phase 
 was then extracted with etoac ( 2  10 ml ) , and the combined organic 
 fractions were washed with brine ( 5 ml ) . the solvent was removed under 
 reduced pressure to dry the product directly onto silica gel . flash 
 chromatography ( 05% v / v meoh in etoac ) yielded 3,4-bis(4-methoxyphenyl)-n-(pyridin-4-ylmethyl)-1h - pyrazol-5-amine 27a as a yellow amorphous solid ( 226 mg , 0.59 mmol , 78% ) , rf 0.13 ( 5% v / v meoh in etoac ) . lcms ( esi+ ) m / z 386.6 [ m + h ] , retention 
 time 1.49 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c23h23n4o2 [ m 
 + h ] = 387.1821 ) , obsd 387.1819 ( ppm = 0.5 ) . h nmr ( 500 mhz , cdcl3 )  8.53 ( d , j = 5.5 hz , 2h ) , 7.30 ( d , j = 5.7 hz , 2h ) , 
 7.22 ( m , 4h ) , 6.91 ( d , j = 8.9 hz , 2h ) , 6.83 ( d , j = 8.9 hz , 2h ) , 4.49 ( s , 2h ) , 3.82 ( s , 3h ) , 3.78 ( s , 3h ) 
 ppm . c nmr ( 125 mhz , cdcl3 )  159.8 , 
 158.6 , 154.8 , 149.9 , 140.8 , 131.0 , 128.7 , 124.7 , 122.5 , 122.4 , 114.6 , 
 114.3 , 104.7 , 55.4 ( 2 c ) , 47.1 ppm . c nmr processed with lb = 1 hz . 
 ir ( thin film ) vmax 3414 ( br , w , n h ) , 
 3001 , 2934 , 2835 ( w , c  h ) , 1604 ( w ) , 1525 , 1511 ( s ) , 1461 , 
 1361 ( m ) , 1285 ( s , c n ) , 1245 , 1032 ( m , c  o ) , 834 ( s ) , 
 801 ( m ) cm . prepared according 
 to general procedure h. the crude product was purified by flash chromatography 
 ( 020% v / v meoh in dcm ) to yield 27b as a white 
 amorphous solid ( 484 mg , 1.29 mmol , 95% ) . lcms ( esi+ ) m / z 376.3 [ m + h ] , retention time 1.61 
 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd 
 c21h22n5o2 [ m + h ] = 376.1773 ) , obsd 376.1766 (  ppm = 2.1 ) . h nmr ( 500 mhz , meod )  7.75 ( br , d , j = 1.5 
 hz , 1h ) , 7.24 ( d , j = 8.8 hz , 2h ) , 7.12 ( d , j = 8.8 hz , 2h ) , 7.00 ( br , d , j = 0.9 hz , 
 1h ) , 6.89 ( d , j = 8.7 hz , 2h ) , 6.84 ( d , j = 8.8 hz , 2h ) , 4.34 ( s , 2h ) , 3.78 ( s , 3h ) , 3.77 ( s , 3h ) ppm . c nmr ( 125 mhz , meod )  161.0 , 159.9 , 155.2 , 142.8 , 
 137.2 , 136.2 , 132.2 , 130.0 , 126.3 , 124.2 , 118.2 , 115.3 , 114.9 , 105.6 , 
 55.7 , 55.7 , 42.0 ppm . h ) , 
 2902 , 2835 ( w , c  h ) , 1612 , 1590 ( w , c = c ) , 1526 , 1511 
 ( s , n h ) , 1462 ( m , c = c ) , 1246 ( s , c  o , c n ) , 
 1177 , 1031 ( m , c  o ) , 834 ( m , c  h ) cm . prepared 
 according to general procedure h except that 4,4-(5-amino-1h - pyrazole-3,4-diyl)diphenol 5 was used as 
 the amine component . the crude product was purified by flash chromatography 
 ( 80100% v / v etoac in petroleum ether , followed by 05% 
 v / v meoh in etoac ) to yield 26f as a white solid ( 93 
 mg , 0.24 mmol , 47% ) , rf 0.45 ( 10% v / v 
 meoh in etoac ) . lcms ( esi+ ) m / z 398.1 
 [ m + h ] , retention time 1.48 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c23h20n5o2 [ m + h ] = 398.1617 ) , obsd 
 398.1612 (  ppm = 1.3 ) . h nmr ( 500 mhz , meod )  
 8.11 ( d , j = 1.0 hz , 1h ) , 7.41 ( d , j = 8.4 hz , 1h ) , 7.30 ( dd , j = 8.4 , 6.9 hz , 1h ) , 
 7.16 ( d , j = 8.7 hz , 2h ) , 7.09 ( dd , j = 6.9 , 1.0 hz , 1h ) , 7.01 ( d , j = 8.7 hz , 2h ) , 6.75 
 ( d , j = 8.6 hz , 2h ) , 6.70 ( d , j = 
 8.7 hz , 2h ) , 4.72 ( s , 2h ) ppm . c nmr ( 125 mhz , meod ) 
  158.7 , 157.3 , 155.7 ( br ) , 142.8 ( br ) , 141.9 , 134.9 , 133.7 , 
 132.3 , 129.9 , 127.9 , 125.2 , 123.1 , 123.0 ( br ) , 116.7 , 116.2 , 109.9 , 
 105.7 , 47.6 ( br ) ppm . ir ( solid ) vmax 3432 , 3443 ( m , n  h , 
 o  h ) , 2986 , 2797 , m , c  h ) , 1612 , 1589 ( m , c = c ) , 
 1528 , 1509 ( s , c = c , n h ) , 1450 , 1383 , 1262 , 1240 ( s , 
 c  o , c n ) , 1173 , 1086 , 1003 ( m , c  o , c n ) , 
 949 ( m ) , 821 ( s , c  h ) , 777 , 739 ( m , c  h ) cm . benzoyl chloride 
 ( 31 l , 0.263 mmol ) and triethylamine ( 42 l , 0.3 mmol ) 
 were added sequentially to a stirred solution of 3,4-bis(4-methoxyphenyl)-1h - pyrazol-5-amine 13a in anhydrous dcm ( 1.25 
 ml ) at 0 c . the reaction was stirred at 0 c for 5 min 
 and then allowed to warm to room temperature and stirred for 3.5 h. 
 the reaction was then diluted with dcm ( 10 ml ) , washed with water 
 ( 3 ml ) and brine ( 3 ml ) , and the solvent removed under reduced pressure . the crude product was purified by flash chromatography ( 05% 
 v / v meoh in dcm ) to yield 28a as a white solid ( 31 mg , 
 0.08 mmol , 31% ) , rf 0.16 ( 5% v / v meoh 
 in dcm ) . lcms ( esi+ ) m / z 400.2 [ m 
 + h ] , retention time 2.03 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c24h22n3o3 [ m + h ] = 400.1661 ) , obsd 
 400.1662 (  ppm = 0.2 ) . h nmr ( 500 mhz , dmso - d6 )  13.00 ( s , 1h ) , 9.95 ( s , 1h ) , 7.88 
 ( d , j = 6.3 hz , 2h ) , 7.56 ( app t , j = 7.5 , 6.9 hz , 1h ) , 7.47 ( appt , j = 7.5 , 6.9 hz , 
 2h ) , 7.29 ( d , j = 8.4 hz , 2h ) , 7.14 ( d , j = 8.4 hz , 2h ) , 6.96 ( d , j = 8.4 hz , 2h ) , 6.84 ( d , j = 8.4 hz , 2h ) , 3.76 ( s , 3h ) , 3.69 ( s , 3h ) ppm . c nmr ( 125 mhz , dmso - d6 )  166.8 , 
 159.1 , 157.9 , 144.2 , 139.3 , 134.0 , 131.6 , 130.3 , 128.6 , 128.4 , 127.5 , 
 124.6 , 122.1 , 114.2 , 113.8 , 113.7 ( partially obscured ) , 55.2 , 54.9 
 ppm . h ) , 3004 , 2932 , 2836 
 ( w , c  h ) , 1657 ( m , c = o ) , 1602 , 1579 ( m , c = c ) , 
 1509 ( s , n h ) , 1463 ( m , c = c ) , 1247 ( s , c n ) , 
 1178 , 1027 ( m , c  o ) , 835 ( m , c  h ) , 79 ( m ) cm . 4-methoxybenzoyl 
 chloride ( 90 mg , 0.525 mmol ) and triethylamine ( 84 l , 0.6 mmol ) 
 were added sequentially to a stirred solution of 3,4-bis(4-methoxyphenyl)-1h - pyrazol-5-amine 13a in anhydrous dcm ( 2.5 
 ml ) at 0 c . the reaction was stirred at 0 c for 5 min 
 and then allowed to warm to room temperature and stirred for 5 h. 
 the reaction was then diluted with dcm ( 10 ml ) , washed with water 
 ( 5 ml ) and brine ( 5 ml ) , dried over anhydrous mgso4 , and 
 the solvent removed under reduced pressure . the crude product was 
 purified by flash chromatography ( 80100% v / v etoac in petroleum 
 ether ) to yield 28b as an orange solid ( 58 mg , 0.13 mmol , 
 27% ) , rf 0.49 ( 5% v / v meoh in etoac ) . lcms ( esi+ ) m / z 430.3 [ m + h ] , retention time 2.02 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c25h24n3o4 [ m + h ] = 430.1767 ) , obsd 430.1778 (  
 ppm = 2.6 ) . h nmr ( 500 mhz , dmso - d6 )  12.98 ( s , 1h ) , 9.80 ( s , 1h ) , 7.88 ( d , j = 8.3 hz , 2h ) , 7.29 ( d , j = 8.6 hz , 2h ) , 7.13 ( d , j = 8.8 hz , 2h ) , 7.01 ( d , j = 8.4 hz , 2h ) , 
 6.95 ( d , j = 7.1 hz , 2h ) , 6.83 ( d , j = 8.2 hz , 2h ) , 3.81 ( s , 3h ) , 3.76 ( s , 3h ) , 3.69 ( s , 3h ) ppm . c nmr ( 125 mhz , dmso - d6 )  
 166.3 , 161.9 , 159.1 , 157.9 , 144.5 , 139.3 , 130.4 , 129.6 , 128.7 , 126.2 , 
 124.7 , 122.2 , 114.2 , 113.8 , 113.7 , 55.5 , 55.2 , 55.0 ppm . ir ( solid ) vmax 3294 , 3154 ( w , br , 
 n h ) , 2938 , 2837 ( w , c  h ) , 1738 , 1669 , 1644 , 1605 ( w , 
 c = c ) , 1501 ( m , n  h , c  h ) , 1241 , 1177 ( s , c  n , 
 c  o ) , 1019 ( m ) , 961 ( w ) , 834 ( s , c  h ) , 759 , 693 ( w , 
 c  h ) cm isonicotinic 
 acid ( 38 mg , 0.31 mmol ) was suspended in anhydrous dcm ( 2 ml ) . the 
 solution was cooled to 0 c , and oxalyl chloride ( 29 l , 
 0.34 mmol ) was added along with a catalytic amount of dmf . the reaction 
 was allowed to come to room temperature and stirred for 2 h. the volatiles 
 were removed under reduced pressure , and the resulting residue was 
 resuspended in anhydrous dcm ( 1.25 ml ) . the solution was cooled to 
 0 c and the aminopyrazole 5 ( 74 mg , 0.25 mmol ) 
 was added , followed by et3n ( 122 l , 0.875 mmol ) . the reaction was stirred at 0 c for 1 h , then allowed to warm 
 to room temperature and stirred overnight or until all of the aminopyrazole 
 had been consumed . the reaction was then diluted with dcm ( 20 ml ) , 
 washed with water ( 5 ml ) , then brine ( 3 ml ) , dried over anhydrous 
 mgso4 , and the solvent evaporated under reduced pressure . the crude product was purified by silica gel chromatography ( 010% 
 meoh in dcm ) to yield 30 as a white solid ( 34 mg , 0.086 
 mmol , 34% ) , rf 0.20 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) : m / z 401.2 [ m + h ] , retention time 1.76 min ( 98% ) . hrms ( esi+ ) : m / z ( calcd c23h21n4o3 [ m + h ] = 401.1614 ) , obsd 401.1617 (  
 ppm = 0.7 ) . h nmr ( 500 mhz , cdcl3 )  
 8.76 ( d , j = 4.8 hz , 2h ) , 8.23 ( s , 1h ) , 7.59 ( d , j = 4.4 hz , 2h ) , 7.36 ( d , j = 8.2 hz , 2h ) , 
 7.20 ( d , j = 8.1 hz , 2h ) , 6.95 ( d , j = 8.2 hz , 2h ) , 6.83 ( d , j = 8.8 hz , 2h ) , 3.84 ( s , 
 3h ) , 3.79 ( s , 3h ) ppm . c nmr ( 125 mhz , cdcl3 )  163.7 , 159.7 , 159.2 , 151.0 , 145.9 ( br ) , 140.2 , 138.6 ( br ) , 
 131.0 , 129.1 , 124.2 ( br ) , 123.7 , 120.9 , 115.0 , 114.1 , 108.0 ( br ) , 
 55.4 , 55.4 ppm . h ) , 2967 , 
 2836 , 2371 ( w , c  h ) 1663 ( m ) , 1613 ( w ) , 1525 , 1508 , 1297 ( m , 
 c = c ) , 1248 ( s , c n ) , 1025 ( w , c  o ) , 835 ( m ) cm . a solution of 1 m bbr3 in dcm 
 ( 21 ml , 21 mmol ) was added dropwise over a period of 30 min to a stirred 
 solution of 3,4-bis(4-methoxyphenyl)-1h - pyrazol-5-amine 13a ( 886 mg , 3.0 mmol ) in anhydrous dcm ( 75 ml ) at 0 c . the reaction was stirred at 0 c for 3.524 h , or until 
 all starting material had been consumed . the reaction was quenched 
 with saturated nahco3 at 0 c , and the resulting precipitate 
 was extracted into etoac ( 50 ml ) . the organic phase was collected , 
 and the aqueous phase was re - extracted with etoac ( 3  25 ml ) . the organic fractions were combined , washed with brine ( 5 ml ) , dried 
 over anhydrous mgso4 or na2so4 , and 
 the solvent removed under reduced pressure . the crude product was 
 purified by flash chromatography ( 010% v / v meoh in dcm ) to 
 yield 4,4-(5-amino-1h - pyrazole-3,4-diyl)diphenol 5 as a pale - brown solid ( 795 mg , 2.97 mmol , 99% ) , rf 0.27 ( 10% v / v meoh in etoac ) . lcms ( esi+ ) : m / z 269.2 [ m + h ] ; retention 
 time 1.19 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c15h14n3o2 [ m 
 + h ] = 268.1086 ) , obsd 268.1075 (  ppm = 4.1 ) . h nmr ( 500 mhz , dmso - d6 )  
 11.64 ( br , s , 1h ) , 9.52 ( br , s , 1h ) , 9.30 ( s , 1h ) , 7.10 ( d , j = 8.6 hz , 2h ) , 6.97 ( d , j = 8.2 hz , 2h ) , 
 6.71 ( d , j = 8.4 hz , 2h ) , 6.67 ( d , j = 8.4 hz , 2h ) ppm . c nmr ( 125 mhz , dmso - d6 )  156.8 , 155.4 , 153.1 , 139.0 , 130.5 , 128.3 , 124.2 , 
 121.9 , 115.5 , 115.2 , 103.8 ppm . ir ( thin film ) vmax 35002700 
 ( br , o  h , n h ) , 2797 , 2670 , 2591 ( w ) , 1613 ( m ) , 1513 
 ( s ) , 1488 , 1435 ( m ) , 1374 ( w ) , 1261 , 1234 ( s ) , 1172 ( m ) , 1101 ( w ) , 
 1028 , 969 ( w ) , 834 ( s ) , 756 ( w ) cm . prepared according to general procedure 
 i as a pale - brown foam in quantitative yield ( 157 mg , 0.5 mmol ) and 
 used without further purification . lcms ( esi+ ) m / z 268.2 [ m + h ] , retention time 1.25 min ( 95% ) . hrms ( esi+ ) m / z ( calcd c15h14n3o2 [ m + h ] = 268.1086 ) , 
 obsd 268.1075 (  ppm = 4.1 ) . h nmr ( 500 mhz , meod ) 
  7.11 ( app t , j = 7.9 , 7.9 hz , 1h ) , 7.07 ( d , j = 8.6 hz , 2h ) , 6.82 ( ddd , j = 8.7 , 1.2 , 
 1.0 hz , 1h ) , 6.79 ( m , 3h ) , 6.71 ( ddd , j = 8.2 , 2.6 , 
 1.0 hz , 1h ) ppm . c nmr ( 125 mhz , meod )  158.5 , 
 157.3 , 153.0 , 143.3 , 133.3 , 132.0 , 130.5 , 125.0 , 119.9 , 116.7 , 116.1 , 
 115.5 , 106.6 ppm . ir ( thin film ) vmax 33963290 
 ( w , br , o  h , n h ) , 2923 , 2853 ( s , c  h ) , 1714 , 
 1606 , 1579 ( w ) , 1455 ( m ) , 1377 ( w ) , 1260 ( m , c ( m , c  o ) , 874 ( w ) , 802 ( m ) cm . prepared according to general procedure 
 i , except that only 3.5 equiv of 1.0 m bbr3 in dcm were 
 used . the crude product was purified by flash chromatography ( 010% 
 v / v meoh in dcm ) to yield 14c as a cream - colored amorphous 
 solid ( 255 mg , 0.95 mmol , 95% ) , rf 0.22 
 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 270.2 [ m + h ] , retention time 1.56 min ( 100% ) . hrms 
 ( esi+ ) : m / z ( calcd c15h13fn3o [ m + h ] = 270.1037 ) , obsd 
 270.1024 (  ppm = 1.9 ) . h nmr ( 500 mhz , meod )  
 7.28 ( m , 1h ) , 7.15 ( d , j = 7.7 hz , 1h ) , 7.06 ( m , 
 3h ) , 6.98 ( app t , j = 10.0 hz , 1h ) , 6.81 ( d , j = 8.6 hz , 2h ) ppm . c nmr ( 125 mhz , meod ) 
  164.1 ( jc  f = 242.8 hz ) , 
 157.6 , 154.8 ( br ) , 140.1 ( br ) , 133.8 , 132.5 , 131.3 , 124.6 , 124.3 , 
 116.8 , 115.6 , 115.0 ( jc  f = 22.5 
 hz ) , 107.7 ppm . h ) , 
 2962 , 2936 , 2856 , 2794 ( w , c  h ) , 1614 , 1578 , 1524 ( s , c = c ) , 
 1488 , 1445 ( m ) , 1265 , 1236 , 1215 ( s , c n ) , 1171 ( m , c  o ) ) , 
 867 , 838 , 788 , 686 ( m ) cm . prepared 
 according to general procedure i , except that only 3.5 equiv of 1.0 
 m bbr3 in dcm were used . the crude product was purified 
 by flash chromatography ( 210% v / v meoh in dcm ) to yield 25a as a brown amorphous solid ( 30 mg , 0.089 mmol , 50% ) , rf 0.05 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 343.3 [ m + h ] , retention 
 time 1.37 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c21h19n4o [ m + h ] = 343.1553 ) , obsd 343.1541 (  ppm = 3.6 ) . h nmr 
 ( 500 mhz , meod ) 7.467.40 ( m , 2h ) , 7.38 ( app t , j = 7.6 hz , 1h ) , 7.22 ( d , j = 8.0 hz , 1h ) , 7.20 ( d , j = 8.6 hz , 2h ) , 7.11 ( app t , j = 7.7 hz , 
 1h ) , 6.86 ( s , 1h ) , 6.83 ( d , j = 7.7 hz , 1h ) , 6.75 
 ( d , j = 8.6 hz , 2h ) , 6.68 ( dd , j = 8.0 , 2.2 hz , 1h ) ppm . c nmr ( 125 mhz , meod )  
 158.9 , 154.2 ( br ) , 149.0 , 143.2 , 142.4 ( br ) , 134.9 , 130.4 , 130.3 , 
 130.1 , 129.8 , 129.43 , 129.35 , 125.8 , 122.7 ( br ) , 118.1 , 116.4 , 115.7 , 
 115.2 , 106.4 ppm . ir 
 ( thin film ) vmax 3341 , 3206 ( br , m , n h , 
 o  h ) , 2984 ( m , c  h ) , 1604 ( s , n  h , c = c ) , 
 1522 , 1505 ( c = c ) , 1470 ( m , c = c ) , 1266 , 1242 ( c  o ) , 
 1173 ( w , c  h ) , 837 ( m , c  h ) , 786 ( m , c  h ) , 701 
 ( m , c  h ) cm . prepared according to general procedure i. the crude product 
 was 
 purified by flash chromatography ( 010% v / v meoh in dcm ) to 
 yield 25b as a white solid ( 25 mg , 0.07 mmol , 46% ) , rf 0.07 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 359.2 [ m + h ] , retention 
 time 1.21 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c21h19n4o2 [ m 
 + h ] = 359.1508 ) , obsd 359.1505 (  ppm = 0.8 ) . h nmr ( 500 mhz , meod )  7.21 ( d , j = 
 8.7 hz , 2h ) , 7.127.06 ( m , 2h ) , 7.04 ( dd , j = 8.2 , 2.2 hz , 1h ) , 6.916.85 ( m , 2h ) , 6.82 ( d. app t , j = 7.6 , 1.4 hz , 1h ) , 6.75 ( d , j = 8.7 
 hz , 2h ) , 6.66 ( ddd , j = 7.9 , 2.3 , 1.0 hz , 1h ) ppm . c nmr ( 125 mhz , meod )  158.7 , 154.3 ( br ) , 154.0 , 148.1 , 
 142.1 ( br ) , 140.8 , 133.2 , 130.6 , 130.1 , 129.7 , 125.7 , 123.6 ( br ) , 
 120.7 , 117.9 , 117.4 , 116.3 , 115.3 , 106.0 ( br ) ppm . ir ( thin film ) vmax 33743200 ( br , s , o  h , n h ) , 
 1612 , 1510 ( s , c = c ) , 1442 , 1399 ( m , c n ) , 
 1174 ( m , c  o ) , 1111 , 1032 ( w , c  o ) , 836 ( s ) , 700 ( w ) 
 cm . prepared 
 according to general procedure i , except that the reaction was allowed 
 to warm to room temperature and stirred for 2 h before being quenched 
 with saturated nahco3 solution . the crude product was purified 
 by flash chromatography ( 010% v / v meoh in dcm ) to yield 26 g as a white solid ( 51 mg , 0.14 mmol , 61% ) , rf 0.10 ( 10% v / v meoh in dcm ) . lcms ( esi+ ) m / z 359.2 [ m + h ] , retention time 1.65 
 min ( 100% ) . hrms ( esi+ ) m / z ( calcd 
 c21h19n4o2 [ m + h ] = 359.1508 ) , obsd 359.1508 ( ppm = 0.0 ) . h nmr ( 500 mhz , dmso - d6 )  11.63 
 ( s , 1h ) , 9.54 ( s , br , 1h ) , 9.32 ( s , br , 1h ) , 8.44 ( d , j = 6.0 hz , 2h ) , 7.32 ( d , j = 5.6 hz , 2h ) , 7.08 ( d , j = 8.4 hz , 2h ) , 7.03 ( d , j = 8.4 hz , 2h ) , 
 6.75 ( d , j = 8.4 hz , 2h ) , 6.67 ( d , j = 8.4 hz , 2h ) , 5.21 ( t , j = 5.6 hz , 1h ) , 4.31 ( d , j = 6.0 hz , 2h ) ppm . c nmr ( 125 mhz , dmso - d6 )  155.7 , 155.8 , 153.9 , 151.3 , 149.2 , 
 139.2 , 130.8 , 128.4 , 123.8 , 122.5 , 121.4 , 115.6 , 115.3 , 103.4 , 46.1 
 ppm . h ) , 
 2905 , 2585 ( w , c  h ) , 1602 , 1575 ( m , c = c ) , 1537 , 1513 , 
 1479 ( s , n  h , c  h ) , 1437 , 1418 ( m ) , 1385 ( w , c  o ) , 
 1246 ( s , c  o , c n ) , 1157 ( m , c  o ) , 1026 , 1006 
 ( m ) , 835 ( s , c  h ) 799 , 661 ( m ) cm . prepared according to general procedure 
 i. the crude product was 
 purified by flash chromatography ( 120% v / v meoh in dcm ) to 
 yield 26h as a white solid ( 118 mg , 0.34 mmol , 68% ) . lcms ( esi+ ) m / z 348.2 [ m + h ] , retention time 1.26 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c19h18n5o2 [ m + h ] = 348.1461 ) , obsd 348.1468 (  
 ppm = 2.0 ) . h nmr ( 500 mhz , meod )  7.60 ( d , j = 1.2 hz , 1h ) , 7.16 ( d , j = 8.7 hz , 2h ) , 
 7.02 ( d , j = 8.6 hz , 2h ) , 6.94 ( d , j = 1.2 hz , 1h ) , 6.76 ( d , j = 8.6 hz , 2h ) , 6.70 ( d , j = 8.7 hz , 2h ) , 4.31 ( s , 2h , 14-ch2 ) ppm . c nmr ( 125 mhz , meod )  158.7 , 157.3 , 155.3 ( br ) , 142.8 
 ( br ) , 137.3 , 136.2 , 132.2 , 129.9 , 125.1 , 123.1 , 118.2 ( br ) , 116.7 , 
 116.2 , 105.8 , 41.9 ppm . ir ( thin film ) vmax 3228 ( s , br , o  h , 
 n  h ) , 2929 ( m , br , c  h ) , 2471 ( m ) , 1613 , 1589 ( m , c = c ) , 
 1527 , 1511 ( s ) , 1445 , 1379 ( m , br , c n ) , 
 1171 , 1104 ( m , c  o ) , 974 ( m ) , 836 ( s ) , 739 ( w ) cm . prepared as a 
 tan powder ( 43 mg , 0.105 mmol , quant ) according to general procedure 
 i. lcms ( esi+ ) m / z 372.2 [ m + h ] , retention 
 time 1.58 min ( 100% ) . hrms ( esi+ ) : m / z ( calcd c22h18n3o3 [ m 
 + h ] = 372.1348 ) , obsd 372.1344 (  ppm = 1.1 ) . h nmr ( 500 mhz , meod )  7.87 ( d , j = 
 7.7 hz , 2h ) , 7.56 ( t , j = 7.4 hz , 1h ) , 7.47 ( app 
 t , j = 7.6 hz , 2h ) , 7.24 ( d , j = 
 8.6 hz , 2h ) , 7.10 ( d , j = 8.1 hz , 2h ) , 6.76 ( d , j = 8.7 hz , 2h ) , 6.71 ( d , j = 8.6 hz , 2h ) 
 ppm . c nmr ( 125 mhz , meod )  170.8 , 159.0 , 157.5 , 
 145.1 ( br ) , 142.4 ( br ) , 135.2 , 133.1 , 131.9 , 130.2 , 129.6 , 128.7 , 
 124.5 , 122.5 ( br ) , 116.4 , 116.3 , 115.7 ( br ) ppm . ir ( thin film ) vmax 3254 ( s , br , o  h , n h ) , 2973 , 2927 ( w , c  h ) , 
 1656 ( m , c = o ) , 1614 ( m , c = c ) , 1510 ( s , br , n h ) , 
 1439 ( m , c = c ) , 1371 ( m ) , 1263 ( s , br , c  o , c n ) , 
 1173 ( m , c  o ) , 836 ( m , c  h ) , 710 ( w ) cm . prepared 
 according to general procedure i. the crude product was purified by 
 flash chromatography ( 110% v / v meoh in dcm ) to yield 29b as a cream - colored amorphous solid ( 29 mg , 0.074 mmol , 
 81% ) . lcms ( esi+ ) m / z 372.2 [ m + 
 h ] , retention time 1.59 min ( 100% ) . hrms ( esi+ ) m / z ( calcd c22h18n3o4 [ m + h ] = 388.1297 ) , obsd 
 388.1286 (  ppm = 2.8 ) . h nmr ( 500 mhz , meod )  
 7.76 ( d , j = 8.4 hz , 2h ) , 7.23 ( d , j = 8.8 hz , 2h ) , 7.09 ( d , j = 8.8 hz , 2h ) , 6.83 ( d , j = 8.4 hz , 2h ) , 6.75 ( d , j = 8.4 hz , 2h ) , 
 6.71 ( d , j = 8.8 hz , 2h ) ppm . c nmr 
 ( 125 mhz , meod )  170.4 , 162.5 , 158.9 , 157.48 , 144.8 ( br ) , 143.0 
 ( br ) , 131.9 , 130.9 , 130.2 , 125.9 , 124.6 , 122.8 ( br ) , 116.4 , 116.3 , 
 116.1 , 115.5 ( br ) ppm . ir ( thin film ) vmax 3204 ( br , m , o  h , 
 n  h ) , 1648 , 1607 , 1587 ( m , c = c ) , 1529 ( s ) , 1441 ( m ) , 
 1261 , 1236 , 1172 ( s , c n , c  o ) , 1022 , 1010 ( w , c  o ) , 
 837 ( m ) , 764 ( w ) cm prepared 
 according to general procedure i , except that 1.0 m bbr3 in dcm was added at 78 c and then the reaction was 
 allowed to warm slowly to 0 c and then stirred overnight at 
 room temperature . the crude product was purified by flash chromatography 
 ( 110% v / v meoh in etoac ) to yield 29c as an off - white 
 solid ( 42.3 mg , 0.114 mmol , 87% ) . lcms ( esi+ ) m / z 373.2 [ m + h ] , retention time 1.30 min ( 98% ) . 
 hrms ( esi+ ) : m / z ( calcd c21h17n4o3 [ m + h ] = 373.1301 ) , 
 obsd 373.1306 (  ppm = 1.3 ) . h nmr ( 500 mhz , meod ) 
  8.72 ( s , br , 2h ) , 7.82 ( d , j = 4.5 hz , 2h ) , 
 7.23 ( d , j = 8.5 hz , 2h ) , 7.08 ( d , j = 8.5 hz , 2h ) , 6.76 ( d , j = 8.5 hz , 2h ) , 6.72 ( d , j = 8.5 hz , 2h ) ppm . c nmr ( 125 mhz , meod ) 
  168.3 , 159.1 , 157.7 , 150.9 , 143.6 ( br ) , 143.0 ( br ) , 131.9 , 
 130.2 , 124.3 , 123.2 , 122.4 ( br ) , 116.5 , 116.4 , 115.6 ( br ) ppm . ir ( thin film ) vmax 34403230 
 ( br , s , o  h , n ( c = c ) , 
 1415 ( m ) , 1264 ( s , c n ) , 1175 ( m , c  o ) , 837 ( s ) , 751 , 
 685 ( w ) cm the cyp121 ( rv2276 ) gene was expressed 
 using a phat2/cyp121 expression clone in escherichia coli c41(de3 ) cells as the his6-tagged construct , following 
 previously described conditions , with minor modifications . c41 ( de3 ) e. coli cells were transformed with phat / cyp121 and cultured overnight in lb medium containing ampicillin ( 100 mg / l ) . mg / l ) were inoculated using an overnight culture of the transformant 
 cells ( 0.4% v / v ) and grown at 37 c , 200 rpm until an od600 = 0.50.6 , at which point the temperature was reduced 
 to 23 c . cyp121 gene expression was induced 
 with iptg ( 0.15 mm ) , and heme synthesis was supported with -aminolevulinic 
 acid ( 0.1 mm ) . cultures were incubated for 24 h , and then cells were 
 harvested by centrifugation ( 9000 g , 4 c , 20 
 min ) and stored at 20 c until purification . pellets 
 were defrosted and resuspended in chilled 50 mm potassium phosphate 
 buffer ( ph 8.0 ) , containing 50 mm kcl , 10%v / v glycerol , and commercial 
 protease inhibitors ( complete edta - free protease inhibitor cocktail 
 tablets , roche ( 1 tablet/50 ml ) , pmsf ( 1 mm ) , and benzamidine hydrochloride 
 ( 1 mm ) ) ( buffer a ) . cells were lysed by sonication at 0 c ( 30 
 s on/60 s off , 12 cycles ) using a sonics vibra - cell 505 sonicator 
 ( sonics and materials inc . , cell debris was pelleted by 
 centrifugation ( 40000 g , 30 min , 4 c ) , and the 
 supernatant was immediately loaded onto a freshly recharged histrap 
 ff affinity column ( ge healthcare , little chalfont uk ) . affinity chromatography 
 was performed using an kta purifier ( ge healthcare ) at 18 
 c . protein and p450 heme absorbance ( a280 and a417 , respectively ) were monitored , and cyp121 was eluted with 
 a linear gradient of imidazole ( 0200 mm ) in buffer a. the 
 purity of fractions collected was assessed by sds - page . pure fractions 
 were combined and dialyzed into 50 mm tris - hcl ( ph 7.2 ) , containing 
 500 mm kcl and 1 mm edta ( buffer b ) over 2448 h using a slide - a - lyser 
 ( thermoscientific , hemel hempstead uk ) dialysis cassette . dialyzed 
 protein was concentrated using a vivaspin ( 10000 mwco ) ultrafiltration 
 column at 6000 g , 4 c , and then stored at 80 
 c until further use . impure protein fractions were pooled , dialyzed 
 into buffer a to remove imidazole , and repurified by a second histrap 
 ff affinity column , as described above . protein concentration and 
 purity was determined by sds - page and amino acid analysis ( protein 
 and nucleic acid chemistry facility [ pnac ] , department of biochemistry , 
 university of cambridge ) . repeated affinity chromatography steps meant 
 that the previously cited anion exchange and gel filtration chromatography 
 steps were not required to obtain sufficiently pure protein . plasmid vectors encoding n - terminal his6-tagged cyp125 ( pet15b / rv3545c ) and cyp126 ( pet15b / rv0778 ) were provided by dr . cyp125 and cyp126 proteins were expressed and purified 
 in e. coli c41(de3 ) cells according 
 to the literature . cyp142 , cyp143 , and 
 cyp144-trv proteins were prepared according 
 to the literature and provided by dr . vis screening 
 was performed using a cary400 uv  vis 
 spectrophotometer ( varian , ca , usa ) . ligands were prepared as dmso - d6 stock solutions ( 1100 mm ) and added 
 to protein ( 5 m ) solutions ( 198 l ) or to buffer alone 
 to give a final dmso - d6 concentration 
 of 1% v / v . cyp121 was prepared in 50 mm tris - hcl ( ph 7.2 ) , 1 mm edta 
 buffer . cyp125 , cyp126 , cyp142 , cyp143 , and cyp144 were prepared in 
 50 mm tris - hcl ( ph 7.5 ) , 100 mm kcl buffer . cyp124 was prepared in 
 100 mm potassium phosphate ( ph 7.0 ) , 100 mm kcl buffer . any spectral interference 
 from the inherent absorbance of added ligands / solvent was removed 
 by subtracting spectra collected for protein - free samples of the same 
 ligands / solvent in buffer . the difference in soret wavelength maximum 
 ( max ) of the respective p450s soret band 
 ( max ) in the presence of ligands compared to that 
 obtained for a 1% v / v dmso - d6 control 
 was used to identify type i or type ii heme - binding interactions . competition assays were performed to detect the binding 
 interactions of ligands that did not perturb that heme cofactor . a 
 known type ii inhibitor , typically clotrimazole ( 550 m ) 
 or econazole ( 550 m ) , was added to assay solutions 
 to achieve a final dmso - d6 concentration 
 of 1% v / v . the difference in the red - shift of the soret band between 
 competition assays and that found for the known type ii ligand in 
 the absence of competing ligands was used to determine competitive 
 binding . buffer control spectra were collected to account for any 
 inherent absorbance of added ligands / solvent and all solutions were 
 inspected and found to be free of precipitate . all spectra were generated 
 using origin software ( originlab , northampton , ma ) and processed using 
 microsoft excel ( microsoft office , 2010 ) and graphpad prism 5.01 ( graphpad 
 software , san diego , usa ) . optical titrations to determine kd values 
 were carried out a varian cary 400 
 uv  vis spectrophotometer ( varian , ca , usa ) according to a previously 
 described procedure . assays were performed 
 in reduced volume ( 200 l ) quartz cuvettes with a path length 
 of 1 cm ( starna , essex , uk ) . ligands were prepared as dmso - d6 stock solutions , and proteins ( 5 m 
 final concentration ) were diluted in the corresponding buffer as described 
 in the ligand screening by uv  vis spectroscopy protocol above . aliquots ( 0.21.0 l ) of ligand stock solutions were 
 added directly to cuvettes containing either protein solutions , or 
 buffer alone , to achieve final concentrations of 100 nm to 1 mm . the 
 final dmso - d6 concentration did not exceed 
 1% v / v of the assay solution . spectra were recorded between 800 and 
 250 nm at 25 c after the addition of each aliquot of ligand . buffer control spectra were subtracted manually from protein spectra 
 during processing or saved as a baseline for automatic subtraction 
 from protein spectra . difference spectra were generated by subtracting 
 the initial ligand - free protein spectrum from each successive titration 
 spectrum . the maximum change in absorbance for each difference spectrum 
 was then plotted against ligand concentration and fitted using either 
 a one - site binding model hyperbolic eq ( eq 1 ) , or a modified version of the morrison eq 
 ( eq 2 ) for tight binding 
 inhibitors . all spectral analysis was 
 done using origin software ( originlab , northampton , ma ) , and data 
 were processed using microsoft excel ( microsoft office , 2010 ) . plots 
 and curve fitting were performed using graphpad prism 5.01 ( graphpad 
 software , san diego , usa).12 in eqs 1 and 2 , aobs is the observed 
 change in absorbance , amax is the maximum 
 absorbance change at saturation , e is the total enzyme 
 concentration , and l is the concentration of ligand . the kd values of non - heme binding ligands 
 were calculated from the change in the apparent kd of a known type ii ligand , typically 4,4-(5-(((1h - imidazol-4-yl)methyl)amino)-1h - pyrazole-3,4-diyl)diphenol 26h , when it was titrated into proteins ( 5 m ) in the 
 presence of the non - heme binding ligand ( 50 m ) . optical titrations 
 were performed and processed as described above , except that the non - heme 
 binding ligand was added initially to the ligand - free protein and 
 buffer control cuvettes . data were fitted using either eqs 1 or 2 to 
 determine the kd ( apparent ) of 26h , and then the kd of competitive ligand 
 was calculated according to eq 3.3 in eq 3 , kd(app ) is 
 the apparent binding affinity calculated from the competition titration , kd is the binding affinity of the 
 known inhibitor , kd is the 
 binding affinity of the non - heme binding ligand , and [ b ] is the concentration 
 of the non - heme binding ligand . itc experiments were 
 performed on a microcal itc200 ( malvern instruments , malvern uk ) instrument 
 at 25 c . ligands were prepared as stock solutions in dmso - d6 , diluted to 0.52.5 mm depending on 
 solubility , and titrated into cyp121 ( 50 m ) . both ligands and 
 cyp121 were prepared in the same 50 mm tris - hcl ( ph 7.5 ) buffer , containing 
 1 mm edta and a final concentration of 10% v / v dmso - d6 . titrations were comprised of 19  2.0 l 
 injections of ligand solution at 6090 s intervals . an initial control titrations were also performed ( ligand into buffer ) to measure 
 any heats of dilution or buffer mismatch . binding 
 isotherms were integrated to give the enthalpy change of each injection 
 and plotted against the molar ratio of ligand added to the sample 
 cell . titrations were fitted using a one - site binding model using 
 origin analysis software , setting the stoichiometry to n = 1 for weak binding compounds , or allowing n to vary for potent 
 compounds which showed high goodness of fit . the following experimental 
 protocol was followed for ligands 6 , 19a , and 25a : protein stock solutions ( 10 m ) were 
 prepared by dilution of purified proteins ( 300500 m ) 
 in 200 mm ammonium acetate buffer ( ph 7.0 ) . samples were buffer exchanged 
 by size exclusion chromatography using micro biospin 6 columns , molecular 
 weight cutoff 6 kda ( biorad , hemel hempstead , uk ) . ligands were prepared 
 as stock solutions in dmso - d6 at 50 , 100 , 
 and 2500 m concentrations . protein samples were 
 prepared by diluting protein stocks ( 10 l ) and ligand stocks 
 ( 1 l ) with ammonium acetate buffer ( 9 l ) to give final 
 concentrations of 5 m cyp121 , 2.5125 m ligand , 
 and 5% v / v dmso - d6 . an identical protocol 
 was followed for ligands 24a , 25b , and 26a , except that concentrations of cyp121 ( 8.7 m ) and 
 dmso - d6 ( 2.175% v / v ) were altered . the 
 concentrations of ligands was adjusted to maintain the same ligand - to - protein 
 ratios . mass spectra were recorded on a synapt hdms instrument ( waters 
 uk ltd . , capillary tips were cut under 
 a stereomicroscope to give inner diameters of 15 m 
 and then loaded with 2.5 l of sample solutions . given below however , conditions were optimized over the course of each 
 experiment to preserve the noncovalent protein  ligand complex 
 and minimize nonspecific interactions . all measurements were carried 
 out in a positive ion mode with ion source temperature of 20 c . 
 a capillary voltage of 1.501.75 kv , cone voltage of 6080 
 v , and extraction cone voltage of 2.34.1 v were applied to 
 perform nanoesi . all reported spectra were collected with a trap collision 
 energy 1218 v , transfer collision energy 12 v , ims pressure 
 5.02  10 mbar , and tof analyzer pressure 
 1.17  10 mbar . external calibration of the 
 spectra was achieved using cesium iodide at 100 mg ml in water . mass differences resulting from ligand binding were 
 calculated relative to the relevant 5% v / v dmso - d6 spectra of cyp121 ( 5 m ) . mass differences were 
 divided by the molecular weight of the ligand to calculate binding 
 stoichiometry . the percentage of monomeric cyp121 was calculated from 
 the sum of the intensities of peaks corresponding to monomeric cyp121 
 ( m / z 35004750 ) divided by 
 the sum of peak intensities corresponding to both monomeric and dimeric 
 ( m / z 52506750 ) cyp121 . untagged cyp121 protein and crystals 
 were prepared as described previously with the following adaptations . crystals were prepared by vapor diffusion in 
 1.52.1 m ammonium sulfate and 0.1 m sodium mes or cacodylate 
 from ph 5.56.15 . additional crystallization was also conducted 
 on the nanolitre scale using a mosquito pipetting robot ( molecular 
 dimensions , newmarket , uk ) , and crystals were produced in the morpheus 
 crystallization screen ( molecular dimensions ) in 800 nl drops with 
 protein - to - mother liquor at a ratio of 1:1 . ligand soaks were carried 
 out either by directly dissolving solid ligand at saturation or by 
 the addition of 25 mm ligand solution in dmso to the mother 
 liquor and soaking was carried out for 24 h. crystals were cryoprotected 
 and frozen as described previously except from morpheus conditions 
 that were frozen directly . data were collected on beamline i24 ( wavelength 
 0.9173/1.0118  ) and i02 ( wavelength 0.9795  ) at the diamond 
 light source facility ( oxfordshire , uk ) . cyp121ligand 
 cocrystal structures are deposited in the protein data bank ( http://www.rcsb.org/pdb/ ) under the accession codes : 6 , 5ibj , 7 ; 5edt , 19a ; 5ibf , 24a ; 5ibd , 25a ; 5ibe , 25b ; 5ibg , 26a ; 5ibi , and 26h ; 5ibh . images for presentation were rendered using an academic 
 version of the pymol molecular graphics system , version 1.3 , 2010 , 
 schrdinger , llc . experimental 
 determination of 
 ic50 values for the inhibition of human p450s by 19a , 25a , and 26a were performed 
 by cyprotex ( cheshire , uk ) . inhibition assays of cyp1a , cyp2c9 , cyp2c19 , 
 cyp2d6 , and cyp3a4 were performed in human liver microsomes in quadruplicate , 
 over a 7-point ligand concentration range ( 025 m ) . the formation of metabolites from the turnover of isoform - specific 
 substrates ( cyp2c9 , tolbutamide ; cyp2c19 , mephenytoin ; cyp2d6 , dextromethorphan ; 
 and cyp3a4 , midazolam ) was monitored by lc / ms . activities 
 reported for cyp1a refer to the combined inhibition of cyp1a1 and 
 cyp1a2 isoforms . ic50 values for known inhibitors 
 ( -naphthoflavone , sulfaphenazole , tranylcypromine , quinidine , 
 ketoconazole ) were determined as a positive control and were found 
 to be within the expected range ( table s3 , supporting information ) . three m. tuberculosis ( mtb ) h37rv 
 cultures were prepared 
 in 7h9 liquid medium supplemented with 10% oadc enrichment ( sigma - aldrich , 
 poole uk ) . , the bacterial suspensions 
 were washed twice with fresh 7h9 medium and then used in the antimycobacterial 
 activity assays . the minimum inhibitory concentration ( mic90 ) of the compounds was measure in triplicate in a 24 well plate - based 
 format . plates were prepared with 2 ml of 7h10 solid medium supplemented 
 with 10% oadc enrichment ( sigma - aldrich ) per well . test compounds 
 were prepared as stock solutions in dmso and diluted into the 7h10 
 medium to achieve final concentrations of 100 , 50 , 25 , 12.5 , and 6.25 
 m of cyp121 inhibitors and a final 1% v / v dmso concentration . rifampicin was used as a positive control and tested at 3.00 , 1.50 , 
 0.75 , 0.37 , and 0.18 m concentrations . prepared mtb suspensions ( od600 = 1.0 , approximately 3.00  105 
 cfu ) were aliquoted ( 10 l ) onto the compound - infused solid 
 medium and incubated at 37 c for 2 weeks . the mic90 was determined as the lowest concentration of compound that resulted 
 in 90% inhibition of bacterial growth compared to the negative control 
 wells , which were not treated with compounds and contained either 
 0% or 1% v / v dmso . lead 
 compound 2 , retrofragments 5 and 6 , and all 
 ar1 , ar2 , and ar3 analogues were prepared using the ligprep v3.2 and 
 epik v3.0 functions of schrdinger suite software ( schrdinger 
 llc , ny ) , selecting to include metal binding states when generating 
 ligand ionization states . ligands were docking into the crystal structure 
 of cyp121 bound to lead 2 ( pdb 4ktl ) or the previously 
 reported ligand 4-(1h-1,2,4-triazol-1-yl)quinolin-6-amine 
 ( pdb 4g1x ) . proteins were prepared using the internal protein preparation function 
 of the schrdinger suite software . ionization states were generated 
 to be compatible with metal - binding interactions , and the heme - iron 
 was manually adjusted to the ferric ( + 3 ) oxidation state . duplicate energy 
 minimized ( opls 2005 ) structures of pdb 4ktl were prepared either with all water molecules 
 removed or retaining the axial heme ar1 
 and ar3 analogues were docked using core constraints to replicate 
 the position of the aminopyrazole ring of lead 2 . ar2 
 analogues were docked using core constraints to replicate the heme 
 binding interactions of the 4-(1h-1,2,4-triazol-1-yl)quinolin-6-amine 
 ligand in pdb 4g1x . images were generated using the pymol molecular graphics system , 
 version 1.3 , 2010 ( schrdinger , llc ) .
the 
 essential enzyme cyp121 is a target for drug development against 
 antibiotic resistant strains of mycobacterium tuberculosis . 
 a triazol-1-yl phenol fragment 1 was identified to 
 bind to cyp121 using a cascade of biophysical assays . 
 synthetic merging 
 and optimization of 1 produced a 100-fold improvement 
 in binding affinity , yielding lead compound 2 ( kd = 15 m ) . 
 deconstruction of 2 into its component retrofragments allowed the group efficiency of 
 structural motifs to be assessed , the identification of more le scaffolds 
 for optimization and highlighted binding affinity hotspots . 
 structure - guided 
 addition of a metal - binding pharmacophore onto le retrofragment scaffolds 
 produced low nanomolar ( kd = 15 nm ) cyp121 
 ligands . 
 elaboration of these compounds to target binding hotspots 
 in the distal active site afforded compounds with excellent selectivity 
 against human drug - metabolizing p450s . 
 analysis of the factors governing 
 ligand potency and selectivity using x - ray crystallography , uv  vis 
 spectroscopy , and native mass spectrometry provides insight for subsequent 
 drug development .