nitric oxide ( no ) is an important biological 
 second messenger in humans , which plays a critical role in cell and 
 neuronal signaling , blood pressure regulation , and the immune response .
no is produced from oxidation of l - arginine 
 ( l - arg ) in the presence of nadph by a class of heme - dependent 
 enzymes , nitric oxide synthases ( nos ) .
mammals have three dominant isoforms of nos : constitutively expressed 
 neuronal nos ( nnos ) , present throughout the nervous system and skeletal 
 muscles , endothelial nos ( enos ) , also a constitutive enzyme located 
 in the endothelium and functioning in regulation of blood pressure 
 and blood flow , and inducible nos ( inos ) , which is associated with 
 the immune response . in the brain , low nanomolar concentrations 
 of no produced by nnos are neuroprotective , and downstream no , along 
 with cyclic guanosine 5-monophosphate ( cgmp ) in the protein 
 kinase g ( pkg ) signaling pathway , plays an important role in neurotransmission 
 and other metabolic processes .
however , 
 overexpression and overactivation of nnos following neuronal damage 
 causes no levels to jump several orders of magnitude , which is neurotoxic .
such no - mediated neurotoxicity leads 
 to protein degradation , misfolding , and aggregation through tyrosine - nitration , s - nitrosylation , and oxidative stress damage through formation of reactive 
 oxygen species ( ros ) and reactive nitrogen species ( rns ) .
this neurotoxicity has been implicated in several 
 neurodegenerative disorders that include alzheimer s , parkinson s , 
 and huntington s diseases and amyotrophic lateral sclerosis 
 ( als ) .
furthermore , progressive neuronal 
 damage and loss of neural tissue associated with no overproduction 
 are seen in cerebral palsy , stroke , ischemic brain damage , and migraine 
 headaches .
therefore , with the increasing 
 human and economic costs associated with neurodegenerative diseases , 
 and the lack of existent treatments , there is an urgent need for the 
 development of new therapeutics that would prevent , cure , or attenuate 
 neurodegeneration . with high levels of no implicated in these neurodegenerative 
 conditions , and target validation linking nnos to these pathological 
 conditions
all nos isoforms are active only as homodimeric 
 enzymes , where each monomer contains an n - terminal oxygenase domain 
 and a c - terminal reductase domain .
the 
 reductase domain contains the fmn , fad , and nadph binding - sites , while the oxygenase domain binds the cofactor 
 ( 6r)-5,6,7,8-tetrahydrobiopterin ( h4b ) 
 and the substrate l - arg at the heme catalytic site . upon 
 dimerization and activation of nos by ca - mediated calmodulin 
 binding ,
electrons flow from nadph to 
 fad and fmn in the reductase domain to the heme in the oxygenase domain , 
 where conversion of l - arg to l - citrulline and no 
 takes place in the presence of oxygen .
therefore , quite predictably , the major approach in the development 
 of nos inhibitors involves the utilization of arginine mimetics as 
 competitive nnos inhibitors .
however , in addition 
 to potent inhibition of nnos , there are many challenges associated 
 in designing nnos inhibitors .
first , selectivity of nnos inhibitors 
 over enos and inos ( nos isoforms share nearly identical active site ) 
 is essential to minimize undesired side effects .
second , as arginine isosteres , these inhibitors are highly 
 polar ; therefore , they suffer from poor bioavailabilty . hence , the 
 design of potent nnos inhibitors with improved pharmacokinetic properties 
 that address high isoform selectivity , blood  brain barrier 
 permeability , and minimal off - target efficacy is crucial . in 
 this respect ,
continuing efforts from our laboratories , guided by 
 structure - based drug design and fragment hopping , have resulted in 
 a series of highly potent and selective small molecule nnos inhibitors 
 based on pyrrolidinomethyl-2-aminopyridine scaffolds ( figure 1 , compounds 1 and 2 ) .
compounds 1 and 2 showed 
 high potency and excellent selectivity over inos and enos , while 2 , when administered intravenously to a pregnant rabbit dam 
 with induced uterine hypoxia , showed a complete reversal of hypoxia 
ischemia 
 induced death in the newborn kits . however , 
 further development of these compounds stalled because of their inability 
 to cross the blood  brain barrier .
this was presumably the result 
 of the hydrophilic nature of the molecule ( too many basic amines and 
 hydrogen - bond donors , high polar surface area , and a large number 
 of rotatable bonds ) .
modifications of 
 these pyrrolidinomethyl-2-aminopyridine scaffolds , such as reducing 
 the number of polar charges and basicity by alkylation , fluorination , and 
 intramolecular hydrogen bonding , met 
 with either diminished potency or selectivity or without notable improvement 
 in cellular permeability .
furthermore , synthesis of these pyrrolidinomethyl-2-aminopyridine 
 scaffolds involved more than 12 steps , difficult chiral resolutions , 
 and diastereomer separations . later modifications of simplified double - headed 
 2-aminopyridine scaffolds displayed good potency and selectivity ; however , they still suffered from poor permeability 
 in a caco-2 assay , which is used to estimate intestinal cellular permeability 
 and also reflects potential brain permeation .
therefore , as bioavailability 
 was a major challenge in these nnos inhibitors , one avenue we explored 
 to improve the cellular permeability of the nos inhibitors , while 
 maintaining a good potency and selectivity , was replacement of the 
 more basic arginine isosteres with fewer basic groups that engage 
 in heme - coordination in the active site of nos to arrest l - arginine turnover ( for example , the pka of conjugate acid of 2-aminopyridine is 7.1 ) .
compared to many inhibitors 
 designed as arginine mimetics , only a few reports exist that explore 
 the heme - coordination of nos . however , 
 imidazole ( pka of its conjugate acid is 
 6.9 ) is known to weakly bind to nnos ( ic50 200 m ) , 
 while the less - donating 1-phenylimidazole shows nearly 10-fold improved 
 potency ( ic50 25 m ) .
furthermore , 2-(1-imidazolyl)pyrimidine scaffolds , such as 3 , have shown very good potency as inos dimerization inhibitors .
in addition , 3 demonstrated good 
 cellular permeability and in vivo efficacy against inos in rats . toward this end , we designed simplified 
 compounds 4 and 5 with improved predicted 
 physicochemical properties by hybridizing selective molecular fragments 
 from inhibitors 1 and 3 .
the key hydrophobic 
 tail of 1 was incorporated into structures 4 and 5 based on previous precedence , where contacts 
 between 1 and residues lining a hydrophobic pocket adjacent 
 to the substrate access channel were 
 implicated in improving isoform selectivity . on the basis of inhibitory assay results , docking studies , and crystallographic 
 analysis , further modifications on 5 , such as changing 
 the linker length between the two aromatic heads ,
the number of secondary 
 amines , and utilizing various substitutions on the aromatic rings , 
 were employed to maximize rat and human nnos potency , improve isoform 
 selectivity , enable cellular permeability , and minimize off - target 
 effects .
figure 2 summarizes the different 
 nnos inhibitors designed and studied in this series . through these 
 structure 
these compounds showed improved 
 cellular permeability , high selectivity against a panel of central 
 nervous system ( cns ) receptors , and attenuated cyp inhibition .
synthesis of the 2,4-disubstituted pyrimidine 
 scaffolds in this study was designed to utilize common intermediates 
 and proceed in relatively few steps .
thus , the synthesis of the 2,4-disubstituted 
 pyrimidine cores in 4 and 5 was obtained 
 by following a modified procedure reported by davey et al .
alkylation of n - boc piperazine 
 ( 21 ) by 3-fluorophenethyl bromide gave 22 , which underwent a sequential one - pot substitution on 4-chloro-2-methanesulfonyl 
 pyrimidine by itself and imidazole , respectively , to provide target 
 compound 4 ( scheme 1 ) .
similar 
 substitutions on 4-chloro-2-methanesulfonyl pyrimidine by 26 and imidazole , respectively , gave the primary framework as a boc - protected 
 precursor of 5 , which was subsequently boc - deprotected 
 with trifluoroacetic acid and obtained as a trihydrochloride salt 
 upon precipitation from methanolic hcl .
compound 25 was 
 synthesized following a reported procedure , from where a mitsunobu reaction with diphenylphosphoryl azide gave 
 the corresponding azide , which was subsequently reduced to give the 
 primary amine 26 .
reagents and conditions : ( a ) ( i ) 
 nah , thf , 0 c , 1 h , then 3-fluorophenethyl bromide , tbai , 60 
 c , 48 h , 56% , ( ii ) tfa , ch2cl2 , 0 c 
  rt , 3 h , 85% ; ( b ) 4-chloro-2-methanesulfonyl pyrimidine , 
 k2co3 , mecn , 40 c , 19 h , then imidazole , 
 65 c , 30 h , 82% ; ( c ) 3-fluorophenyl acetaldehyde , nabh(oac)3 , rt , 14 h , 91% ; ( d ) ( i ) h2 , pd / c , meoh , 12 h , 
 86% , ( ii ) boc2o , ch2cl2 , 3 h , 76% ; 
 ( e ) ( i ) diad , pph3 , dppa , thf , 12 h , ( ii ) pph3 , thf / h2o , 41% ; ( f ) ( i ) same as ( b ) , 60% , ( ii ) tfa , ch2cl2 , 2 h , ( iii ) hcl in meoh , 10 min , 83% . in 6 and 7 , where the secondary 
 amine was benzylic to the pyrimidine , a reductive amination to form 
 the c 
n bond with an aromatic aldehyde was conceived as a favorable 
 disconnection ( scheme 2 ) . therefore , 2-methylthio-4-pyrimidine 
 carboxaldehyde ( 30 ) was first obtained by condensation 
 between pyruvaldehyde dimethylacetal ( 27 ) and thiourea 
 ( 28 ) and then acetal deprotection .
this intermediate underwent efficient reductive amination 
 with 3-fluorophenylethylamine and 3-fluorophenylpropylamine to form 
 secondary amines 33 and 34 , respectively
. 
 then boc protection of the secondary amine and oxidation of the thioether 
 group to methanesulfonyl by mcpba enabled the successive displacement 
 by imidazole to get the main structural frameworks of 6 and 7 . finally , deprotection of the boc group under 
 acidic conditions and subsequent precipitation from methanolic hcl 
 gave 6 and 7 as dihydrochloride salts .
reagents 
 and conditions : ( a ) ( i ) n , n - dimethylformamide 
 dimethyl acetal , 110 c , 8 h , ( ii ) naome , meoh , rt , 16 h , ( iii ) 
 mei , 60 c , 12 h , 87% ; ( b ) hcl , 85 c , 6 h , 58% ; ( c ) nabh(oac)3 , cat .
acoh , 3  sieves , ch2cl2 , 14 h , 8589% ; ( d ) ( i ) boc2o , thf , 12 h , 9799% , 
 ( ii ) mcpba , ch2cl2 , 12 h , 7589% ; ( e ) 
 imidazole , k2co3 , mecn , 60 c , 4 h , 8493% ; 
 ( f ) ( i ) tfa , ch2cl2 , 3 h , ( ii ) hcl in meoh , 
 10 min , 7993% .
2,4-disubstituted pyrimidine 
 scaffolds possessing two methylene units between the secondary amine 
 and the pyrimidine ring ( 814 ) were 
 synthesized via a michael addition on the electron - deficient vinyl 
 pyrimidine ring ( scheme 3 ) .
therefore , the 
 2-methylthio-4-vinyl pyrimidine ( 40 ) was synthesized 
 by a stille coupling between commercially available 4-chloro-2-methylthio 
 pyrimidine ( 39 ) and tributylvinyltin in the presence 
 of tetrakistriphenylphosphine pd(0 ) .
a 
 michael addition between 40 and the particular primary 
 amines gave the corresponding homobenzylic amines ( 4147 ) .
the primary amines ( 3132 , s3s12 ( see supporting information ) , 62 ) were 
 synthesized either from commercially available bromides or carboxylic 
 acids in 23 steps , as elaborated in the supporting information .
note that in the case of the primary 
 amines containing a cyclopropyl group , the cis and trans isomers were completely separable by silica gel column 
 chromatography ( see supporting information for further details ) , and the trans isomer was 
 carried forward for the michael addition reaction .
reagents and conditions : ( a ) tributylvinyl tin , pd(pph3)4 , dce , 70 c , 48 h , 92% ; ( b ) r - nh2 , 
 cat .
acoh , etoh , 848 h , 6297% ; ( c ) ( i ) boc2o , thf , 3 h , 8095% , ( ii ) mcpba , ch2cl2 , 3 h , 6591% ( note in case of 46 , oxone was 
 used instead of mcpba in a 1:1 thf / h2o mixture for 4 h ) ; 
 ( d ) imidazole , k2co3 , mecn , 65 c , 510 
 h , 7692% ; ( e ) ( i ) tfa , ch2cl2 , 3 h , 
 ( ii ) hcl in meoh , 10 min , 8099% . following 
 a similar route
as illustrated for the synthesis of 6 and 7 , final compounds 814 were obtained ( scheme 3 ) .
however , 
 in the case of 46 , after boc protection , mcpba oxidation 
 ( regardless of conditions ) always led to undesired oxidation of the 
 pyridine ring to the n - oxide .
finally , oxidation 
 of 46 in a 1:1 mixture of thf and h2o by oxone 
 at room temperature for 4 h gave desired sulfone 53 .
similar imidazole substitution , boc deprotection , 
 and acidification gave final compound 13 as a pure trihydrochloride 
 salt .
when separation of the two enantiomers of 12 became crucial , the trans - isomer of 2-(3-fluorophenyl)-1-cyclopropyl 
 amine ( 62 ) was subjected to dcc - mediated amidation with 
 ( s)-(+)--methoxyphenylacetic acid ( scheme 4 ) .
the diastereomers , 63a and 63b , were obtained pure by silica gel 
 column chromatographic separation .
finally , hydrolysis of the auxiliary 
 under refluxing ethanolic hcl gave the enantioenriched amines 62a and 62b in > 95% enantiopurity .
these amines 
 were then independently converted to the final enantiomerically enriched 
 isomers ( r , r)-12 and 
 ( s , s)-12 by following 
 the exact same route as used to synthesize 12 from 62 and 2-methylthio-4-vinylpyrimidine ( 40 ) .
reagents and conditions : ( a ) ( i ) 
 ( s)-(+)--methoxyphenylacetic acid , dcc , ch2cl2 , 20 c to rt , 12 h , ( ii ) chiral 
 resolution of diastereomers on silica gel column ; ( b ) hcl in etoh , 
 reflux , 12 h. when the 2,4-disubstituted pyrimidine 
 was replaced by a 3,5-disubstituted pyridine ring , as in 15 , synthesis was initiated from commercially available 5-bromo nicotinic 
 acid ( 64 ) by reduction to alcohol 65 ( scheme 5 ) .
homologation by one methylene unit was obtained 
 by conversion of the alcohol to a nitrile ( 66 ) and then 
 to its aldehyde by dibal .
as the intermediate aldehyde was photosensitive 
 and unstable , it was synthesized in the dark , and the crude reaction , 
 after work up , was immediately subjected to reductive amination with 
 3-fluorophenethyl amine ( 31 ) to provide 67 in a 41% yield ( over two steps ) .
boc protection of the secondary 
 amine , followed by a cu - catalyzed amination of the 3-bromopyridine 
 group , gave 68 , which was 
 boc deprotected and treated with methanolic hcl to give final compound 15 as a trihydrochloride salt .
reagents and conditions : ( a ) ( i ) 
 isobutyl chloroformate , et3n , thf , rt , 1 h , ( ii ) nabh4 , h2o , 12 h , 43% ; ( b ) ( i ) socl2 , ch2cl2 , 2 h , ( ii ) kcn , dmf , rt , 12 h , 70% ; ( c ) ( i ) 
 dibal , ch2cl2 , 78 c , 1 h , ( ii ) 
 3-fluorophenethylamine , cat .
acoh , mgso4 , nabh(oac)3 , 12 h , 41% ; ( d ) ( i ) boc2o , thf , 12 h , 92% , ( ii ) 
 cubr , 8-acetyl-5,6,7,8-tetrahydroquinoline , imidazole , cs2co3 , dmso , 100 c , 12 h , 67% ; ( e ) ( i ) tfa , ch2cl2 , 3 h , ( ii ) hcl in meoh , 10 min , 88% .
the different aromatic substitutions at the 2-position 
 of the pyrimidine in 1620 were synthesized 
 from advanced intermediate 49 by substituting the sulfone 
 with the different methyl imidazoles or triazoles ( scheme 6 ) .
thereafter , a similar tfa - mediated boc deprotection 
 and salt formation gave the compounds 1620 .
however , when 4-methylimidazole was used , both 4- and 
 5-methyl-2-imidazolyl pyrimidines 72 and 73 were obtained , which were inseparable by chromatographic conditions . 

therefore , a subsequent boc deprotection of the mixture of 72 and 73 , followed by a chromatographic separation , provided 
 the free bases of 19 and 20 .
these were 
 subsequently treated with methanolic hcl to obtain pure dihydrochloride 
 salts 19 and 20 .
reagents 
 and conditions : ( a ) imidazole / triazole , k2co3 , mecn , 65 c , 524 h , 8288% ; ( b ) ( i ) tfa , ch2cl2 , 1 h , ( ii ) hcl in meoh , 10 min , 8799% .
table 1 summarizes the binding affinity of 420 in the in vitro enzyme inhibitory assays 
 against several isoforms of nos .
an oxyhemoglobin no capture assay 
 was used to determine the ic50 value of inhibitors against 
 the purified rat and human nnos , murine macrophage inos , and bovine 
 enos .
the apparent ki values of the inhibitors were determined from ic50 and substrate km values using 
 the cheng  prusoff equation , and the corresponding isoform selectivities 
 as ratios of their respective ki values .
compounds 420 were assayed in vitro against four purified nos isoforms : 
 rat nnos ( rnnos ) , bovine enos ( enos ) , murine inos ( inos ) , and human 
 nnos ( hnnos ) using known literature methods .
prusoff equation 
 directly from ic50 values ( see experimental 
 section for details ) .
ic50 values are the average 
 of at least two replicates with nine data points ; all experimental 
 standard error values are less than 15% , and all correlation coefficients 
 are > 0.9 .
selectivity values are the ratios of respective ki values . nt = not tested . between the two initial inhibitors 
 designed , 4 and 5 ,
compound 4 only weakly inhibited nnos ( ki  
 5 m ) while 5 displayed a stronger inhibition of 
 nnos ( ki 0.368 m ) .
this nearly 
 10-fold difference in potency can be attributed to the key structural 
 difference in the linker between the pyrimidine and 3-fluorophenyl 
 ring in 4 and 5 . while a piperazine ring 
 may be sterically favorable in a more open active site , in case of 3 binding to an inos monomer ( and hence preventing dimerization )
, the same may cause unfavorable steric clashes 
 with the peptide backbone in the nnos dimer .
furthermore , the open 
 and flexible linker in 5 can also engage in favorable 
 interactions via the secondary amines , thus orienting the hydrophobic 
 end of the molecule properly . to gain more insight into the structural 
 basis for potencies and selectivity , we determined the crystal structure 
 of 5 bound to nnos and enos .
indeed , the crystal structure 
 of 5 bound to nnos ( figure 3a ) 
 shows that while the 2-imidazolyl pyrimidine head in the molecule 
 ligates to the heme fe , the void left 
 between the imidazole ring and glu592 is filled with a water molecule . 

the secondary amine next to the pyrimidine ring is engaged in a salt 
 bridge with heme propionate a , while the other secondary amine makes 
 a hydrogen bond with a water bridging in between h4b and 
 the heme .
this also orients the rest of the linker toward the hydrophobic 
 pocket lined by tyr706 , met336 , leu337 , and trp306 ( from the other 
 monomer ) in rat nnos .
the aromatic ring 
 of 5 engages in quite a few van der waals interactions 
 with these four residues at distances from 3.5 to 4.0  , except 
 for the closer distance ( 3.3  ) from the fluorine atom of 5 to the carbonyl oxygen of met336 .
this also represents the 
 first structure of nnos with a type ii imidazole - based heme - bound 
 inhibitor .
the crystal structure of 5 bound to enos ( figure 3b ) reveals two major differences compared to its 
 binding conformation seen in nnos .
first , similar to nnos , the tail 
 fluorophenyl ring can still make van der waals contacts with tyr477 , 
 leu107 , and trp76 ( the other monomer ) , but the fluorine atom is no 
 longer inserted into the pocket as in the nnos case .
instead , the 
 aromatic ring simply caps the pocket at its edge with the f atom pointing 
 sideways .
the reason for this binding orientation is very likely an 
 amino acid variation , val106 ( enos ) vs met336 ( nnos ) , which is also 
 part of this hydrophobic pocket .
the more degrees of freedom of met336 
 in nnos can more readily adapt to inhibitor binding than val106 in 
 enos . as a result ,
the cg1 atom of val106 in enos would clash with 
 the aromatic ring of 5 if it were in its position seen 
 in nnos .
consequently , in enos the secondary amine in the linker of 5 makes a hydrogen bond with heme propionate d , rather than 
 with a water molecule as in nnos .
the val / met variation is , therefore , 
 the structural basis for the observed 100-fold selectivity for 5 ( table 1 ) . active site structure 
 of 5 bound to rat nnos ( ( a ) pdb 4d3b ) and bovine enos 
 ( ( b ) pdb 4d33 ) .
key hydrogen bonds are shown by dashed lines , and distances are 
 in  .
fc map for the ligand is contoured at 2.5 . 
 the heme pyrrole rings are labeled in order to identify the propionate 
 positions . all structural figures were prepared with pymol ( www.pymol.org ) . because the part of inhibitor 5 positioned directly over the heme appeared optimal , we sought 
 to improve the enzyme  inhibitor interactions beyond the active 
 site ( compounds 69 ) , utilizing different 
 linker lengths between the pyrimidine and 3-fluorophenyl rings .
the 
 rationale behind the design of the shorter 4-atom - linker , as in 6 , was to avoid the twisting of methylene units on top of 
 each other , as seen in the conformation of 5 , but still 
 enable the aromatic end to reach the hydrophobic pocket . also , we 
 reasoned that moving the secondary amine away from the pyrimidine 
 by one or two methylene units might enable it to hydrogen bond with 
 both the heme propionates and improve the potency of the molecule . 
 with this amine moving up along the linker ,
the other secondary amine 
 in 5 becomes obsolete and thus can be removed to reduce 
 the basicity of the inhibitors . from evaluation of the binding 
 affinity of these inhibitors against nnos ( table 1 )
, we found two crucial factors that contribute to their potency 
 and selectivity : the position of the secondary amine and the length 
 of the linker .
moving the secondary amine by one more methylene unit 
 from the pyrimidine ring ( 8 and 9 ) dramatically 
 decreased the ki values relative to 6 and 7 ( 8.7 and 2.7 m , respectively ) . 

the structure of 7 bound to nnos ( figure 4a ) reveals that the secondary amine can no longer make a hydrogen 
 bond with either of the heme propionates , thus suggesting that this 
 interaction is critical for imparting potency to these pharmacophores .
this polar interaction was regained in 8 and 9 , where the secondary amine is one carbon farther away from 
 the pyrimidine than in 6 and 7 .
the structure 
 of 8 bound to nnos ( figure 4b ) 
 indicates that this position of the amine enables it to engage in 
 dual salt bridges with both the heme propionates , with the secondary 
 amine positioned equidistant between the two .
however , the five - atom 
 linker of 8 is not long enough to bring the fluorophenyl 
 ring into tighter van der waals contacts with the hydrophobic pocket . 

this , in fact was attained with the six - atom linker between the fluorophenyl 
 and the pyrimidine to provide maximum potency .
thus , 9 is one of the most potent nnos inhibitors in this series of 2-imidazolylpyrimidine 
 scaffolds ( ki = 0.019 m ) .
indeed , 
 the structure of nnos9 ( figure 5a ) demonstrates the structural basis for this good potency . 
 like 8 ,
the secondary amine of 9 can establish 
 dual salt bridges with both heme propionates .
in addition , owing to 
 the longer linker of 9 , the fluorine atom of the aromatic 
 ring can be inserted into the hydrophobic pocket , as in the case of 5 .
extensive van der waals contacts provided by the inhibitor s 
 aromatic ring should be responsible for the 7-fold higher potency 
 when comparing 9 with 8 ( figure 5a vs figure 4b ) .
furthermore , 9 displayed 260-fold selectivity over enos , with an improved 
 41-fold selectivity over inos .
the differences in interactions with 
 residues lining the hydrophobic pocket also contribute to isoform 
 selectivity .
as shown in figure 5b , even though 9 in enos can retain the dual salt bridges from its secondary 
 amine in the linker , its fluorophenyl ring can not insert into the 
 hydrophobic pocket as it does in nnos .
this is the result of an amino 
 acid variation ( val106 in enos vs met336 in nnos ) , as postulated for 5 .
the lack of these van der waals contacts reflects in the 
 poorer potency of 9 to enos , which is supported by the 
 fact that both 8 and 9 share similar potency 
 to enos ( see figure s1 in the supporting information for structure of 8 bound to enos ) .
active site structures 
 of 7 ( ( a ) pdb 4v3v ) and 8 ( ( b ) pdb 4v3w ) bound to rat nnos . 

key hydrogen bonds are shown by dashed lines and distances are in 
  . the omit fo 
fc map for the ligand is contoured at 2.5 . active site structures of 9 bound 
 to rat nnos ( ( a ) pdb 4v3x ) and bovine enos ( ( b ) pdb 4d35 ) .
fc map for the ligand is contoured at 2.5 . encouraged by the good potency 
 and selectivity of 9 , we sought to replace the fluorophenyl 
 with a chlorophenyl ring at the hydrophobic end of 10 .
prior studies have shown that halogen replacements could result 
 in enhanced inhibitory potency and selectivity from contacts between 
 the inhibitor and residues in the hydrophobic pocket . however , in our case , this change results in slightly diminished 
 inhibitor potency in nnos ( 32 nm for 10 vs 19 nm for 9 ) .
the structure of nnos10 ( figure s2 in supporting information ) shows that 
 because of the bulkiness of the chlorine atom , the aromatic ring actually 
 retreats from the hydrophobic pocket , leading to less extensive van 
 der waals contacts with the protein compared to 9 .
as 
 expected , the chlorophenyl ring of 10 in the enos10 structure ( figure s3 in supporting 
 information ) is entirely outside of the pocket , similar to 
 the situation in enos9 ( figure 5b ) , and therefore 10 still maintains good n / e 
 selectivity ( 253-fold ) .
while 9 showed a high level 
 of selectivity against enos , we looked deeper into further improving 
 its inos selectivity .
the three nos isoforms have a 5060% 
 overall sequence identity and highly conserved , almost identical active 
 site sequences
. therefore , specificity 
 among these nos isoforms have relied on key differences in residues 
 and their conformational flexibility along the substrate access channel 
 that connects the active site to the hydrophobic pocket ( vide supra ) 
 lying at the farthest end of this channel .
previous designs of nnos 
 inhibitors have shown that contacts between the inhibitor and residues 
 of this pocket could be in part responsible for imparting isoform 
 selectivity .
in murine inos , for example , 
 a polar asn115 replaces leu337 of rat nos in this pocket , which would 
 strongly disfavor hydrophobic interactions .
in addition , superimposition 
 of the nnos9 and inos crystal structures reveals 
 subtle differences in spatial orientations of residues along the substrate 
 access channel .
we reasoned that conformational restrictions and an 
 increase of steric bulk in 9 , by introduction of cyclopropane 
 rings along the methylene linker , might impose steric clashes and 
 thereby lower binding affinity to inos .
furthermore , installing a 
 cyclopropane ring as in 11 increases the metabolic stability 
 of a more labile benzylic methylene group in 9 , while 
 the electron - withdrawing nature of the ring can reduce the basicity 
 of the secondary amine as in 12 , thereby increasing the 
 probability of blood  brain barrier permeation .
although both 11 and 12 displayed a slightly lower binding 
 affinity for nnos , they displayed a weaker inhibitory effect on inos 
 than parent compound 9 .
in fact , 12 demonstrated 
 an improved 73-fold selectivity over inos , more than the previous 
 lead , and the highest n / e selectivity ( 363-fold ) obtained so far in 
 this series of scaffolds . to gain more concrete evidence
, we 
 determined the crystal structures of both 11 and 12 in nnos ( parts a and b of figure 6 , respectively ) and enos ( supporting information , 
 figures s4 and s5 , respectively ) .
because of the rigidity of 
 the linker s cyclopropyl ring , the fluorophenyl ring of both 
 inhibitors in nnos retreats from the deeper position seen for 9 ( figure 5a ) but is oriented similarly 
 to 10 ( supporting information , figure 
 s2 ) .
again , less extensive van der waals contacts for 11 and 12 relative to those by 9 are the reason for the slightly weaker potency of these two inhibitors 
 ( table 1 ) .
interestingly , although racemic 
 samples of 11 and 12 were used for the crystal 
 preparations , the electron density indicated that only one enantiomer 
 preferably binds in each case : ( r , r)-11 in nnos11 and ( r , r)-12 in nnos12 .
similarly , the same enantiomer also dominates the binding of 11 or 12 to enos ( supporting 
 information , figures s4 and s5 , respectively ) ; however , in 
 both cases , the fluorophenyl ring caps at the edge of the hydrophobic 
 pocket .
inhibitor contacts 
 are the origin of poorer binding affinity for 11 and 12 toward enos . owing to a different location of the cyclopropane 
 in the linker ,
the kink caused by the cyclopropyl ring in 12 pulls the fluorophenyl ring away from the protein more severely 
 than that observed in 11 .
this explains the even poorer 
 affinity of 12 to enos , which results in better n / e selectivity 
 ( table 1 ) .
active site structures of 11 ( ( a ) pdb 4v3z ) and 12 ( ( b ) pdb 4d2y ) bound to nnos . there is preferential 
 binding of one enantiomer : ( r , r)-11 and ( r , r)-12 for 11 and 12 , respectively .
key hydrogen 
 bonds are shown by dashed lines , and distances are in  .
fc map for the ligand is contoured at 2.5 . the observation that one enantiomer of 12 dominates the binding prompted us to synthesize and test the two 
 enantiomers of 12 separately to identify the more potent 
 enantiomer in the pair and determine the effect of chirality on the 
 binding of the inhibitor to the nos active site .
table 2 summarizes the binding affinity to noss by the two enantiomers 
 of 12 . while the  ring up  ( r , r)-12 shows a much tighter binding 
 to nnos , comparable to the affinity of 9 , this particular 
 enantiomer also preferably binds to enos and inos .
however , the very 
 low ki for nnos ( 18 nm ) compared to that 
 for enos and inos makes ( r , r)-12 the best inhibitor in this series with 573- and 119-fold 
 selectivities against enos and inos , respectively .
ic50 values 
 are the average of at least two replicates with nine data points ; 
 all experimental standard error values are less than 10% , and all 
 correlation coefficients are > 0.95 .
these results are also in agreement 
 with the structural studies . the nnos structure with ( r
, r)-12 bound confirms that this enantiomer 
 is indeed the one that dominates the binding to nnos when racemic 12 was used for crystal preparation ( figure 6b ) .
it is surprising that the structure of ( s , s)-12 enantiomer bound to nnos ( figure s6 in supporting information ) also shows 
 a binding mode that is not that much different from that of ( r , r)-12 ; both the positions 
 of the secondary amine and the fluorine of the aromatic ring more 
 or less overlap between the two enantiomers , even though different 
 chiralities at the cyclopropane lead to differences in the linker 
 conformation and phenyl ring orientation ( figure 
 s6 in supporting information ) .
therefore , the differences in 
 linker conformation and position of the cyclopropane ring may result 
 in the 8-fold variation in the nnos affinities between these two enantiomers 
 ( table 2 ) . alongside determining the 
 potencies of these inhibitors in the three lower animal isoforms of 
 nos , we assayed these compounds with the human nnos enzyme to see 
 if the potency and selectivity ratios are also reflected similarly 
 in the human isoform .
prior work on isosteric arginine mimetics developed 
 from our lab , such as the highly potent and selective inhibitor 1 , showed a human nnos potency of 0.070 m , with a 5-fold 
 selectivity difference between the human and rat isoform .
this is 
 one of the most potent human nnos inhibitors developed in our lab 
 to date .
when compounds 812 were 
 assayed against human nnos , we were pleased to see that inhibitors 912 displayed good potency in human nnos , 
 and a similar 410-fold selectivity difference between the 
 human and rat isoform of nnos , as seen with previously reported inhibitors 
 from our lab .
the human nnos exhibits an exact sequence identity with 
 the rat isoform , except for the hydrophobic pocket , where a hydrophilic 
 his342 replaces the leu337 present in rat nnos .
thus , the pocket in 
 human nnos is smaller and more hydrophilic on one side .
therefore , 
 the modifications that we made in our chemical structures to improve 
 nnos potency , although generally well tolerated in human nnos , can 
 be modified to improve their human nnos potency and lower the rat / human 
 selectivity . for this target
, we designed and tested compounds 1314 , with smaller and polar aromatic 
 ends that may interact effectively with his342 residue in the pocket 
 in human nnos .
indeed , we found that 13 is a potent inhibitor 
 of human nnos ( 0.125 m ) and rat nnos ( 0.054 m ) , where 
 the selectivity between the two has dropped down to 2.3-fold ( table 1 ) .
the structure of the human nnos13 complex ( figure 7a ) shows , as expected , 
 that the nitrogen of the pyridine ring indeed makes a hydrogen bond 
 with the side chain of his342 .
although the bond distances vary from 
 2.8 to 3.2  in the four independent copies of 13 , all are well ordered in structure .
in contrast , the polar pyridine 
 ring of 13 does not behave as well in the structure of 
 rat nnos13 ( figure 7b ) , 
 where the ring shows the sign of disordering with weak electron density . 

the more hydrophobic pocket in rat nnos prefers a phenyl ring over 
 the pyridine ring for interactions .
active site structures of 13 bound to human nnos ( ( a ) pdb 4v3u ) or rat nnos ( ( b ) pdb 4d30 ) .
key hydrogen bonds 
 are shown by dashed lines and distances are in  . the omit fo 
fc map 
 for the ligand is contoured at 2.5 . compound 10 ( with a 3-chlorophenyl tail ) also 
 displayed potent human nnos inhibition similar to 13 . 
 with a 3-chloro substitution being bigger than a 3-fluoro , it is difficult 
 to explain why 10 is a more potent human nnos inhibitor 
 than 9 . with the known behavior of 10 in 
 rat nnos ( figure s2 in supporting information ) ,
the bulkier chlorophenyl ring in human nnos is likely pulled out 
 a bit from the depth of the hydrophobic pocket compared to the position 
 of a fluorophenyl ring in 9 .
this movement may allow 
 the 3-chlorophenyl ring to fit better in the pocket , which might contribute 
 to the slightly better affinity of 10 than 9 to human nnos .
on the other hand , it is easier to explain the preference 
 for more polar aromatic heads being tolerated better in human nnos 
 with 14 , where a polar 3-cyanophenyl with a shorter 4-atom 
 linker between the arene and the pyrimidine head , demonstrated a 138 
 nm binding affinity for human nnos , while a 183 nm binding affinity 
 for rat nnos .
thus , this is the first case of a potent human nnos 
 inhibitor from our lab , where the selectivity is reversed in preference 
 toward its human isoform potency over the rat isoform . with a shorter 
 linker ,
the cyanophenyl ring of 14 locates right outside 
 the hydrophobic pocket in rat nnos ( figure s7 
 in supporting information ) .
however , in human nnos , the larger 
 side chain of his342 is at the right distance for a hydrogen bond , 
 as shown in figure s8 in supporting information , which makes 14 a better inhibitor for human nnos than 
 rat nnos .
therefore , these inhibitors clearly demonstrate that we 
 can utilize the same pocket in the substrate access channel for both 
 potency and selectivity determining factors in designing the nos inhibitors .
finally , with compounds 1520 , 
 we investigated variations in the 2-imidazolylpyrimidine part of the 
 scaffold that may result in improving potency or minimizing off - target 
 affinity . to this end
, preliminary docking studies indicated that 
 a 3,5-disubstituted pyridine could replace the 2,4-disubstituted pyrimidine 
 in 8 and engage in a salt bridge formation with the glu592 
 in the active site of nnos , the same residue that forms crucial hydrogen 
 bonds with the guanidine group of l - arginine or other arginine 
 mimetics .
however , when 15 was assayed against nnos , it showed a poor 5.5 m inhibition 
 against nnos .
therefore , further variations on the imidazole ring 
 fragment were made based on 9 , keeping the pyrimidine 
 ring intact .
we were interested from the literature examples of using 
 of heme - nitrogenous ligands as inhibitors of aromatase enzymes for 
 the treatment of breast cancer , and inhibitors of lanosterol demethylase 
 enzymes as antifungal drugs . among the 
 triazoles and methyl - substituted imidazoles
incorporated as 2-pyrimidyl 
 substituents in compounds 1620 , 
 both the 1,2,4- and 1,2,3-triazole - substituted scaffolds were poor 
 inhibitors of nnos , presumably because of a major depletion of the 
 heme - fe binding affinity by the electron - deficient triazoles . among 
 the substituted imidazoles ,
only ( 5-methyl)-2-imidazolylpyrimidine 
 ( 20 ) retained very high potency against nnos , and along 
 with high selectivity against enos , it demonstrated a remarkably high 
 400-fold selectivity against inos as well , in sharp contrast to all 
 the imidazole - substituted inhibitors mentioned previously .
this presumably 
 arises from the residues lining the active site over the heme - porphyrin , 
 which makes a smaller and less flexible pocket in inos compared to 
 nnos .
this pocket is surrounded by pro565 , 
 val567 , and phe584 from two -strands in rat nnos ( figure 8a ) , but by pro344 , val346 , and phe363 in murine 
 inos . while residues facing the heme are conserved , the residue that 
 stabilizes the second -strand is ser585 in nnos and asn364 
 in inos .
asn364 is involved in more hydrogen bonds than ser585 , which 
 should help rigidify this part of the structure ; however , the asn364 
 polypeptide chain in inos also shrinks the active site pocket to accommodate 
 its larger side chain compared to ser585 in nnos .
the crystal structure 
 of 20 bound to nnos ( figure 8a ) 
 shows that the methyl group sticks in to this pocket just described , 
 and the imidazole ring is bent away from the pyrimidine ring plane 
 at a larger angle than usual because of steric hindrance between the 
 pyrimidine ring and the methyl group .
therefore , the methyl group 
 might create some clashes in the active site pocket of inos , thereby 
 making it a very poor inos inhibitor . on the other hand ,
the 257-fold 
 selectivity of 20 against enos stems from different structural 
 features at the other end of inhibitor .
similar to parent compound 9 , the fluorophenyl ring of 20 in enos ( figure 8b ) can not insert into the hydrophobic pocket on 
 the far end of the substrate access channel .
thus , lack of good van 
 der waals contacts in the pocket in enos compared to those encountered 
 by 20 in nnos leads to poorer affinity for enos
. active site 
 structures of 20 bound to rat nnos ( ( a ) pdb 4d32 ) and bovine enos 
 ( ( b ) pdb 4d3a ) .
key hydrogen bonds are shown by dashed lines and distances are 
 in  . the omit fo 
fc map for ligand is contoured at 2.5 . encouraged by the high nnos affinity 
 and good isoform selectivities of several compounds in this series , 
 as one of the lead nnos inhibitors , 9 was subjected to 
 a caco-2 monolayer permeability assay ( table 3 ) .
the caco-2 cell line is a human intestinal epithelial line used 
 to approximate the compound s permeabililty in the gastrointestinal 
 tract as well as the blood 
compound 9 showed good cellular permeability , 
 thus reflecting a better potential for oral bioavailability .
in addition , 
 the efflux ratio of 9 was below 2 , which indicates that 
 it is likely not a favorable substrate for p - gp or any active transport 
 system that might shuttle it out of the cells .
we also sought to determine the off - target effects 
 of 9 against a panel of 50 cns receptors , which included 
 g - protein coupled receptors such as the serotonin , adrenergic , dopamine , 
 and histamine receptors , as well as muscarinic and  receptors .
compound 9 showed significant inhibition 
 ( > 75% ) at a high 10 m concentration in a primary binding 
 assay only at the following targets : human serotonin 5-ht1a and 5-ht2a 
 ( 83 and 88% ) , adrenergic -2c ( 95% ) , -1 and -2 receptors 
 ( 94 and 88% ) , and dopamine d3 receptor ( 87% ) .
therefore , a consecutive 
 secondary binding assay on these targets were evaluated , which revealed 
 a binding affinity ( ki ) of 0.2 
 m only for -2c and  receptors , while the rest 
 were 0.5 m or higher .
so , even a 0.2 m off - target affinity 
 holds 10-fold selectivity ( relative to the nnos ki ) , which nonetheless is about 7% of the total receptors 
 assayed .
thus , overall 9 displays a good safety profile , 
 which is very promising for further development of this class of scaffolds .
finally , the presence of a heme - coordinating group increases the 
 likelihood of these compound s ability to inhibit cytochromes 
 p450 ( cyps ) , the xenobiotic - metabolizing enzymes in humans . therefore ,
9 was evaluated against five major human liver microsomal 
 p450s and , at 10 m concentration , was found to decrease the 
 activity of cyp2c19 , cyp2d6 , and cyp3a4 by more than 70% . because 
 cyp3a4 is the major liver and intestinal p450 that metabolizes the 
 majority of drugs
, we determined ic50 values for 9 , ( r , r)-12 , 13 , and 20 using an 
 in vitro cyp3a4 activity assay and 7-benzyloxy-4-trifluoromethylcoumarin 
 as a substrate ( table 4 ) .
compound 9 displayed moderate inhibitory potency for cyp3a4 ( ic50 of 2.5 m ) and maintained > 130-fold selectivity toward 
 nnos .
when hydrophobicity in the molecules is increased through the 
 cyclopropane ring insertion or methyl substitution of the imidazole 
 in ( r , r)-12 and 20 , respectively , the cyp3a4 inhibitory potency is substantially 
 increased ( ic50 < 1 m ) .
this is understandable 
 given the bigger and more hydrophobic active site of cyp3a4 that preferably 
 binds large , nonpolar molecules .
the more polar and potent human nnos 
 inhibitor 13 acts as a weak cyp3a4 inactivator ( ic50 of 70 m ) , which suggests that modulation of hydrophobicity 
 and bulkiness of compounds containing the heme - coordinating group 
 can attenuate cyp inhibition , even with the presence of heme - ligating 
 groups like imidazole .
ic50 values are the average of at least two replicates 
 with nine data points ; all experimental standard error values are 
 less than 10% , and all correlation coefficients are > 0.95 .
we have designed and 
 synthesized a new series of 2,4-disubstituted pyrimidine scaffolds 
 by exploiting the much less - explored heme coordinating ability of 
 inhibitors in the active site of nos .
this design was based on the 
 rationale that 2-imidazolylpyrimidines might bind and inhibit nnos 
 with groups that are less polar and less basic than the 2-aminopyridines 
 and therefore more bioavailable .
we also speculated that by modulating 
 other parts of the inhibitor molecule , we would be able to incorporate 
 selectivity into the inhibitor against other nos isozymes and heme - containing 
 cyp enzymes .
indeed , we were able to obtain 2,4-disusbtituted pyrimidines 
 that are highly potent inhibitors of nnos , as shown by its low nanomolar 
 binding affinity to both rat and human nnos , and > 200-fold and 
 > 100-fold selectivity over enos and inos , respectively .
crystal 
 structures of the compounds bound to both rat and human nnos indicate 
 heme - fe coordination by the 2-imidazolyl fragment , and the noncoordinating 
 aryl rings are stabilized in a hydrophobic pocket at the far end of 
 the substrate access channel .
access to this pocket is important for 
 gaining inhibitory potency in both rat and human nnos isoforms ( via 
 extensive van der waals contacts and polar interactions ) and isoform 
 selectivity ( owing to the sequence diversity in the pocket among nos 
 isoforms ) .
very promising compounds that came from this study were 9 , ( r , r)-12 , and 13 , which showed good caco-2 permeability , minimal 
 off - target binding efficacy , and good selectivity .
even with the presence 
 of imidazole rings , cyp3a4 inhibition could be attenuated by modifications 
 to other parts of the molecule .
therefore , these results exhibit high 
 potential among these compounds to be orally bioavailable and brain 
 permeable . hence , further developments of these compounds are currently 
 in progress .
anhydrous solvents were purified by passage through 
 a solvent column composed of activated alumina and a supported copper 
 redox catalyst .
all remaining solvents and reagents were purchased 
 from commercial vendors and used without further purification .
moisture 
 or oxygen - sensitive reactions were performed under an atmosphere of 
 dry n2 or argon .
analytical thin - layer chromatography was 
 performed on silicycle precoated silica gel 60  f254 plates . 

an agilent 971-fp flash purification system with various siliasep 
 ( silicycle , 4063 m , 60  ) prepacked silica gel 
 cartridges was used for flash column chromatography .
h 
 nmr and c nmr spectra were recorded at 500 and 126 mhz 
 respectively on a bruker avance - iii instrument .
low - resolution esims 
 was performed on a thermo finnigan lcq or bruker amazon sl mass spectrometer 
 consisting of an electrospray ionization ( esi ) source .
high - resolution 
 mass spectral data were obtained at the integrated molecular structure 
 education and research facility ( northwestern university ) on an agilent 
 6210a tof mass spectrometer in positive ion mode using electrospray 
 ionization , with an agilent g1312a hplc pump and an agilent g1367b 
 autoinjector .
the purity of the compounds was evaluated on a beckman 
 gold reverse phase analytical hplc system using a phenonemex gemini 
 c-18 ( 4.6 mm  250 mm , 5 m ) reverse phase column with 
 uv absorbance and evaporative light scattering detection .
purities 
 of all final compounds that were subjected to enzymatic assays were 
 found to be > 95% .
preparative hplc was performed at the northwestern 
 university center for molecular innovation and drug discovery chemcore 
 lab , using an agilent 1200 series hplc and agilent 6120 quadrupole 
 mass spectrometer ( api - ms mode ) , and a phenomenex gemini - nx 5 m 
 c18 column ( 150 mm  21.2 mm ) .
chiral hplc to determine enantiopurity 
 of precursors of racemic and enantiomers of 11 was performed 
 on an agilent 1260 series hplc using a 0.46 cm  25 cm chiralpak 
 ad - h column , with hexanes and 2-propanol ( isocratic 10% 2-propanol 
 in hexanes ) as the mobile phases , and the flow rate of 0.5 ml / min 
 with uv detection .
4-chloro-2-methanesulfonyl pyrimidine,24 , 25 , 26,40,65 , and 66(43 ) were synthesized following 
 previously reported procedures .
syntheses of the remaining primary 
 amines are detailed in supporting information . to a solution of 22 ( 0.152 
 g , 0.729 mmol ) in mecn ( 4 ml ) , 4-chloro-2-methanesulfonyl pyrimidine 
 ( 0.14 g , 0.729 mmol ) and k2co3 ( 0.21 g , 1.522 
 mmol ) were added .
the mixture was heated at 40 c for 19 h and 
 monitored by tlc for complete consumption of 22 .
then , 
 imidazole ( 0.248 g , 3.645 mmol ) was added and the resulting mixture 
 heated at 65 c for an additional 30 h. the mixture was cooled , 
 diluted in ch2cl2 ( 40 ml ) , washed with h2o ( 2  30 ml ) , and the organic layer dried over anhydrous 
 sodium sulfate .
the solution was concentrated , and the resulting crude 
 oil was purified by flash chromatography ( ch2cl2/meoh ) to yield 4 as clear sticky oil ( 0.289 g , 82% ) .
h nmr ( 500 mhz ; cdcl3 ) :  8.51 ( s , 1 h ) , 
 8.13 ( d , j = 6.2 hz , 1 h ) , 7.80 ( s , 1 h ) , 7.267.21 
 ( m , 1 h ) , 7.10 ( s , 1 h ) , 6.98 ( d , j = 7.8 hz , 1 h ) , 
 6.946.86 ( m , 2 h ) , 6.36 ( d , j = 6.2 hz , 1 
 h ) , 3.71 ( s , 4 h ) , 2.82 ( t , j = 10.5 , 8.6 hz , 2 h ) , 
 2.64 ( dd , j = 10.1 , 8.3 hz , 2 h ) , 2.59 ( t , j = 5.2 hz , 4 h ) .
c nmr ( 126 mhz ; cdcl3 ) :  ( 163.74 , 161.79 , d , j = 245.9 
 hz , 1 c ) , 162.1 , 156.7 , 154.1 , ( 142.43 , 142.37 , d , j = 7.3 hz , 1 c ) , 136.0 , 129.9 , ( 129.81 , 129.74 , d , j = 8.4 hz , 1 c ) , ( 124.27 , 124.25 , d , j = 2.5 hz , 
 1 c ) , 116.5 , ( 115.53 , 115.36 , d , j = 21.0 hz , 1 c ) , 
 ( 113.08 , 112.92 , d , j = 21.0 hz , 1 c ) , 100.5 , 59.7 , 
 53.4 , 43.8 , 33.2 . hrms ( esi ) : calcd for c19h22fn6 [ m + h ] , 353.1884 ; found , 353.1887 . compound 26 ( 0.146 g , 0.516 
 mmol ) was diluted with mecn ( 3 ml ) , followed by addition of 4-chloro-2-methanesulfonyl 
 pyrimidine ( 0.1 g , 0.516 mmol ) and k2co3 ( 0.143 
 g , 1.033 mmol ) and heated at 40 c for 19 h. imidazole ( 0.176 
 g , 2.583 mmol ) was added to the resulting mixture , and heating continued 
 at 65 c for another 30 h. the mixture was cooled , diluted in 
 ch2cl2 ( 30 ml ) , washed with h2o ( 2 
  20 ml ) , and the organic layer dried over anhydrous sodium sulfate . 

the solution was concentrated , and the resulting crude oil was purified 
 by flash chromatography ( etoac / meoh ) to yield the boc - protected precursor 
 of 5 as a clear sticky oil ( 0.132 g , 60% ) .
this intermediate 
 boc - protected carbamate was dissolved in ch2cl2 ( 3 ml ) , cooled to 0 c , and trifluoroacetic acid was added 
 ( 1.5 ml ) to it .
the resulting solution was stirred at room temperature 
 for 2 h , when it was diluted with more ch2cl2 ( 15 ml ) , and treated slowly with saturated k2co3 ( 10 ml ) .
the layers were separated , and the aqueous layer was extracted 
 with ch2cl2 ( 2  10 ml ) . the combined organic 
 layers were dried with anhydrous sodium sulfate , concentrated , and 
 chromatographed with etoac / meoh to give an oily residue .
methanolic 
 hcl ( 2 m , 2 ml ) was added to the residue , stirred for 10 min , 
 concentrated to 1 ml , and treated with excess et2o , when 
 a white crystalline precipitate formed .
the solid was collected by 
 filtration and dried to obtain 5 as a white crystalline 
 solid ( 0.11 g , 83% ) ; mp = 208210 c .
h nmr 
 ( 500 mhz ; dmso - d6 ) :  10.32 ( s , 
 1 h ) , 9.49 ( s , 2 h ) , 8.66 ( t , j = 6.0 hz , 1 h ) , 8.52 
 ( t , j = 1.8 hz , 1 h ) , 8.22 ( d , j = 6.0 hz , 1 h ) , 7.89 ( t , j = 1.7 hz , 1 h ) , 7.39 
 ( td , j = 7.9 , 6.3 hz , 1 h ) , 7.197.07 ( m , 
 3 h ) , 6.69 ( d , j = 6.0 hz , 1 h ) , 3.903.86 
 ( m , 2 h ) , 3.23 ( dq , j = 11.4 , 6.3 hz , 2 h ) , 3.183.14 
 ( m , 2 h ) , 3.06 ( dd , j = 9.7 , 6.5 hz , 2 h ) , 1.30 ( dd , j = 15.1 , 6.8 hz , 2 h ) .
c nmr ( 126 mhz ; dmso - d6 ) :  ( 164.16 , 162.23 , d , j = 243.9 hz , 1 c ) , 164.1 , 155.8 , 153.1 , ( 141.09 , 141.00 , d , j = 11.3 hz , 1 c ) , 136.2 , ( 131.52 , 131.46 , d , j = 7.6 hz , 1 c ) , ( 125.79 , 125.78 , d , j = 1.3 hz , 
 1 c ) , 122.1 , 119.7 , ( 116.48 , 116.31 , d , j = 21.4 
 hz , 1 c ) , ( 114.69 , 114.48 , j = 26.4 hz , 1 c ) , 107.7 , 
 48.4 , 46.7 , 37.2 , 32.0 . hrms ( esi ) : calcd for c17h20fn6 [ m + h ] , 327.1728 ; found , 327.1731 .
general method a for trifluoroacetic 
 acid mediated boc - group deprotection : compound 37 ( 0.257 
 g , 0.647 mmol ) was diluted in ch2cl2 ( 5.5 ml ) 
 and cooled to 0 c .
trifluoroacetic acid ( 2.75 ml ) was added 
 to the resulting solution and stirred at room temperature for 3 h. 
 the reaction mixture was diluted with more ch2cl2 ( 25 ml ) and treated with saturated k2co3 ( 15 
 ml ) .
the layers were separated , and the aqueous layer was extracted 
 again with ch2cl2 ( 2  15 ml ) .
the resulting 
 oily residue was purified by flash column chromatography ( ch2cl2/meoh ) to give the free base of 6 as a 
 yellow oil .
the oil was treated with methanolic hcl ( 2 m , 
 2 ml ) for 10 min , when a white precipitate started forming .
this suspension 
 was concentrated to 0.5 ml and treated with excess et2o and sonicated .
the white solid was filtered , washed twice 
 with et2o , and dried to give a white amorphous solid of 6 as a dihydrochloride salt ( 0.223 g , 93% ) ; mp = 220222 
 c .
h nmr ( 500 mhz , dmso - d6 ) :  10.07 ( s , 2 h ) , 10.02 ( s , 1 h ) 9.05 ( d , j = 5.1 hz , 1 h ) , 8.57 ( s , 1 h ) , 7.78 ( d , j = 5.0 
 hz , 2 h ) , 7.447.37 ( m , 1 h ) , 7.217.09 ( m , 3 h ) , 4.61 
 ( t , j = 5.2 hz , 2 h ) , 4.01 ( br s , 1 h ) , 3.393.28 
 ( m , 2 h ) , 3.243.15 ( m , 2 h ) .
c nmr ( 126 mhz ; 
 dmso - d6 ) :  164.8 , ( 164.17 , 162.23 , 
 d , j = 244.4 hz , 1 c ) , 161.2 , 153.4 , ( 141.05 , 140.99 , j = 7.6 hz , 1 c ) , 136.9 , ( 131.51 , 131.44 , d , j = 8.8 hz , 1 c ) , ( 125.80 , 125.78 , d , j = 2.5 hz , 
 1 c ) , 125.1 , 120.7 , 119.3 , ( 116.48 , 116.31 , d , j = 
 21.4 hz , 1 c ) , ( 114.68 , 114.45 , d , j = 29 hz , 1 c ) , 
 49.7 , 48.2 , 31.9 . hrms ( esi ) : calcd for c16h17fn5 [ m + h ] , 298.1463 ; found , 298.1462 .
compound 7 was 
 obtained as a white solid in its dihydrochloride salt form ( 0.37 g , 
 79% ) from 38 ( 0.502 g , 1.22 mmol ) following general method 
 a ; mp = 153155 c .
h nmr ( 500 mhz , dmso - d6 ) :  9.61 ( br s , 3 h ) , 9.01 ( d , j = 4.9 hz , 1 h ) , 8.40 ( s , 1 h ) , 7.69 ( d , j = 4.6 hz , 1 h ) , 7.60 ( s , 1 h ) , 7.407.34 ( m , 1 h ) ,
7.147.05 
 ( m , 3 h ) , 4.57 ( t , j = 5.5 hz , 2 h ) , 3.69 ( br s , 
 1 h ) , 3.093.03 ( m , 2 h ) , 2.74 ( t , j = 7.6 
 hz , 2 h ) , 2.162.00 ( m , 2 h ) .
c nmr ( 126 mhz ; 
 dmso - d6 ) :  168.0 , 164.9 , ( 164.26 , 
 162.32 , d , j = 244.44 , 1 c ) , 161.3 , 153.7 , ( 144.75 , 
 144.69 , d , j = 7.56 hz , 1 c ) , 137.1 , ( 131.38 , 131.31 , 
 d , j = 8.8 hz , 1 c ) , ( 125.52 , 125.5 , d , j = 2.5 hz , 1 c ) , 120.3 , 119.0 , ( 116.14 , 115.97 , d , j = 21.42 hz , 1 c ) , ( 114.03 , 113.86 , d , j = 21.42 
 hz , 1 c ) , 49.7 , 47.3 , 32.5 , 27.7 . hrms ( esi ) : calcd for c17h19fn5 [ m + h ] , 312.1619 ; found , 
 312.1625 .
compound 8 was obtained as a pale 
 cream - colored amorphous solid ( 0.182 g , 80% ) from 55 ( 0.244 
 g , 0.594 mmol ) following general method a ; mp = 200201 c .
h nmr ( 500 mhz , dmso - d6 ) :  
 9.88 ( s , 1 h ) , 9.39 ( s , 2 h ) , 8.94 ( d , j = 5.1 hz , 
 1 h ) , 8.44 ( s , 1 h ) , 7.82 ( s , 1 h ) , 7.70 ( d , j = 
 5.1 hz , 1 h ) , 7.42 ( q , j = 7.8 hz , 1 h ) , 7.287.05 
 ( m , 3 h ) , 4.08 ( br s , 1 h ) , 3.563.53 ( m , 2 h ) , 3.39 ( t , j = 6.9 hz , 2 h ) , 3.313.25 ( m , 2 h ) , 3.133.03 
 ( m , 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  170.1 , ( 164.26 , 162.32 , d , j = 
 244.44 hz , 1 c ) , 160.5 , 153.5 , ( 141.21 , 141.15 , d , j = 7.56 hz , 1 c ) , 136.6 , ( 131.63 , 131.56 , d , j = 
 8.82 hz , 1 c ) , 125.9 , 125.8 , ( 122.23 , 122.19 , d , j = 5.0 hz , 1 c ) , 119.4 , ( 116.57 , 116.40 , d , j = 
 21.42 hz , 1 c ) , ( 114.74 , 114.57 , d , j = 21.42 hz , 
 1 c ) , 48.5 , 44.9 , 33.4 , 32.0 . hrms ( esi ) : calcd for c17h19fn5 [ m + h ] , 312.1619 ; found , 
 312.1622 . title compound 9 was obtained as a white amorphous solid ( 0.385 g , 89% ) from 56 ( 0.462 g , 1.086 mmol ) following general method a ; mp = 
 196198 c .
h nmr ( 500 mhz , dmso - d6 ) :  9.94 ( s , 1 h ) , 9.39 ( s , 2 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.45 ( s , 1 h ) , 7.85 ( s , 1 h ) , 7.70 ( d , j = 5.1 hz , 1 h ) , 7.37 ( q , j = 7.9 hz , 
 1 h ) , 7.12 ( t , j = 8.4 hz , 2 h ) , 7.06 ( td , j = 8.9 , 2.3 hz , 1 h ) , 4.25 ( br s , 1 h ) , 3.533.48 
 ( m , 2 h ) , 3.433.37 ( m , 2 h ) , 3.02.94 ( m , 2 h ) , 2.74 
 ( t , j = 7.6 hz , 2 h ) , 2.03 ( p , j = 8.2 hz , 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  170.2 , ( 164.17 , 162.24 , d , j = 243.18 hz , 1 c ) , 160.4 , 153.3 , ( 144.72 , 144.66 , d , j = 7.56 hz , 1 c ) , 136.5 , ( 131.29 , 131.22 , d , j = 
 8.82 hz , 1 c ) , ( 125.44 , 125.42 , d , j = 2.52 hz , 1 
 c ) , 123.9 , 122.3 , 119.5 , ( 116.07 , 115.91 , d , j = 
 20.16 hz , 1 c ) , ( 113.92 , 113.75 , d , j = 21.42 hz , 
 1 c ) , 47.3 , 44.7 , 33.4 , 32.6 , 27.7 . hrms ( esi ) : calcd for c18h21fn5 [ m + h ] , 326.1776 ; found , 
 326.1779 .
compound 10 was synthesized as a white amorphous solid ( 0.5 g , 99% ) from 57 ( 0.541 g , 1.224 mmol ) following general method a ; mp = 
 184186 c .
h nmr ( 500 mhz , dmso - d6 ) :  10.03 ( s , 1 h ) , 9.59 ( s , 2 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.47 ( s , 1 h ) , 7.88 ( s , 1 h ) , 7.71 ( d , j = 5.1 hz , 1 h ) , 7.397.32 ( m , 2 h ) , 7.29 ( d , j = 8.2 hz , 1 h ) , 7.24 ( d , j = 7.6 hz , 
 1 h ) , 4.01 ( s , 1 h ) , 3.533.47 ( m , 2 h ) , 3.453.36 ( m , 
 2 h ) , 2.992.93 ( m , 2 h ) , 2.73 ( t , j = 7.6 
 hz , 2 h ) , 2.04 ( p , j = 7.8 hz , 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  170.2 , 
 160.4 , 153.2 , 144.4 , 136.3 , 133.9 , 131.2 , 129.1 , 128.0 , 127.0 , 123.2 , 
 122.4 , 119.6 , 47.3 , 44.7 , 33.4 , 32.5 , 27.6 .
hrms ( esi ) : calcd for 
 c18h21cln5 [ m + h ] , 342.1480 ; 
 found , 342.1483 .
compound 11 was prepared from 58 ( 0.25 g , 0.571 mmol ) as an off - white amorphous dihydrochloride 
 salt ( 0.206 g , 88% ) following general method a ; mp = 198200 
 c .
h nmr ( 500 mhz , dmso - d6 ) :  10.00 ( s , 1 h ) , 9.58 ( s , 1 h ) , 9.49 ( s , 1 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.43 ( s , 1 h ) , 7.88 ( s , 1 h ) , 7.71 ( d , j = 5.1 hz , 1 h ) , 7.31 ( q , j = 7.9 hz , 
 1 h ) , 7.076.97 ( m , 3 h ) , 4.48 ( br s , 1 h ) , 3.593.50 
 ( m , 2 h ) , 3.443.39 ( m , 2 h ) , 3.183.15 ( m , 1 h ) , 3.012.96 
 ( m , 1 h ) , 2.21 ( dt , j = 9.2 , 5.2 hz , 1 h ) , 1.49 ( dq , j = 12.5 , 7.9 hz , 1 h ) , 1.221.09 ( m , 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  
 170.2 , ( 164.21 , 162.28 , d , j = 243.18 hz , 1 c ) , 160.4 , 
 153.2 , ( 145.83 , 145.77 , d , j = 7.56 hz , 1 c ) , 136.3 , 
 ( 131.0 , 130.93 , d , j = 8.82 hz , 1 c ) , 123.4 , ( 123.03 , 
 123.01 , d , j = 2.52 hz , 1 c ) , 122.4 , 119.5 , ( 113.63 , 
 113.46 , d , j = 21.42 hz , 1 c ) , ( 113.40 , 113.24 , d , j = 20.16 hz , 1 c ) , 51.2 , 44.2 , 33.3 , 22.6 , 20.0 , 15.4 . 
 hrms ( esi ) : calcd for c19h21fn5 [ m 
 + h ] , 338.1776 ; found , 338.1778 .
compound 12 was synthesized as a white amorphous solid ( 0.256 g , 93% ) from 59 ( 0.294 g , 0.672 mmol ) following general method a ; mp = 
 210212 c .
h nmr ( 500 mhz , dmso - d6 ) :  9.85 ( s , 1 h ) , 9.69 ( s , 1 h ) , 9.60 ( s , 1 h ) , 
 8.93 ( d , j = 5.1 hz , 1 h ) , 8.39 ( s , 1 h ) , 7.82 ( s , 
 1 h ) , 7.65 ( d , j = 5.1 hz , 1 h ) , 7.37 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.227.14 ( m , 2 h ) , 7.05 ( td , j = 8.4 , 2.3 hz , 1 h ) , 4.2 ( br s , 1 h ) , 3.553.42 
 ( m , 2 h ) , 3.33 ( t , j = 7.2 hz , 2 h ) , 2.77 ( m , 1 h ) , 
 2.682.59 ( m , 2 h ) , 1.731.66 ( m , 1 h ) , 1.23 ( ddd , j = 9.7 , 5.7 , 4.0 hz , 1 h ) , 0.84 ( q , j = 
 6.1 hz , 1 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  170.0 , ( 164.11 , 162.17 , d , j = 244.44 , 1 c ) , 160.4 , 153.4 , ( 144.24 , 144.18 , d , j = 7.56 hz , 1 c ) , 136.4 , ( 131.21 , 131.14 , d , j = 
 8.82 hz , 1 c ) , 125.5 , 124.2 , 122.1 , 119.3 , ( 116.16 , 115.99 , d , j = 21.42 hz , 1 c ) , ( 113.97 , 113.80 , d , j = 21.42 hz , 1 c ) , 45.3 , 36.7 , 35.8 , 33.0 , 18.6 , 10.7 .
hrms ( esi ) : 
 calcd for c19h21fn5 [ m + h ] , 338.1776 ; found , 338.1776 .
title compound 13 was obtained as a trihydrochloride salt , and as a pale - green amorphous 
 solid ( 0.26 g , 91% ) from 60 ( 0.28 g , 0.684 mmol ) following 
 general method a ; mp = 212214 c .
h nmr ( 500 
 mhz , dmso - d6 ) :  10.12 ( s , 1 h ) , 
 9.77 ( s , 2 h ) , 8.97 ( s , 1 h ) , 8.95 ( d , j = 5.1 hz , 
 1 h ) , 8.85 ( d , j = 5.4 hz , 1 h ) , 8.61 ( d , j = 7.8 hz , 1 h ) , 8.50 ( s , 1 h ) , 8.07 ( dd , j = 6 , 7.5 hz , 1 h ) , 7.92 ( s , 1 h ) , 7.73 ( d , j = 
 5.1 hz , 1 h ) , 3.563.39 ( m , 4 h ) , 3.022.99 ( m , 4 h ) , 
 2.15 ( p , j = 7.4 hz , 2 h ) . note that the pyridinium 
 and the imidazolium protons appear very broad on the baseline due 
 to exchange with solvent .
c nmr ( 126 mhz , dmso - d6 ) :  170.3 , 160.4 , 153.1 , 147.0 , 141.9 , 
 141.8 , 140.4 , 136.3 , 127.9 , 122.7 , 122.5 , 119.7 , 46.8 , 44.7 , 33.3 , 
 29.5 , 26.9 . hrms ( esi ) : calcd for c17h21n6 [ m + h ] , 309.1822 ; found , 309.1824 .
compound 14 was synthesized as a white amorphous solid ( 0.277 g , 95% ) from 61 ( 0.313 g , 0.774 mmol ) following general method a ; mp = 
 220222 c .
h nmr ( 500 mhz , dmso - d6 ) :  9.93 ( br s , 3 h ) , 8.93 ( d , j = 5.1 hz , 1 h ) , 8.41 ( s , 1 h ) , 8.17 ( s , 1 h ) , 8.02 ( d , j = 7.9 hz , 1 h ) , 7.92 ( d , j = 7.8 hz , 1 h ) , 7.85 
 ( s , 1 h ) , 7.727.66 ( m , 2 h ) , 4.33 ( t , j = 
 5.3 hz , 2 h ) , 3.583.49 ( m , 2 h ) , 3.44 ( t , j = 7.0 hz , 2 h ) .
note that the imidazolium proton appears very broad 
 on the baseline due to exchange with solvent .
c nmr ( 126 
 mhz , dmso - d6 ) :  170.0 , 160.4 , 153.3 , 
 136.4 , 136.1 , 134.9 , 134.5 , 133.5 , 130.7 , 123.8 , 122.3 , 119.4 , 119.3 , 
 112.3 , 50.0 , 44.5 , 33.2 .
hrms ( esi ) : calcd for c17h17n6 [ m + h ] , 305.1509 ; found , 305.1512 .
compound 15 was obtained as a white amorphous 
 solid ( 0.260 g , 88% ) from 68 ( 0.288 g , 0.704 mmol ) following 
 general method a ; mp = 230232 c .
h nmr ( 500 
 mhz , dmso - d6 ) :  9.96 ( s , 1 h ) , 
 9.61 ( s , 2 h ) , 9.02 ( d , j = 2.4 hz , 1 h ) , 8.73 ( d , j = 1.4 hz , 1 h ) , 8.47 ( t , j = 1.6 hz , 
 1 h ) , 8.42 ( t , j = 2.0 hz , 1 h ) , 8.02 ( s , 1 h ) , 7.42 
 ( td , j = 7.9 , 6.5 hz , 1 h ) , 7.19 ( dd , j = 10.0 , 1.9 hz , 1 h ) , 7.16 ( d , j = 7.7 hz , 1 h ) , 
 7.13 ( td , j = 8.7 , 2.5 hz , 1 h ) , 5.57 ( s , 2 h ) , 3.36 
 ( dt , j = 11.9 , 6.8 hz , 2 h ) , 3.273.19 ( m , 
 4 h ) , 3.153.03 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) :  ( 164.19 , 162.25 , d , j = 244.44 hz , 1 c ) , 151.3 , 142.2 , ( 141.18 , 141.12 , d , j = 7.56 hz , 1 c ) , 136.0 , 135.2 , 132.6 , 131.6 , ( 131.56 , 131.49 , d , j = 8.82 hz , 1 c ) , 125.8 , 122.1 , 121.9 , ( 116.50 , 116.34 , 
 d , j = 20.16 hz , 1 c ) , ( 114.65 , 114.49 , d , j = 20.16 hz , 1 c ) , 48.3 , 47.4 , 31.9 , 29.1 . hrms ( esi ) : 
 calcd for c18h20fn4 [ m + h ] , 311.1667 ; found , 311.1673 . title compound 16 was obtained as a pale - yellow hygroscopic solid ( 0.186 g , 89% ) from 69 ( 0.224 g , 0.525 mmol ) following general method a. h nmr ( 500 mhz , dmso - d6 ) :  
 8.94 ( d , j = 5.0 hz , 1 h ) , 8.69 ( br s , 2 h ) , 8.25 
 ( s , 2 h ) , 7.64 ( d , j = 5.0 hz , 1 h ) , 7.38 ( q , j = 7.9 hz , 1 h ) , 7.167.00 ( m , 3 h ) , 3.62 ( br s , 
 1 h ) , 3.543.47 ( m , 2 h ) , 3.303.27 ( m , 2 h ) , 3.053.02 
 ( m , 2 h ) , 2.742.70 ( m , 2 h ) , 1.991.96 ( m , 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  
 169.6 , ( 164.16 , 162.22 , d , j = 244.44 hz , 1 c ) , 160.5 , 
 155.7 , ( 144.56 , 144.52 , d , j = 5.04 hz , 1 c ) , 138.9 , 
 ( 131.27 , 131.21 , d , j = 7.56 hz , 1 c ) , ( 125.37 , 125.35 , 
 d , j = 2.52 hz , 1 c ) , 121.5 , ( 116.00 , 115.83 , d , j = 21.42 hz , 1 c ) , ( 113.92 , 113.75 , d , j = 21.42 hz , 1 c ) , 47.3 , 45.5 , 33.6 , 32.4 , 27.8 . hrms ( esi ) : calcd 
 for c17h20fn6 [ m + h ] , 327.1728 ; found , 327.1735 .
compound 17 was obtained as a white amorphous solid in its dihydrochloride form 
 ( 0.125 g , 99% ) from 70 ( 0.135 g , 0.316 mmol ) following 
 general method a ; mp = 177179 c .
h nmr ( 500 
 mhz , dmso - d6 ) :  9.56 ( s , 1 h ) , 
 9.20 ( s , 2 h ) , 8.89 ( d , j = 5.1 hz , 1 h ) , 8.35 ( s , 
 1 h ) , 7.60 ( d , j = 5.1 hz , 1 h ) , 7.37 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.157.08 ( m , 2 h ) , 7.06 ( td , j = 8.8 , 2.5 hz , 1 h ) , 6.29 ( br s , 1 h ) , 3.46 ( p , j = 7.0 hz , 2 h ) , 3.33 ( t , j = 7.2 hz , 
 2 h ) , 2.99 ( dt , j = 12.3 , 6.3 hz , 2 h ) , 2.74 ( t , j = 7.6 hz , 2 h ) , 2.01 ( p , j = 7.8 hz , 
 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  169.7 , ( 164.15 , 162.22 , d , j = 243.18 , 
 1 c ) , 160.4 , 154.6 , 154.3 , 145.6 , ( 144.67 , 144.61 , d , j = 7.56 hz , 1 c ) , ( 131.24 , 131.18 , d , j = 7.56 hz , 
 1 c ) , ( 125.38 , 125.36 , d , j = 2.52 hz , 1 c ) , 121.3 , 
 ( 116.02 , 115.85 , d , j = 21.42 hz , 1 c ) , ( 113.88 , 
 113.71 , d , j = 21.42 hz , 1 c0 , 47.2 , 45.0 , 33.4 , 
 32.5 , 27.7 . hrms ( esi ) : calcd for c17h20fn6 [ m + h ] , 327.1728 ; found , 327.1728 .
compound 18 was prepared from 71 ( 0.141 g , 0.32 mmol ) following 
 general method a to give the dihydrochloride salt , which was obtained 
 as an off - white hygroscopic solid ( 0.115 g , 87% ) .
h nmr 
 ( 500 mhz , dmso - d6 ) :  9.33 ( s , 2 
 h ) , 8.98 ( d , j = 5.1 hz , 1 h ) , 8.36 ( d , j = 2.2 hz , 1 h ) , 7.79 ( d , j = 2.1 hz , 1 h ) , 7.73 
 ( d , j = 5.1 hz , 1 h ) , 7.38 ( td , j = 8.0 , 6.5 hz , 1 h ) , 7.167.09 ( m , 2 h ) , 7.07 ( td , j = 8.8 , 2.3 hz , 1 h ) , 3.51 ( br s , 1 h ) , 3.403.37 
 ( m , 4 h ) , 3.02 ( s , 3 h ) , 2.992.94 ( m , 2 h ) , 2.74 ( t , j = 7.6 hz , 2 h ) , 2.01 ( p , j = 7.8 hz , 
 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  169.9 , ( 164.14 , 162.21 , d , j = 243.18 , 
 1 c ) , 160.3 , 154.5 , 147.4 , ( 144.65 , 144.59 , d , j = 
 7.56 hz , 1 c ) , ( 131.25 , 131.18 , d , j = 8.82 hz , 1 
 c ) , 125.4 , 122.0 , 121.3 , 119.7 , ( 116.01 , 115.84 , d , j = 21.42 hz , 1 c ) , ( 113.89 , 113.72 , d , j = 21.42 , 
 1 c ) , 47.2 , 45.0 , 33.5 , 32.5 , 27.6 , 15.8 . hrms ( esi ) : calcd for c19h23fn5 [ m + h ] , 340.1932 ; 
 found , 340.1933 .
compounds 19 and 3-(3-fluorophenyl)-n-2-[2-(5-methyl-1h - imidazol-1-yl)pyrimidin-4-yl]ethylpropan-1-amine 
 dihydrochloride ( 20 ) were obtained in an inseparable 
 15:1 ratio ( by h nmr ) from the reaction of 15:1 mixture 
 of compounds 72 and 73 ( 0.668 g , 1.52 mmol ) 
 following general method a , as a white amorphous solid of the corresponding 
 dihydrochloride salts ( 0.62 g , 99% total ) .
this mixture was separated 
 by preparative hplc on an agilent 1200 series instrument using a phenomenex 
 gemini - nx 5 m c18 column ( 150 mm  21.2 mm ) , using a gradient 
 of 115% mecn in h2o with 0.1% formic acid , to isolate 19 and 20 separately in > 95% purity . following 
 this ,
19 and 20 were converted to their 
 corresponding dihydrochloride salts by similar precipitation with 
 methanolic hcl and excess et2o .
white amorphous solid ; mp = 2 03205 c . h 
 nmr ( 500 mhz , dmso - d6 ) :  9.53 ( s , 
 1 h ) , 8.95 ( s , 2 h ) , 8.90 ( d , j = 5.1 hz , 1 h ) , 8.07 
 ( s , 1 h ) , 7.62 ( d , j = 5.1 hz , 1 h ) , 7.38 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.157.09 ( m , 2 h ) , 7.07 ( td , j = 8.9 , 2.2 hz , 1 h ) , 3.63 ( br s , 1 h ) , 3.50 ( dt , j = 12.8 , 7.1 hz , 2 h ) , 3.31 ( t , j = 7.2 
 hz , 2 h ) , 3.052.95 ( m , 2 h ) , 2.73 ( t , j = 
 7.6 hz , 2 h ) , 2.34 ( s , 3 h ) , 1.99 ( p , j = 7.7 hz , 
 2 h ) .
c nmr ( 126 mhz , dmso - d6 ) :  170.0 , ( 164.14 , 162.21 , d , j = 243.18 
 hz , 1 c ) , 160.4 , 153.2 , ( 144.65 , 144.60 , d , j = 6.3 
 hz , 1 c ) , 135.6 , 133.4 , ( 131.25 , 131.18 , d , j = 8.82 
 hz , 1 c0 , 125.4 , 122.0 , ( 116.01 , 115.85 , d , j = 20.16 
 hz , 1 c ) , 115.8 , ( 113.88 , 113.71 , d , j = 21.42 hz , 
 1 c ) , 47.3 , 44.7 , 33.4 , 32.5 , 27.6 , 11.3 . hrms ( esi ) : calcd for c19h23fn5 [ m + h ] , 340.1932 ; 
 found , 340.1937 .
h nmr ( 500 mhz , dmso - d6 ) :  9.74 ( s , 1 h ) , 9.25 ( s , 2 h ) , 8.96 ( d , j = 5.1 hz , 1 h ) , 7.72 ( d , j = 5.1 hz , 1 h ) , 7.59 
 ( s , 1 h ) , 7.437.32 ( m , 1 h ) , 7.11 ( t , j = 
 7.4 hz , 2 h ) , 7.097.03 ( m , 1 h ) , 3.68 ( br s , 1 h ) , 3.483.43 
 ( m , 2 h ) , 3.37 ( q , j = 6.7 hz , 2 h ) , 2.97 ( dt , j = 14.9 , 9.5 hz , 2 h ) , 2.73 ( t , j = 7.6 
 hz , 2 h ) , 2.65 ( s , 3 h ) , 2.051.97 ( m , 2 h ) . c 
 nmr ( 126 mhz , dmso - d6 ) :  169.9 , 
 ( 164.14 , 162.21 , d , j = 243.18 hz , 1 c ) , 160.3 , 154.7 , 
 ( 144.63 , 144.57 , d , j = 7.56 hz , 1 c ) , 137.5 , ( 131.25 , 
 131.18 , d , j = 8.82 hz , 1 c ) , 131.1 , ( 125.37 , 125.35 , 
 d , j = 2.52 hz , 1 c ) , 122.1 , 121.1 , ( 116.00 , 115.84 , 
 d , j = 20.16 hz , 1 c ) , ( 113.89 , 113.72 , d , j = 21.42 hz , 1 c ) , 47.2 , 45.0 , 33.5 , 32.5 , 27.6 , 13.13 . 
 hrms ( esi ) : calcd for c19h23fn5 [ m 
 + h ] , 340.1932 ; found , 340.1937 . to a suspension of nah ( 0.107 g , 2.686 
 mmol ) in thf ( 4 ml ) cooled to 0 c , a solution of 21 ( 0.5 g , 2.686 mmol ) in thf ( 2.5 ml ) was added dropwise .
the reaction 
 was stirred at room temperature for 1 h followed by the addition of 
 2-(3-fluorophenyl)ethyl bromide ( 0.66 g , 3.242 mmol ) in thf ( 2 ml ) 
 and a pinch of tetrabutylammonium iodide ( 0.05 g , 0.13 mmol ) . 

the resulting solution was heated at 60 c for 48 h , when it 
 was cooled to room temperature and diluted with ethyl acetate ( 20 
 ml ) and water ( 20 ml ) .
the layers were separated , and the aqueous 
 layer was washed with etoac ( 2  10 ml ) .
the combined organic 
 layers were washed with brine ( 20 ml ) , dried over sodium sulfate , 
 and concentrated .
the residue was purified by flash column chromatography 
 ( hexanes / etoac ) , and the resulting oil ( 0.464 g , 1.504 mmol , 56% ) 
 was diluted with ch2cl2 ( 12 ml ) .
this solution 
 was cooled to 0 c and treated with trifluoroacetic acid ( 6 ml ) . 

the reaction was stirred for 3 h at room temperature , after which 
 it was concentrated .
the residue was diluted with etoac ( 20 ml ) and 
 treated with saturated k2co3 ( 15 ml ) .
the organic 
 layer was extracted , and the aqueous layer was re - extracted with more 
 etoac ( 2  10 ml ) .
the combined organic layers were dried and 
 concentrated to yield a colorless oil ( 0.266 g , 85% ) , clean by nmr .
h nmr ( 500 mhz , cdcl3 ) :  7.22 ( q , j = 7.2 hz , 1h ) , 6.96 ( d , j = 7.7 hz , 1h ) , 
 6.926.85 ( m , 2h ) , 3.98 ( s , 1h ) , 2.96 ( t , j = 5.0 hz , 4h ) , 2.78 ( dd , j = 11.2 , 8.3 hz , 2h ) , 
 2.58 ( dd , j = 11.0 , 8.5 hz , 2h ) , 2.53 ( s , 4h ) .
c nmr ( 126 mhz , cdcl3 ) :  ( 163.75 , 161.80 , 
 d , j = 245.7 hz , 1 c ) , ( 142.71 , 142.65 , d , j = 7.56 hz , 1 c ) , ( 129.75 , 129.69 , d , j = 7.56 hz , 1 c ) , ( 124.29 , 124.27 , d , j = 2.52 hz , 
 1 c ) , ( 115.55 , 115.38 , d , j = 21.42 hz , 1 c ) , ( 112.98 , 
 112.81 , d , j = 21.42 hz , 1 c ) , 60.4 , 53.5 , 45.5 , 
 33.0 . compound 30 ( 0.2 g , 1.3 mmol ) 
 was added in ch2cl2 ( 20 ml ) to a flask containing 
 oven - dried 3  molecular sieves .
compound 31 ( 0.17 
 ml , 0.18 g , 1.3 mmol ) was added as a solution in ch2cl2 ( 6 ml ) , followed by glacial acetic acid ( 20 l ) .
the 
 mixture was stirred for 1.5 h and cooled to 0 c , and sodium 
 triacetoxyborohydride ( 0.413 g , 1.95 mmol ) was added in one portion . 

the mixture was stirred at room temperature for 14 h , after which 
 it was diluted with ch2cl2 ( 30 ml ) and filtered . 

the filtrate was washed with saturated aqueous nahco3 ( 30 
 ml ) , and the aqueous layer was extracted with ch2cl2 ( 2  20 ml ) .
the organic phase was washed with brine 
 ( 40 ml ) , dried over anhydrous sodium sulfate , and concentrated .
the 
 resulting residue was purified by flash column chromatography , eluting 
 with a gradient of ch2cl2/meoh to yield the 
 intermediate 33 ( 0.306 g , 85% ) as an off - white sticky 
 oil .
h nmr ( 500 mhz , cdcl3 ) :  8.42 ( d , j = 5.0 hz , 1 h ) , 7.25 ( q , j = 7.5 , hz , 
 1 h ) , 6.98 ( d , j = 7.6 hz , 1 h ) , 6.946.88 
 ( m , 3 h ) , 3.83 ( s , 2 h ) , 2.90 ( t , j = 6.6 hz , 2 h ) , 
 2.83 ( t , j = 6.7 hz , 2 h ) , 2.50 ( s , 3 h ) , 2.13 ( s , 
 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  172.4 , 
 168.6 , ( 163.84 , 161.88 , d , j = 246.96 hz , 1 c ) , 157.1 , 
 ( 142.29 , 142.23 , d , j = 7.56 hz , 1 c ) , ( 129.92 , 129.85 , 
 d , j = 8.82 hz , 1 c ) , ( 124.36 , 124.34 , d , j = 2.52 hz , 1 c ) , ( 115.60 , 115.43 , d , j = 21.42 hz , 1 c ) , 114.2 , ( 113.21 , 113.04 , d , j = 
 21.42 hz , 1 c ) , 53.9 , 50.2 , 36.1 , 14.0 . ms ( esi ) m / z [ m + h ] : calcd , 278.1 ; found , 278.06 . compound 34 ( 0.594 g , 89% ) 
 was prepared from 30 ( 0.352 g , 2.285 mmol ) and 32 ( 0.35 g , 2.285 mmol ) by the exact procedure used to prepare 33 .
h nmr ( 500 mhz , cdcl3 ) :  
 8.42 ( d , j = 5.0 hz , 1 h ) , 7.21 ( q , j = 7.5 hz , 1 h ) , 6.96 ( d , j = 5.0 hz , 1 h ) , 6.93 
 ( d , j = 7.6 hz , 1 h ) , 6.886.83 ( m , 2 h ) , 
 3.80 ( s , 2 h ) , 2.682.63 ( m , 4 h ) , 2.54 ( s , 3 h ) , 2.22 ( s , 
 1 h ) , 1.83 ( p , j = 7.3 hz , 2 h ) .
c nmr 
 ( 126 mhz , cdcl3 ) :  172.3 , 168.7 , ( 163.77 , 161.82 , 
 d , j = 245.7 hz , 1 c ) , 157.1 , ( 144.47 , 144.41 , d , j = 7.56 hz , 1 c ) , ( 129.69 , 129.63 , d , j = 7.56 hz , 1 c ) , ( 123.94 , 123.92 , d , j = 2.52 hz , 
 1 c ) , ( 115.16 , 115.0 , d , j = 20.16 hz , 1 c ) , 114.3 , 
 ( 112.70 , 112.54 , d , j = 20.16 hz , 1 c ) , 54.0 , 48.7 , 
 33.1 , 31.3 , 14.1 . ms ( esi ) m / z [ m 
 + na ] : calcd , 314.11 ; found 314.22 .
compound 33 ( 0.306 g , 1.103 
 mmol ) was dissolved in thf ( 10 ml ) , and a solution of di - tert - butyl dicarbonate ( 0.265 g , 1.213 mmol ) in thf ( 6 ml ) was added . 

the resulting solution was stirred overnight , and thereafter , it was 
 diluted with etoac / h2o ( 30 ml , 1:1 ) . the layers were separated 
 and the aqueous layer extracted with etoac ( 2  10 ml ) .
the combined 
 organic layers were dried , concentrated , and purified by column chromatography 
 ( hexanes / etoac ) . the resulting oil
( 0.412 g , 1.091 mmol , 99% ) was 
 dissolved in ch2cl2 ( 2 ml ) and added to a solution 
 of m - chloroperbenzoic acid ( 0.69 g , 4.0 mmol ) in 
 ch2cl2 ( 4 ml ) at 0 c .
the reaction was 
 gradually warmed to room temperature while it was stirred overnight . 

the resulting suspension was then filtered and the white solid washed 
 with cold ch2cl2 ( 5 ml ) .
the collected 
 filtrate was washed with 10% aqueous k2co3 ( 5 
 ml ) and the organic fraction dried , concentrated , and purified by 
 flash column chromatography to give a sticky white solid ( 0.335 g , 
 75% ) .
h nmr ( 500 mhz , cdcl3 ) :  ( 8.81 
 ( d , j = 5.0 hz ) , 8.79 ( d , j = 4.9 
 hz ) , 2:3 , 1 h ) , ( 7.39 ( d , j = 4.8 hz ) , 7.32 ( d , j = 4.0 hz ) , 3:2 , 1 h ) , 7.23 ( q , j = 7.8 
 hz , 1 h ) , 6.966.83 ( m , 3 h ) , ( 4.53 ( s ) , 4.46 ( s ) , 3:2 , 2 h ) , 
 3.573.52 ( m , 2 h ) , 3.33 ( s , 3 h ) , ( 2.89 ( t , j = 6.7 hz ) , 2.84 ( t , j = 7 hz ) , 2:3 , 2 h ) , ( 1.45 
 ( s ) , 1.35 ( s ) , 3:2 , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) :  ( 170.85 , 170.17 , 1 c ) , ( 165.82 , 165.61 , 1 c ) , ( 163.78 , 
 161.83 , d , j = 245.7 hz , 1 c ) , 158.51 , ( 155.53 , 154.77 , 
 1 c ) , ( 140.92 , 140.87 , d , j = 6.3 hz , 1 c ) , ( 130.09 , 
 130.03 , d , j = 7.56 hz , 1 c ) , 124.48 , ( 121.18 , 120.17 , 
 1 c ) , ( 115.72 , 115.55 , d , j = 21.42 hz , 1 c ) , ( 113.54 , 
 113.37 , d , j = 21.42 hz , 1 c ) , ( 81.03 , 80.93 , 1 c ) , 
 ( 52.99 , 52.17 , 1 c ) , ( 49.90 , 49.83 , 1 c ) , ( 39.12 , 39.03 , 1 c ) , ( 34.70 , 
 34.20 , 1 c ) , 28.20 .
ms ( esi ) m / z [ m + na ] : calcd , 432.14 ; found , 432.10 .
compound 36 ( 0.745 g , 89% ) 
 was synthesized from 34 in two steps , following the exact 
 same procedure as used to synthesize 35 .
h nmr ( 500 mhz , cdcl3 ) :  ( 8.85 ( d , j = 6.0 hz ) , 8.83 ( d , j = 4.5 hz , 4:5 , 1 h ) , ( 7.45 
 ( d , j = 4.5 hz ) , 7.40 ( d , j = 4.5 
 hz , 5:4 , 1 h ) , 7.23 ( q , j = 6.9 hz , 1 h ) , 6.936.84 
 ( m , 3 h ) , ( 4.60 ( s ) , 4.56 ( s ) , 5:4 , 2 h ) , ( 3.37 ( t , j = 7 hz ) , 3.28 ( t , j = 7 hz ) , 4:5 , 2 h ) , 3.33 ( s , 
 3 h ) , 2.652.55 ( m , 2 h ) , 1.87 ( p , j = 7.4 
 hz , 2 h ) , ( 1.45 ( s ) , 1.35 ( s ) , 5:4 , 9 h ) .
c nmr ( 126 
 mhz , cdcl3 ) :  ( 171.07 , 170.43 , 1 c ) , ( 165.86 ,
165.66 , 
 1 c ) , ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 158.59 , 
 ( 155.71 , 154.90 , 1 c ) , ( 143.69 , 143.58 , d , j = 13.8 
 hz , 1 c ) , ( 129.92 , 129.85 , d , j = 8.82 hz , 1 c ) , 
 123.85 , ( 121.16 , 120.17 , 1 c ) , ( 115.11 , 114.95 , d , j = 20.16 hz , 1 c ) , ( 113.01 , 112.84 , d , j = 21.42 
 hz , 1 c ) , ( 80.92 , 80.82 , 1 c ) , ( 52.30 , 51.98 , 1 c ) , ( 47.98 ,
47.81 , 
 1 c ) , ( 39.11 , 39.03 , 1 c ) , ( 32.78 , 32.64 , 1 c ) , ( 29.66 , 29.59 , 1 c ) , 
 28.25 .
ms ( esi ) m / z [ 2 m + na ] : calcd , 869.31 ; found , 869.23 .
compound 35 ( 0.292 g , 0.713 
 mmol ) was dissolved in mecn ( 4 ml ) , and imidazole ( 0.243 g , 3.567 
 mmol ) and k2co3 ( 0.197 g , 1.427 mmol ) added 
 to the solution .
the resulting mixture was heated at 65 c for 
 4 h , after which it was cooled and diluted with ch2cl2 ( 20 ml ) .
water ( 20 ml ) was added to the organic layer and 
 the layers separated .
the aqueous layer was washed once with ch2cl2 ( 10 ml ) , and the combined organic layers were 
 dried over anhydrous sodium sulfate , concentrated , and purified by 
 flash column chromatography using hexanes / etoac .
compound 37 was obtained as a faint - yellow sticky oil ( 0.263 g , 93% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.59 ( s , 1 h ) , 8.57 ( d , j = 5.2 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.23 ( q , j = 7.8 hz , 1 h ) , 7.15 ( s , 1 h ) , ( 7.07 ( d , j = 4.9 
 hz ) , 7.01 ( d , j = 4.7 hz ) , 3:2 , 1 h ) , 6.996.83 
 ( m , 3 h ) , ( 4.44 ( s ) , 4.36 ( s ) , 3:2 , 2 h ) , ( 3.58 ( t , j = 7.5 hz ) , 3.54 ( t , j = 7.2 hz ) , 2:3 , 2 h ) , ( 2.91 
 ( t , j = 7.2 hz ) , 2.86 ( t , j = 7.1 
 hz ) , 2:3 , 2 h ) , ( 1.46 ( s ) , 1.36 ( s ) , 3:2 , 9 h ) .
c nmr 
 ( 126 mhz , cdcl3 ) :  ( 170.13 , 169.55 , 1 c ) , ( 163.80 , 
 161.85 , d , j = 245.7 hz , 1 c ) , 158.85 , ( 155.58 , 155.00 , 
 1 c ) , 154.43 , ( 141.15 , 141.10 , d , j = 6.3 hz , 1 c ) , 
 136.13 , ( 130.63 , 130.52 , d , j = 13.86 hz , 1 c ) , 130.05 , 
 124.45 , ( 116.49 , 116.09 , 1 c ) , ( 115.71 , 115.55 , d , j = 20.91 hz , 1 c ) , 115.29 , ( 113.49 , 113.32 , d , j = 21.42 hz , 1 c ) , 80.63 , ( 52.81 , 51.91 , 1 c ) , 49.89 , ( 34.83 , 34.35 , 
 1 c ) , 28.26 . ms ( esi ) m / z [ m + h ] : calcd , 398.21 ; found , 398.42 .
compound 38 was synthesized 
 from 36 ( 0.648 g , 1.53 mmol ) following the same procedure 
 as used above to synthesize 37 from 35 . 

compound 38 was obtained as an off - white viscous oil 
 after purification ( 0.531 g , 84% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.61 ( d , j = 4.3 hz , 1 h ) , 8.58 
 ( s , 1 h ) , 7.85 ( s , 1 h ) , 7.21 ( q , j = 6.5 hz , 1 h ) , 
 7.14 ( s , 1 h ) , ( 7.10 ( d , j = 4.5 hz ) , 7.05 ( d , j = 3.9 hz ) , 5:4 , 1 h ) , 6.956.83 ( m , 3 h ) , ( 4.50 
 ( s ) , 4.44 ( s ) , 5:4 , 2 h ) , ( 3.40 ( t , j = 6.9 hz ) , 
 3.31 ( t , j = 7.1 hz ) , 4:5 , 2 h ) , 2.652.59 
 ( m , 2 h ) , 1.961.82 ( m , 2 h ) , ( 1.46 ( s ) , 1.35 ( s ) , 5:4 , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) :  ( 170.28 , 169.76 , 
 1 c ) , ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 158.84 , 
 ( 155.77 , 155.14 , 1 c ) , 154.42 , ( 143.91 , 143.86 , d , j = 6.3 hz , 1 c ) , 136.12 , ( 130.63 , 130.52 , d , j = 
 13.86 hz , 1 c ) , 129.81 , 123.84 , 116.47 , ( 116.07 , 115.27 , 1 c ) , ( 115.10 , 
 114.93 , d , j = 21.42 hz , 1 c ) , ( 112.98 , 112.81 , d , j = 21.42 hz , 1 c ) , 80.50 , ( 52.18 , 51.76 , 1 c ) , 47.84 , ( 32.83 , 
 32.69 , 1 c ) , 29.66 , 28.29 . ms ( esi ) m / z [ m + h ] : calcd , 412.20 ; found , 412.00 . to a solution of 40 ( 0.3 g , 
 1.971 mmol ) in ethanol
( 12 ml ) , 31 ( 1.3 ml , 1.371 g , 
 9.85 mmol ) and acetic acid ( 0.06 ml , 0.06 g , 0.985 mmol ) were added . 

the reaction was stirred at room temperature for 8 h , after which 
 it was diluted with ch2cl2 ( 25 ml ) .
the resulting 
 solution was treated with saturated nahco3 ( 20 ml ) , and 
 the layers were separated . the aqueous layer was washed with ch2cl2 ( 3  10 ml ) , and the combined organic 
 layers were dried over anhydrous sodium sulfate and concentrated . 

the obtained residue was purified by flash column chromatography ( ch2cl2/meoh ) to obtain 41 as a clear 
 oil ( 0.356 g , 62% ) .
h nmr ( 500 mhz , cdcl3 ) : 
  8.37 ( d , j = 5.0 hz , 1 h ) , 7.267.20 
 ( m , 1 h ) , 6.95 ( d , j = 7.6 hz , 1 h ) , 6.936.87 
 ( m , 2 h ) , 6.79 ( d , j = 5.1 hz , 1 h ) , 3.03 ( t , j = 6.8 hz , 2 h ) , 2.91 ( t , j = 7.0 hz , 
 2 h ) , 2.86 ( t , j = 6.8 hz , 2 h ) , 2.79 ( t , j = 7.1 hz , 2 h ) , 2.52 ( s , 3 h ) , 1.61 ( br s , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  172.4 , 168.8 , 
 ( 163.86 , 161.91 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 142.30 , 
 142.25 , d , j = 6.3 hz , 1 c ) , ( 129.92 , 129.85 , d , j = 8.82 hz , 1 c ) , ( 124.29 , 124.27 , d , j = 2.52 hz , 1 c ) , 115.8 , ( 115.52 , 115.36 , d , j = 
 20.16 hz , 1 c ) , ( 113.21 , 113.04 , d , j = 21.42 hz , 
 1 c ) , 50.5 , 47.7 , 37.4 , 35.9 , 14.0 . ms ( esi ) m / z [ m + h ] : calcd , 292.1 ; found , 291.84 .
compounds 4247 were synthesized 
 from 40 and the corresponding primary amines ( 32 , 62 , s3s12 ; see supporting information ) , following the same procedure 
 used to synthesize 41 .
h nmr ( 500 mhz , cdcl3 ) :  8.36 ( d , j = 5.0 hz , 1 h ) , 7.237.18 ( m , 1 h ) , 6.93 ( t , j = 7.7 hz , 1 h ) , 6.886.83 ( m , 2 h ) , 6.81 ( d , j = 5.0 hz , 1 h ) , 2.98 ( t , j = 6.6 hz , 2 h ) , 2.85 
 ( t , j = 6.7 hz , 2 h ) , 2.752.68 ( m , 2 h ) , 
 2.672.59 ( m , 2 h ) , 2.52 ( s , 3 h ) , 1.76 ( p , j = 7.3 hz , 2 h ) , 1.57 ( br s , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  172.3 , 169.1 , ( 163.75 , 161.80 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 144.60 , 144.54 , d , j = 
 7.56 hz , 1 c ) , ( 129.65 , 129.58 , d , j = 8.82 hz , 1 
 c ) , ( 123.93 , 123.91 , d , j = 2.52 hz , 1 c ) , 115.8 , 
 ( 115.13 , 114.97 , d , j = 20.16 hz , 1 c ) , ( 112.62 , 
 112.45 , d , j = 21.42 hz , 1 c ) , 48.9 , 47.9 , 37.6 , 
 33.2 , 31.2 , 14.0 . ms ( esi )
m / z [ m 
 + h ] : calcd , 306.1 ; found , 305.85 .
pale - yellow oil ( 0.591 g , 75% ) . h 
 nmr ( 500 mhz , cdcl3 ) :  8.39 ( d , j = 5.0 hz , 1 h ) , 7.217.14 ( m , 3 h ) , 7.04 ( d , j = 7.2 hz , 1 h ) , 6.82 ( d , j = 5.0 hz , 1 h ) , 3.02 
 ( t , j = 6.6 hz , 2 h ) , 2.88 ( t , j = 6.6 hz , 2 h ) , 2.66 ( d , j = 7.5 hz , 2 h ) , 2.63 
 ( d , j = 8.0 hz , 2 h ) , 2.55 ( s , 3 h ) , 1.90 ( br s , 
 1 h ) , 1.81 ( p , j = 7.5 hz , 3 h ) .
c nmr 
 ( 126 mhz , cdcl3 ) :  172.4 , 169.0 , 156.9 , 144.0 , 134.0 , 
 129.6 , 128.5 , 126.5 , 126.0 , 115.8 , 48.9 , 47.9 , 37.5 , 33.2 , 31.2 , 14.1 . 

ms ( esi ) m / z [ m + h ] : calcd , 322.1 ; found , 321.91 .
h nmr ( 500 mhz , cdcl3 ) :  8.38 ( d , j = 5.0 hz , 1 h ) , 7.18 ( q , j = 7.8 hz , 
 1 h ) , 6.876.78 ( m , 3 h ) , 6.766.68 ( m , 1 h ) , 3.093.02 
 ( m , 2 h ) , 2.89 ( t , j = 6.7 hz , 2 h ) , 2.72 ( t , j = 6.0 hz , 1 h ) , 2.68 ( d , j = 6.5 hz , 
 1 h ) , 2.53 ( s , 3 h ) , 1.80 ( br s , 1 h ) , 1.741.69 ( m , 1 h ) , 
 1.331.28 ( m , 1 h ) , 0.930.86 ( m , 2 h ) .
c nmr ( 126 mhz , cdcl3 ) :  172.4 , 169.0 , ( 163.94 , 
 161.99 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 145.73 , 145.67 , 
 d , j = 7.56 hz , 1 c ) , ( 129.64 , 129.58 , d , j = 7.56 hz , 1 c ) , 121.4 , 115.8 , ( 112.48 , 112.30 , d , j = 22.68 hz , 1 c ) , ( 112.33 , 112.16 , d , j = 21.42 hz , 1 c ) , 53.8 , 47.7 , 37.6 , 23.6 , 21.9 , 15.1 , 14.0 . ms ( esi ) m / z [ 2 m + h ] : calcd , 635.2 ; 
 found , 635.08 .
h nmr ( 500 mhz , cdcl3 ) :  8.37 ( d , j = 5.0 hz , 1 h ) , 7.277.20 ( m , 1 h ) , 6.99 ( d , j = 7.5 hz , 1 h ) , 6.966.91 ( m , 1 h ) , 6.89 ( td , j = 8.6 , 2.2 hz , 1 h ) , 6.77 ( d , j = 5.0 
 hz , 1 h ) , 3.022.97 ( m , 2 h ) , 2.80 ( t , j = 
 6.6 hz , 2 h ) , 2.59 ( dd , j = 14.8 , 6.8 hz , 1 h ) , 2.54 
 ( s , 3 h ) , 2.47 ( dd , j = 14.8 , 7.6 hz , 1 h ) , 2.112.05 
 ( m , 1 h ) , 1.90 ( br s , 1 h ) , 1.070.99 ( m , 1 h ) , 0.690.63 
 ( m , 1 h ) , 0.470.43 ( m , 1 h ) .
c nmr ( 126 mhz , 
 cdcl3 ) :  172.4 , 169.1 , ( 163.82 , 161.87 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 144.17 , 144.12 , d , j = 6.3 hz , 1 c ) , ( 129.73 , 129.67 , d , j = 7.56 hz , 
 1 c ) , ( 123.92 , 123.89 , d , j = 3.78 hz , 1 c ) , 115.7 , 
 ( 115.15 , 114.99 , d , j = 20.16 hz , 1 c ) , ( 112.85 , 
 112.68 , d , j = 21.42 hz , 1 c ) , 47.5 , 38.1 , 37.4 , 
 37.2 , 21.0 , 14.0 , 13.7 . ms ( esi ) m / z [ m + h ] : calcd , 318.1 ; found , 317.9 .
h nmr ( 500 mhz , cdcl3 ) :  8.43 ( s , 1 h ) , 8.41 ( d , j = 6.1 hz , 1 h ) , 8.36 ( d , j = 5.0 hz , 
 1 h ) , 7.47 ( d , j = 7.7 hz , 1 h ) , 7.17 ( dd , j = 7.6 , 4.9 hz , 1 h ) , 6.80 ( d , j = 5.0 
 hz , 1 h ) , 3.02 ( t , j = 6.7 hz , 2 h ) , 2.88 ( t , j = 6.7 hz , 2 h ) , 2.67 ( t , j = 7.0 hz , 
 2 h ) , 2.64 ( t , j = 7.5 hz , 2 h ) , 2.52 ( s , 3 h ) , 2.40 
 ( br s , 1 h ) , 1.81 ( p , j = 7.3 hz , 2 h ) .
c nmr ( 126 mhz , cdcl3 ) :  172.3 , 168.8 , 156.8 , 149.8 , 
 147.4 , 137.0 , 135.7 , 123.2 , 115.8 , 48.7 , 47.7 , 37.2 , 30.9 , 30.5 , 14.0 . 
 ms ( esi ) m / z [ m + h ] : calcd , 289.1 ; found , 289.01 . cream - colored oil ( 0.426 g , 
 83% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.39 
 ( d , j = 5.1 hz , 1 h ) , 7.63 ( s , 1 h ) , 7.55 ( d , j = 8.0 hz , 1 h ) , 7.52 ( d , j = 7.5 hz , 
 1 h ) , 7.41 ( t , j = 7.7 hz , 1 h ) , 6.84 ( d , j = 5.1 hz , 1 h ) , 3.84 ( s , 2 h ) , 3.02 ( t , j = 6.6 hz , 2 h ) , 2.89 ( t , j = 6.6 hz , 2 h ) , 2.52 
 ( s , 3 h ) , 1.77 ( br s , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  172.2 , 168.8 , 156.7 , 141.6 , 132.3 , 131.3 , 130.5 , 
 128.9 , 118.7 , 115.7 , 112.1 , 52.6 , 47.1 , 37.3 , 13.8 . ms ( esi ) m / z [ m + h ] : calcd , 285.1 ; 
 found , 284.91 . to a solution of 41 ( 0.356 g , 1.222 mmol ) in thf ( 10 
 ml ) , a solution of di - tert - butyl dicarbonate ( 0.293 
 g , 1.344 mmol ) in thf ( 7 ml ) was added .
the resulting solution was 
 stirred at room temperature for 3 h and , thereafter , it was diluted 
 with etoac / h2o ( 30 ml , 1:1 ) . the layers were separated , 
 and the aqueous layer extracted with etoac ( 2  10 ml ) .
combined 
 organic layers were dried , concentrated , and purified by column chromatography 
 ( hexanes / etoac ) .
the resulting oil ( 0.39 g , 0.996 mmol , 82% ) was dissolved 
 in ch2cl2 ( 2 ml ) and added to a solution of m - chloroperbenzoic acid ( 0.636 g , 3.685 mmol ) in ch2cl2 ( 3 ml ) at 0 c .
the resulting solution 
 was stirred at room temperature for 3 h. at that point , the reaction 
 was filtered , and the white residue was washed with cold ch2cl2 ( 5 ml ) . the collected filtrate was washed 
 with 10% aqueous k2co3 ( 5 ml ) , the organic layer 
 was dried , concentrated , and purified by flash column chromatography 
 ( hexanes / etoac ) to give a clear viscous oil ( 0.337 g , 80% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.76 ( d , j = 4.1 hz , 1 h ) , ( 7.43 ( s ) , 7.31 ( s ) , 7:3 , 1 h ) , 7.23 ( q , j = 6.5 hz , 1 h ) , 6.976.84 ( m , 3 h ) , ( 3.613.53 
 ( m ) , 3.493.44 ( m ) , 7:3 , 2 h ) , 3.37 ( t , j = 
 7.1 hz , 2 h ) , 3.34 ( s , 3 h ) , ( 3.183.10 ( m ) , 3.082.98 
 ( m ) , 7:3 , 2 h ) , ( ( 2.862.80 ( m ) , 2.782.72 ( m ) , 3:7 , 
 2 h ) , 1.38 ( s , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) :  ( 170.93 , 170.82 , 1 c ) , ( 165.98 , 165.76 , 1 c ) , ( 163.79 , 
 161.84 , d , j = 245.7 hz , 1 c ) , ( 158.01 , 157.88 , 1 
 c ) , ( 155.22 , 154.87 , 1 c ) , 141.38 , ( 130.00 , 129.94 , d , j = 7.56 hz ) , 124.54 , 123.56 , ( 115.75 , 115.59 , d , j = 20.16 hz , 1 c ) , ( 113.37 , 113.21 , d , j = 20.16 
 hz , 1 c ) , 80.05 , ( 49.37 , 49.18 , 1 c ) , ( 46.80 , 46.14 , 1 c ) , 39.09 , 
 ( 36.91 , 36.29 , 1 c ) , ( 34.90 , 34.19 , 1 c ) , 28.25 .
ms ( esi ) m / z [ m + na ] : calcd , 446.15 ; 
 found , 446.06 .
compounds 4952 were synthesized from compounds 4245 following the same procedures used to synthesize 48 .
h nmr ( 500 mhz , cdcl3 ) :  8.818.72 ( m , 1 
 h ) , ( 7.477.43 ( m ) , 7.377.32 ( m ) , 3:2 , 1 h ) , 7.23 ( q , j = 7.4 hz , 1 h ) , 6.93 ( d , j = 7.5 hz , 
 1 h ) , 6.87 ( t , j = 8.0 hz , 2 h ) , 3.663.56 
 ( m , 2 h ) , 3.34 ( s , 3 h ) , 3.243.08 ( m , 4 h ) , 2.58 ( t , j = 7.7 hz , 2 h ) , 1.81 ( p , j = 7 hz , 2 
 h ) , 1.39 ( s , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) : 
  ( 170.97 , 170.87 , 1 c ) , ( 165.98 , 165.76 , 1 c ) , ( 163.81 , 161.86 , 
 d , j = 245.7 hz , 1 c ) , 157.89 , ( 155.39 , 155.02 , 1 
 c ) , ( 143.99 , 143.90 , d , j = 11.34 hz , 1 c ) , ( 129.85 , 
 129.79 , d , j = 7.56 hz , 1 c ) , ( 123.92 , 123.90 , d , j = 2.52 hz , 1 c ) , 123.54 , ( 115.11 , 114.95 , d , j = 20.16 hz , 1 c ) , ( 112.89 , 112.72 , d , j = 21.42 
 hz , 1 c ) , 79.93 , ( 47.38 , 47.04 , 1 c ) , 46.03 , 39.08 , ( 36.94 , 36.37 , 
 1 c ) , 32.76 , ( 29.85 , 29.63 , 1 c ) , 28.30 . ms ( esi ) m / z [ 2 m + na ] : calcd , 897.19 ; found , 
 897.26 . colorless oil ( 0.613 g , 89% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.76 ( d , j = 4.5 hz , 1 h ) , ( 7.45 
 ( s ) , 7.35 ( s ) , 3:2 , 1 h ) , 7.20 ( q , j = 7.5 hz , 1 
 h ) , 7.187.13 ( m , 2 h ) , 7.04 ( d , j = 7.3 hz , 
 1 h ) , 3.673.56 ( m , 2 h ) , 3.34 ( s , 3 h ) , 3.253.06 ( m , 
 4 h ) , 2.56 ( t , j = 7.5 hz , 2 h ) , 1.81 ( p , j = 7.6 hz , 2 h ) , 1.39 ( s , 9 h ) .
c nmr ( 126 
 mhz , cdcl3 ) :  170.96 , ( 166.02 , 165.84 , 1 c ) , 157.91 , 
 155.36 , 143.42 , 134.13 , 129.69 , 128.35 , 126.48 , 126.15 , 123.50 , 79.96 , 
 ( 47.40 , 47.19 , 1 c ) , 46.07 , 39.11 , ( 37.01 , 36.46 , 1 c ) , 32.74 , 29.89 , 
 28.34 .
ms ( esi ) m / z [ m + na ] : calcd , 476.09 ; found , 476.01 .
h nmr ( 500 mhz , cdcl3 ) :  8.66 
 ( d , j = 4.9 hz , 1 h ) , ( 7.35 ( s ) , 7.25 ( s ) , 1:1 , 1 
 h ) , 7.10 ( q , j = 7.7 hz , 1 h ) , 6.796.68 ( m , 
 2 h ) , 6.61 ( dt , j = 2 , 10.3 hz , 1 h ) , 3.683.60 
 ( m , 2 h ) , 3.323.26 ( m , 1 h ) , 3.21 ( s , 3 h ) , 3.183.02 
 ( m , 3 h ) , 1.831.75 ( m , 1 h ) , 1.35 ( s , 9 h ) , 1.201.16 
 ( m , 1 h ) , 0.920.84 ( m , 2 h ) .
c nmr ( 126 mhz , 
 cdcl3 ) :  170.70 , ( 165.68 , 165.55 , 1 c ) , ( 163.69 , 
 161.74 , d , j = 245.7 hz , 1 c ) , 157.74 , 155.01 , 145.08 , 
 ( 129.56 , 129.49 , d , j = 8.82 hz , 1 c ) , 123.33 , 121.26 , 
 ( 112.23 , 112.07 , d , j = 20.16 hz , 1 c ) , ( 112.15 , 
 111.97 , d , j = 22.68 hz , 1 c ) , 79.74 , ( 51.04 , 50.41 , 
 1 c ) , 45.83 , 38.87 , ( 36.67 , 36.06 , 1 c ) , ( 31.29 , 30.58 , 1 c ) , 28.08 , 
 ( 22.66 , 22.35 , 1 c ) , ( 14.37 , 13.82 , 1 c ) .
ms ( esi ) m / z [ m + h ] : calcd , 450.17 ; found , 450.07 .
h nmr ( 500 mhz , cdcl3 ) :  8.70 
 ( d , j = 5.1 hz , 1 h ) , 7.31 ( d , j = 4.0 hz , 1 h ) , 7.18 ( q , j = 8 hz , 1 h ) , 6.92 ( d , j = 7.6 hz , 1 h ) , 6.86 ( d , j = 9.9 hz , 
 1 h ) , 6.81 ( td , j = 8.5 , 2.2 hz , 1 h ) , 3.52 ( s , 1 
 h ) , 3.49 ( t , j = 7 hz , 1 h ) , 3.28 ( s , 3 h ) , 2.992.92 
 ( m , 2 h ) , 2.79 ( dd , j = 14.5 , 5.3 hz , 1 h ) , 2.35 
 ( dd , j = 8 , 13.5 hz , 1 h ) , 2.272.23 ( m , 1 
 h ) , 1.34 ( s , 9 h ) , 0.840.76 ( m , 1 h ) , 0.66 ( q , j = 6.1 hz , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) : 
  170.87 , 165.71 , ( 163.64 , 161.69 , d , j = 245.7 
 hz , 1 c ) , 157.84 , 156.00 , ( 143.07 , 143.01 , d , j = 
 7.56 hz , 1 c ) , ( 129.78 , 129.71 , d , j = 8.82 hz , 1 
 c ) , ( 124.00 , 123.98 , d , j = 2.52 hz , 1 c ) , 123.25 , 
 ( 115.14 , 114.98 , d , j = 20.16 hz , 1 c ) , ( 112.92 , 
 112.75 , d , j = 21.42 hz , 1 c ) , 79.82 , 45.94 , 38.96 , 
 37.46 , 36.06 , 34.84 , 28.20 , ( 22.87 , 22.45 , 1 c ) , 14.84 . ms ( esi ) m / z [ m + h ] : calcd , 450.17 ; 
 found , 450.20 .
compound 46 ( 0.443 
 g , 1.536 mmol ) was dissolved in thf ( 15 ml ) , and a solution of di - tert - butyl dicarbonate ( 0.369 g , 1.689 mmol ) in thf ( 6 ml ) 
 was added .
the resulting solution was stirred at room temperature 
 for 3 h , and thereafter , it was diluted with etoac / h2o 
 ( 40 ml , 1:1 ) .
the layers were separated , and the aqueous layer was 
 extracted with etoac ( 2  15 ml ) . combined organic layers were 
 dried , concentrated , and purified by column chromatography ( hexanes / etoac ) . 
 the resulting oil ( 0.567 g , 1.459 mmol , 95% ) was dissolved in thf 
 ( 12 ml ) .
water ( 12 ml ) and oxone ( 1.345 g , 2.188 mmol ) were sequentially 
 added , and the reaction was stirred at room temperature for 4 h. at 
 that point , the reaction was diluted with etoac / h2o ( 30 
 ml , 1:1 ) and the layers separated . the aqueous layer was basified 
 to ph 10 with 6 n naoh , saturated by addition of solid k2co3 , and extracted with etoac ( 3  15 ml ) . combined 
 organic fractions were dried over sodium sulfate , and concentrated . 

the resulting light - yellow oil ( 0.527 g , 86% ) was clean by nmr and 
 was used in the next step without further purification .
h nmr ( 500 mhz , cdcl3 ) :  8.74 ( d , j = 3.9 hz , 1 h ) , 8.42 ( s , 2 h ) , 7.48 ( d , j = 6.9 
 hz , 1 h ) , ( 7.42 ( s ) , 7.34 ( s ) , 3:2 , 1 h ) , 7.20 ( dd , j = 7.3 , 4.9 hz , 1 h ) , 3.663.54 ( m , 2 h ) , 3.31 ( s , 3 h ) , 3.243.03 
 ( m , 4 h ) , 2.56 ( t , j = 7.8 hz , 2 h ) , 1.80 ( p , j = 7 hz , 2 h ) , 1.36 ( s , 9 h ) .
c nmr ( 126 mhz , 
 cdcl3 ) :  170.87 , 165.81 , 157.85 , 155.27 , 149.53 , 
 147.30 , 136.70 , 135.74 , 123.47 , 123.37 , 79.93 , ( 47.30 , 46.94 , 1 c ) , 
 46.01 , 39.05 , ( 36.87 , 36.33 , 1 c ) , 30.11 , ( 29.84 , 29.58 , 1 c ) , 28.25 . 
 ms ( esi ) m / z [ m + na ] : calcd , 443.16 ; found , 443.03 .
compound 54 ( 0.396 g , 91% ) 
 was obtained as a colorless oil from 47 following the 
 same procedure used to synthesize 48 from 41 .
h nmr ( 500 mhz , cdcl3 ) :  8.76 ( d , j = 4.8 hz , 1 h ) , 7.53 ( d , j = 7.3 hz , 
 1 h ) , 7.487.37 ( m , 4 h ) , 4.46 ( s , 2 h ) , 3.743.61 ( m , 
 2 h ) , 3.33 ( s , 3 h ) , 3.203.07 ( m , 2 h ) , 1.38 ( s , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) :  170.46 , 165.58 , 
 157.79 , ( 155.26 , 154.98 , 1 c ) , ( 139.96 , 139.55 , 1 c ) , ( 131.83 ,
131.28 , 
 1 c ) , 130.75 , 130.23 , 129.26 , 123.42 , 118.36 , 112.36 , 80.49 , ( 50.65 , 
 49.71 , 1 c ) , 45.59 , 38.87 , ( 36.34 , 35.94 , 1 c ) , 28.00 .
ms ( esi ) m / z [ m + na ] : calcd , 439.14 ; 
 found , 438.99 . compound 48 ( 0.335 g , 0.791 
 mmol ) was dissolved in mecn ( 5 ml ) , and imidazole ( 0.269 g , 3.958 
 mmol ) and k2co3 ( 0.22 g , 1.583 mmol ) were added 
 to the solution .
the resulting mixture was heated at 65 c for 
 5 h , after which it was cooled and diluted with ch2cl2 ( 20 ml ) .
water ( 20 ml ) was added to the organic layer and 
 the layers separated .
the aqueous layer was washed once with ch2cl2 ( 15 ml ) , and the combined organics were dried 
 over anhydrous sodium sulfate , concentrated , and purified by flash 
 column chromatography using hexanes / etoac . the desired compound ( 55 ) was obtained as an off - white viscous oil ( 0.247 g , 76% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.61 ( s , 1 h ) , 
 8.53 ( d , j = 4.9 hz , 1 h ) , 7.88 ( s , 1 h ) , 7.24 ( q , j = 6.0 hz , 1 h ) , 7.16 ( s , 1 h ) , ( 7.08 ( s ) , 6.97 ( s ) , 3:2 , 
 1 h ) , 6.946.82 ( m , 3 h ) , ( 3.56 ( t , j = 8.5 
 hz ) , 3.47 ( s ) , 3:2 , 2 h ) , 3.433.36 ( m , 2 h ) , ( 2.99 ( t , j = 8.5 hz ) , 2.90 ( s ) , 3:2 , 2 h ) , ( 2.84 ( s ) , 2.79 ( t , j = 7 hz ) , 2:3 , 2 h ) , 1.38 ( s , 9 h ) .
c nmr 
 ( 126 mhz , cdcl3 ) :  ( 170.39 , 170.27 , 1 c ) , ( 163.81 , 
 161.85 , d , j = 246.96 hz , 1 c ) , ( 158.43 , 158.33 , 
 1 c ) , ( 155.18 , 154.98 , 1 c ) , ( 154.55 , 154.38 , 1 c ) , ( 141.54 , 141.47 , 
 d , j = 8.82 hz , 1 c ) , 136.09 , ( 130.55 , 130.47 , 1 
 c ) , ( 130.00 , 129.94 , 1 c ) , ( 124.48 , 124.46 , d , j = 
 2.52 hz , 1 c ) , 118.49 , 116.48 , ( 115.74 , 115.57 , d , j = 21.42 hz , 1 c ) , ( 113.38 , 113.22 , d , j = 20.16 
 hz , 1 c ) , 79.87 , ( 49.37 , 49.23 , 1 c ) , ( 46.97 , 46.21 , 1 c ) , ( 36.84 , 
 36.07 , 1 c ) , ( 35.00 , 34.29 , 1 c ) , 28.23 .
ms ( esi ) m / z [ m + h ] : calcd , 412.20 ; found , 412.08 .
compounds 5661 were synthesized 
 from compounds 4954 following the 
 same procedures used to synthesize 55 .
h nmr ( 500 mhz , cdcl3 ) :  8.59 ( s , 1 h ) , 
 8.52 ( d , j = 4.1 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.21 ( q , j = 7 hz , 1 h ) , 7.15 ( s , 1 h ) , ( 7.07 ( s ) , 7.00 ( s ) , 3:2 , 
 1 h ) , 6.91 ( d , j = 7.5 hz , 1 h ) , 6.886.81 
 ( m , 2 h ) , 3.663.54 ( m , 2 h ) , 3.243.14 ( m , 2 h ) , 3.042.94 
 ( m , 2 h ) , 2.58 ( t , j = 7.7 hz , 2 h ) , 1.83 ( p , j = 7.6 hz , 2 h ) , 1.38 ( s , 9 h ) .
c nmr ( 126 
 mhz , cdcl3 ) :  ( 170.42 , 170.32 , 1 c ) , ( 163.79 , 161.84 , 
 d , j = 245.7 hz , 1 c ) , ( 158.40 , 158.31 , 1 c ) , ( 155.36 , 
 155.12 , 1 c ) , ( 154.54 , 154.39 , 1 c ) , ( 144.04 , 143.90 , d , j = 17.6 hz , 1 c ) , 136.09 , 130.52 , ( 129.81 , 129.74 , d , j = 8.82 hz , 1 c ) , ( 123.84 , 123.82 , d , j = 2.52 hz , 
 1 c ) , 118.43 , 116.45 , ( 115.08 , 114.91 , d , j = 21.42 
 hz , 1 c ) , ( 112.87 , 112.70 , d , j = 21.42 hz , 1 c ) , 
 79.73 , ( 47.24 , 46.97 , 1 c ) , ( 46.27 , 46.08 , 1 c ) , ( 36.91 , 36.15 , 1 
 c ) , 32.76 , ( 29.86 , 29.60 , 1 c ) , 28.27 .
ms ( esi ) m / z [ 2 m + na ] : calcd , 873.43 ; found , 
 873.39 .
h nmr ( 500 mhz , cdcl3 ) :  8.63 ( s , 1 h ) , 
 8.54 ( d , j = 4.9 hz , 1 h ) , 7.88 ( s , 1 h ) , 7.18 ( q , j = 7.5 hz , 1 h ) , 7.177.14 ( m , 3 h ) , ( 7.09 ( s ) , 
 7.05 ( s ) , 3:2 , 1 h ) , 7.03 ( d , j = 7.1 hz , 1 h ) , 3.653.55 
 ( m , 2 h ) , 3.253.14 ( m , 2 h ) , 3.052.94 ( m , 2 h ) , 2.57 
 ( t , j = 7.7 hz , 2 h ) , 1.83 ( p , j = 7.6 hz , 2 h ) , 1.39 ( s , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) :  170.49 , 158.39 , ( 155.38 , 155.21 , 1 c ) , 154.40 , 
 143.39 , 136.01 , 134.14 , ( 130.34 , 130.21 , 1 c ) , 129.65 , 128.34 , 126.40 , 
 126.15 , 118.55 , 116.54 , 79.80 , ( 47.27 , 47.06 , 1 c ) , ( 46.28 ,
46.12 , 
 1 c ) , ( 36.99 , 36.21 , 1 c ) , 32.77 , ( 29.91 , 29.73 , 1 c ) , 28.32 .
ms ( esi ) m / z [ m + h ] : calcd , 442.19 ; 
 found , 442.06 .
h nmr ( 500 mhz , cdcl3 ) :  8.60 ( s , 1 h ) , 8.50 ( d , j = 5.0 hz , 1 h ) , 7.87 ( s , 1 h ) , 7.17 ( q , j = 8 hz , 1 h ) , 7.14 ( s , 1 h ) , ( 7.05 ( s ) , 6.94 ( d , j = 7.7 h ) , 2:3 , 1 h ) , 6.876.78 ( m , 2 h ) , 6.67 ( dt , j = 2.5 , 10.2 hz , 1 h ) , 3.753.63 ( m , 2 h ) , 3.413.30 
 ( m , 1 h ) , 3.253.16 ( m , 1 h ) , 3.082.95 ( m , 2 h ) , 1.921.78 
 ( m , 1 h ) , 1.41 ( s , 9 h ) , 1.291.26 ( m , 1 h ) , 0.980.93 
 ( m , 2 h ) .
c nmr ( 126 mhz , cdcl3 ) :  
 170.40 , ( 163.93 , 161.98 , d , j = 245.7 hz , 1 c ) , 158.34 , 
 155.26 , 154.47 , 145.25 , 136.05 , 130.44 , ( 129.75 , 129.68 , d , j = 8.82 hz , 1 c ) , 121.37 , 118.41 , 116.46 , ( 112.53 , 112.40 , 
 d , j = 16.38 hz , 1 c ) , ( 112.40 , 112.23 , d , j = 21.42 hz , 1 c ) , 79.89 , ( 51.24 , 50.73 , 1 c ) , 46.24 , ( 36.90 , 
 36.07 , 1 c ) , 28.32 , 22.85 , 22.08 , 14.65 . ms ( esi ) m / z [ m + h ] : calcd , 438.2 ; found , 438.03 .
h nmr ( 500 mhz , cdcl3 ) :  8.62 ( s , 1 h ) , 
 8.52 ( d , j = 5.0 hz , 1 h ) , 7.88 ( s , 1 h ) , 7.257.18 
 ( m , 1 h ) , 7.16 ( s , 1 h ) , 6.99 ( s , 1 h ) , 6.96 ( d , j = 7.6 hz , 1 h ) , 6.91 ( d , j = 9.8 hz , 1 h ) , 6.87 
 ( td , j = 8.5 , 2.3 hz , 1 h ) , ( 3.53 ( t , j = 7 hz ) , 3.50 ( t , j = 7 hz ) , 2:1 , 2 h ) , 2.88 ( t , j = 6.5 hz , 2 h ) , 2.84 ( dd , j = 10 , 5 hz , 
 1 h ) , 2.43 ( dd , j = 14.0 , 8.0 hz , 1 h ) , 2.36 ( dt , j = 7.2 , 3.6 hz , 1 h ) , 1.37 ( s , 9 h ) , 1.231.20 ( m , 
 1 h ) , 0.86 ( dt , j = 9.3 , 5.3 hz , 1 h ) , 0.72 ( q , j = 6.1 hz , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  170.62 , ( 163.80 , 161.85 , d , j = 
 245.7 hz , 1 c ) , 158.30 , 156.21 , 154.36 , ( 143.16 , 143.11 , d , j = 6.3 hz , 1 c ) , 136.06 , 130.30 , ( 129.86 , 129.80 , d , j = 7.56 hz , 1 c ) , ( 124.08 , 124.06 , d , j = 2.52 hz , 1 c ) , 118.39 , 116.53 , ( 115.29 , 115.13 , d , j = 20.16 hz , 1 c ) , ( 113.09 , 112.93 , d , j = 20.16 
 hz , 1 c ) , 79.83 , 46.13 , 37.67 , 35.99 , 34.88 , 28.31 , 23.02 , 15.01 . 
 ms ( esi ) m / z [ 2 m + na ] : calcd , 897.43 ; found , 897.32 . pale - yellow oil ( 0.43 g , 84% ) .
h nmr ( 500 mhz , cdcl3 ) :  8.57 ( s , 1 h ) , 
 8.51 ( d , j = 4.7 hz , 1 h ) , 8.40 ( s , 2 h ) , 7.84 ( s , 
 1 h ) , 7.44 ( s , 1 h ) , 7.17 ( dd , j = 7.3 , 4.9 hz , 1 
 h ) , 7.12 ( s , 1 h ) , ( 7.05 ( s ) , 6.99 ( s ) , 3:2 , 1 h ) , 3.653.52 
 ( m , 2 h ) , 3.253.14 ( m , 2 h ) , 3.052.92 ( m , 2 h ) , 2.56 
 ( t , j = 7.8 hz , 2 h ) , 1.82 ( p , j = 7.6 hz , 2 h ) , 1.36 ( s , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) : 
  170.30 , 158.33 , 155.21 , 154.43 , 149.60 , 147.38 , 136.73 , 136.03 , 
 135.56 , 130.45 , 123.27 , 118.40 , 116.42 , 79.76 , ( 47.16 , 46.97 , 1 c ) , 
 46.22 , ( 36.85 ,
36.09 , 1 c ) , 30.14 , ( 29.83 , 29.58 , 1 c ) , 28.22 .
ms 
 ( esi ) m / z [ m + h ] : calcd , 
 409.22 ; found , 409.08 .
h nmr ( 500 mhz , cdcl3 ) :  8.57 ( s , 1 h ) , 8.54 ( d , j = 5.0 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.52 ( d , j = 7.3 hz , 1 h ) , ( 7.46 ( s ) , 7.42 ( s ) , 1:1 , 2 h ) , 7.40 ( d , j = 7.4 hz , 1 h ) , 7.15 ( s , 1 h ) , ( 7.09 ( s ) , 7.01 ( s ) , 1:1 , 
 1 h ) , 4.45 ( s , 2 h ) , ( 3.71 ( s ) , 3.64 ( s ) , 1:1 , 2 h ) , 3.04 ( s ) , 3.00 
 ( s ) , 1:1 , 2 h ) , ( 1.45 ( s ) , 1.38 ( s ) , 1:1 , 9 h ) . c nmr 
 ( 126 mhz , cdcl3 ) :  169.90 , 158.35 , ( 155.42 , 155.02 , 
 1 c ) , 154.26 , 139.80 , 135.85 , 131.71 , 131.17 , 130.78 , 130.57 , 130.31 , 
 129.21 , 118.38 , 116.31 , 112.47 , 80.52 , ( 50.73 , 49.94 , 1 c ) , 46.09 , 
 ( 36.33 , 35.69 , 1 c ) , 28.07 . ms ( esi ) m / z [ 2 m + na ] : calcd , 831.37 ; found , 831.33 . 2-(3-fluorobenzyl)cyclopropan-1-amine ( 62 ; 0.851 g , 
 5.15 mmol ) was diluted in ch2cl2 ( 20 ml ) and 
 cooled to 20 c .
( s)-(+)--methoxylphenylacetic 
 acid ( 1.0 g , 6.18 mmol ) was added to the resulting solution , followed 
 by the addition of dicyclohexylcarbodiimide ( 1.275 g , 6.18 mmol ) . 

the resulting suspension was filtered , and the 
 white precipitate was washed with cold ch2cl2 ( 15 ml ) . the combined filtrate was concentrated and chromatographed 
 in silica gel using a gradient of hexanes / etoac to obtain the two 
 diastereomers ( r , r , s)-63a and ( s , s , s)-63b .
h nmr ( 500 mhz , cdcl3 ) :  7.427.29 ( m , 5 
 h ) , 7.23 ( q , j = 7.8 hz , 1 h ) , 7.01 ( d , j = 7.5 hz , 1 h ) , 6.95 ( d , j = 10.0 hz , 1 h ) , 6.936.86 
 ( m , 2 h ) , 4.61 ( s , 1 h ) , 3.33 ( s , 3 h ) , 2.91 ( dd , j = 14.9 , 5.8 hz , 1 h ) , 2.67 ( dq , j = 7.2 , 3.6 hz , 
 1 h ) , 2.39 ( dd , j = 14.9 , 8.0 hz , 1 h ) , 1.201.13 
 ( m , 1 h ) , 0.83 ( dt , j = 9.3 , 4.8 hz , 1 h ) , 0.76 ( q , j = 6.1 hz , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  170.8 , ( 163.61 , 161.66 , d , j = 
 245.7 hz , 1 c ) , ( 143.01 , 142.95 , d , j = 7.56 hz , 
 1 c ) , 136.7 , ( 129.54 , 129.47 , d , j = 8.82 hz , 1 c ) , 
 128.3 , 128.2 , 126.8 , ( 123.89 , 123.87 , d , j = 2.52 
 hz , 1 c ) , ( 115.06 , 114.89 , d , j = 21.42 hz , 1 c ) , 
 ( 112.76 , 112.59 , d , j = 21.42 hz , 1 c ) , 83.5 , 56.8 , 
 37.3 , 28.4 , 20.4 , 13.1 .
h nmr ( 500 mhz , cdcl3 ) :  7.437.30 ( m , 5 h ) , 7.27 ( td , j = 7.9 , 6.2 hz , 1 h ) , 7.07 ( d , j = 7.6 hz , 1 h ) , 
 7.036.99 ( m , 1 h ) , 6.91 ( td , j = 8.3 , 2.5 
 hz , 2 h ) , 4.62 ( s , 1 h ) , 3.34 ( s , 3 h ) , 2.87 ( dd , j = 14.9 , 6.3 hz , 1 h ) , 2.68 ( dq , j = 7.4 , 3.7 hz , 
 1 h ) , 2.48 ( dd , j = 14.9 , 7.6 hz , 1 h ) , 1.261.18 
 ( m , 1 h ) , 0.820.69 ( m , 2 h ) .
c nmr ( 126 mhz , 
 cdcl3 ) :  170.7 , ( 163.59 , 161.64 , d , j = 245.7 hz , 1 c ) , ( 143.04 , 142.98 , d , j = 7.56 
 hz , 1 c ) , 136.7 , ( 129.54 , 129.48 , d , j = 7.56 hz , 
 1 c ) , 128.2 , 128.1 , 126.7 , ( 123.89 , 123.87 , d , j = 
 2.52 hz , 1 c ) , ( 115.05 , 114.88 , d , j = 21.42 hz , 
 1 c ) , ( 112.75 , 112.58 , d , j = 21.42 hz , 1 c ) , 83.5 , 
 56.8 , 37.4 , 28.4 , 20.5 , 13.0 . to a solution of 
 ( r , r , s)-63a ( 0.775 g , 2.472 mmol ) in ethanol ( 12 ml ) , 12 n hcl ( 12 ml ) was added , 
 and the reaction was heated to reflux for 12 h. the resulting solution 
 was cooled to room temperature , concentrated , and treated with et2o/6 n hcl ( 30 ml , 1:1 )
. the layers were separated , and the 
 aqueous layer was washed with et2o ( 10 ml ) , basified to 
 ph 1012 with 6
the combined ch2cl2 extracts were dried over sodium sulfate and concentrated to obtain 
 ( r , r)-62a ( 0.347 g , 
 85% ) , which was pure by nmr and used directly in the next step without 
 further purification .
( s , s)-62b was obtained 
 from ( s , s , s)-63b ( 0.756 g , 2.42 mmol ) in 88% yield ( 0.352 g ) following 
 the same procedure used to synthesize ( r , r)-62a . compounds ( r , r)-12 and ( s , s)-12 were synthesized from 62a and 62b , respectively , following the same procedures 
 used to synthesize racemic 12 from 2-methylthio-4-vinylpyrimidine 
 ( 40 ) and 62 . enantiopurities of ( r , r)-59 ( enantiomeric ratio : 
 97:3 ) and ( s , s)-59 ( enantiomeric 
 ratio : 96:4 ) were determined by chiral hplc using a chiralpak ad - h 
 column using an isocratic gradient of 10% 2-propanol / hexanes at flow 
 rate 0.5 ml / min . compound 66 ( 0.55 g , 2.78 mmol ) was dissolved in ch2cl2 ( 50 ml ) and cooled to 78 c .
dibal in thf ( 25 
 wt % in toluene ; 2.8 ml , 2.37 g , 4.17 mmol ) was added dropwise , and 
 the reaction continued at 78 c for 1 h. at this point , 
 sodium sulfate decahydrate was added to the reaction mixture , which 
 was gradually warmed to room temperature over 1 h. the resulting suspension 
 was filtered through celite and washed with ch2cl2 ( 20 ml ) , and the combined organic layers were dried and concentrated . 
 to this crude 2-(5-bromopyridin-3-yl)acetaldehyde ,
chcl3 ( 50 ml ) was added , followed by the addition of anhydrous mgso4 ( 3.0 g ) .
3-fluorophenethylamine ( 31 ; 
 0.36 ml , 0.387 g , 2.78 mmol ) and acetic acid ( 60 l ) were sequentially 
 added and stirred at room temperature for 1 h. the reaction mixture 
 was cooled to 0 c , and sodium triacetoxyborohydride ( 0.71 g , 
 3.336 mmol ) was added in one portion .
the reaction was filtered , 
 and the filtrate was washed with saturated aqueous nahco3 ( 20 ml ) , and the aqueous layer was extracted with chcl3 ( 2  10 ml ) . the organic phase was washed with brine ( 20 ml ) , 
 dried over sodium sulfate , concentrated , and purified by flash column 
 chromatography with ch2cl2/meoh to obtain 67 ( 0.37 g , 41% ) as a pale - yellow oil .
h nmr ( 500 
 mhz , cdcl3 ) :  8.51 ( d , j = 2.1 
 hz , 1 h ) , 8.34 ( d , j = 1.6 hz , 1 h ) , 7.64 ( t , j = 1.8 hz , 1 h ) , 7.257.20 ( m , 1 h ) , 6.93 ( d , j = 7.5 hz , 1 h ) , 6.916.84 ( m , 2 h ) , 2.88 ( td , j = 7.1 , 4.0 hz , 4 h ) , 2.76 ( dt , j = 14.5 , 
 7.1 hz , 4 h ) , 1.31 ( s , 1 h ) .
c nmr ( 126 mhz , cdcl3 ) :  ( 163.81 , 161.86 , d , j = 245.7 
 hz , 1 c ) , 148.7 , 148.2 , ( 142.27 , 142.21 , d , j = 7.56 
 hz , 1 c ) , 138.6 , 137.2 , ( 129.89 , 129.83 , d , j = 7.56 
 hz , 1 c ) , ( 124.26 , 124.24 , d , j = 2.52 hz , 1 c ) , 
 120.6 , ( 115.47 , 115.31 , d , j = 20.16 hz , 1 c ) , ( 113.20 , 
 113.04 , d , j = 20.16 hz , 1 c ) , 50.5 , 50.2 , 36.0 , 
 33.1 . ms ( esi ) m / z [ 1:1 ; ( m + h ] ) ] : calcd , 323.0 , 325.0 ; found , 322.77 , 324.76 .
compound 67 ( 0.37 
 g , 1.145 mmol ) was dissolved in thf ( 10 ml ) , and a solution of di - tert - butyl dicarbonate ( 0.275 g , 1.26 mmol ) in thf ( 6 ml ) 
 was added .
the resulting solution was stirred overnight , and then 
 it was diluted with etoac / h2o ( 30 ml , 1:1 ) . the layers 
 were separated and the aqueous layer extracted with etoac ( 2  
 10 ml ) .
the combined organic layers were dried , concentrated , and 
 purified by column chromatography ( hexanes / etoac ) .
the resulting oil 
 ( 0.445 g , 1.051 mmol , 92% ) was added to a vial along with cubr ( 7.5 
 mg , 0.052 mmol ) and cs2co3 ( 0.685 g , 2.102 mmol ) . 

the reaction vial was evacuated and backfilled with argon ; anhydrous 
 dmso ( previously purged for 5 min with argon ) , 8-acetyl-5,6,7,8-tetrahydroquinoline 
 ( 17 l , 0.018 g , 0.105 mmol ) , and imidazole ( 0.107 g , 1.576 
 mmol ) were sequentially added , the reaction sealed , and heated at 
 100 c for 12 h. it was cooled to room temperature , treated with 
 etoac / h2o ( 30 ml , 1:1 ) , and the layers separated .
the aqueous 
 layer was extracted with etoac ( 2  10 ml ) , and the combined 
 etoac extracts were washed with brine ( 20 ml ) , dried , and concentrated . 

the residue was purified by flash column chromatography using ch2cl2/meoh to obtain 68 ( 0.289 g , 67% ) 
 as a cream - colored oil .
h nmr ( 500 mhz , cdcl3 ) :  8.59 ( s , 1 h ) , 8.43 ( s , 1 h ) , 8.01 ( s , 1 h ) , 7.59 ( s , 
 1 h ) , 7.42 ( s , 2 h ) , 7.24 ( q , j = 6.5 hz , 1 h ) , 7.026.79 
 ( m , 3 h ) , 3.423.33 ( m , 4 h ) , 2.962.69 ( m , 4 h ) , 1.38 
 ( s , 9 h ) .
c nmr ( 126 mhz , cdcl3 ) :  
 ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 155.17 , ( 154.88 , 
 154.85 , 1 c ) , 149.15 , 141.51 , ( 140.86 , 140.71 , 1 c ) , 135.86 , 133.89 , 
 ( 130.02 , 129.97 , d , j = 6.3 hz , 1 c ) , 129.13 , ( 124.49 , 
 124.47 , d , j = 2.52 hz , 1 c ) , ( 115.74 , 115.57 , d , j = 21.42 hz , 1 c ) , ( 113.44 , 113.28 , d , j = 20.16 hz , 1 c ) , 79.98 , ( 49.33 , 49.04 , 1 c ) , 48.19 , ( 34.95 , 34.27 , 
 1 c ) , ( 32.01 , 31.88 , 1 c ) , 28.27
. ms ( esi ) m / z [ m + h ] : calcd , 411.21 ; found , 411.33 .
compounds 6973 were synthesized 
 from 49 following the same procedure used to synthesize 56 .
h nmr ( 500 mhz , cdcl3 ) :  8.69 ( s , 1 h ) , 
 7.93 ( s , 1 h ) , 7.86 ( s , 1 h ) , 7.68 ( s , 1 h ) , 7.14 ( q , j = 7.8 hz , 1 h ) , 6.886.84 ( m , 2 h ) , 6.79 ( d , j = 9.0 hz , 1 h ) , 3.60 ( t , j = 6.8 hz , 2 h ) , 3.223.04 
 ( m , 4 h ) , 2.50 ( t , j = 7.7 hz , 2 h ) , 1.76 ( p , j = 7 hz , 2 h ) , 1.33 ( s , 9 h ) .
c nmr ( 126 mhz , 
 cdcl3 ) :  171.28 , ( 163.71 , 161.76 , d , j = 245.7 hz , 1 c ) , 158.73 , ( 155.23 , 155.08 , 1 c ) , 143.91 , 137.50 , 
 134.86 , ( 129.66 , 129.59 , d , j = 8.82 hz , 1 c ) , 123.89 , 
 ( 123.76 , 123.74 , d , j = 2.52 hz , 1 c ) , 119.95 , ( 114.98 , 
 114.82 , d , j = 201.6 hz , 1 c ) , ( 112.69 , 112.52 , d , j = 21.42 hz , 1 c ) , 79.62 , ( 47.31 , 46.89 , 1 c ) , 46.34 , ( 37.23 , 
 36.62 , 1 c ) , 32.71 , ( 29.78 , 29.53 , 1 c ) , 28.18 . ms ( esi ) m / z [
h nmr ( 500 mhz , cdcl3 ) :  9.22 ( s , 1 h ) , 
 8.66 ( d , j = 4.9 hz , 1 h ) , 8.16 ( s , 1 h ) , ( 7.22 ( s ) , 
 7.13 ( s ) , 1:1 , 1 h ) , 7.20 ( q , j = 7.2 hz , 1 h ) , 6.90 
 ( d , j = 7.5 hz , 1 h ) , 6.886.81 ( m , 2 h ) , 
 3.663.58 ( m , 2 h ) , 3.233.15 ( m , 2 h ) , 3.113.03 
 ( m , 2 h ) , 2.57 ( t , j = 7.7 hz , 2 h ) , 1.82 ( p , j = 7.7 hz , 2 h ) , 1.38 ( s , 9 h ) .
c nmr ( 126 
 mhz , cdcl3 ) :  171.17 , ( 163.82 , 161.87 , d , j = 245.7 hz , 1 c ) , 158.75 , 155.37 , 154.28 , 153.57 , 143.89 , 
 143.73 , ( 129.81 , 129.74 , d , j = 8.82 hz , 1 c ) , ( 123.84 , 
 123.82 , d , j = 2.52 hz , 1 c ) , 119.83 , ( 115.09 , 114.92 , 
 d , j = 21.42 hz , 1 c ) , ( 112.88 , 112.72 , d , j = 20.16 hz , 1 c ) , 79.83 , ( 47.28 , 47.06 , 1 c ) , 46.27 , ( 37.06 , 
 36.45 , 1 c ) , 32.78 , ( 29.86 , 29.65 , 1 c ) , 28.30 . ms ( esi ) m / z [ 2 m + na ] : calcd , 875.39 ; found , 
 875.33 . colorless oil ( 0.141 g , 85% ) .
h nmr ( 500 mhz , 
 cdcl3 ) :  8.53 ( d , j = 4.5 hz , 1 
 h ) , 7.82 ( s , 1 h ) , 7.18 ( q , j = 7.1 hz , 1 h ) , ( 7.03 
 ( s ) , 6.97 ( s ) , 1:1 , 1 h ) , 6.92 ( s , 1 h ) , 6.89 ( d , j = 7.5 hz , 1 h ) , 6.856.79 ( m , 2 h ) , 3.653.52 ( m , 
 2 h ) , 3.233.09 ( m , 2 h ) , 3.012.89 ( m , 2 h ) , 2.78 ( s , 
 3 h ) , 2.55 ( t , j = 7.7 hz , 2 h ) , 1.80 ( p , j = 7 hz , 2 h ) , 1.37 ( s , 9 h ) .
c nmr ( 126 mhz , 
 cdcl3 ) :  169.92 , ( 163.74 , 161.79 , d , j = 245.7 hz , 1 c ) , 158.03 , 155.90 , 155.09 , 146.41 , 143.84 , ( 129.74 , 
 129.68 , d , j = 7.56 hz , 1 c ) , 127.32 , ( 123.77 , 123.75 , 
 d , j = 2.52 hz , 1 c ) , 118.35 , 117.63 , ( 115.00 , 114.84 , 
 d , j = 20.16 hz , 1 c ) , ( 112.79 , 112.62 , d , j = 21.42 hz , 1 c ) , 79.66 , ( 47.17 , 46.90 , 1 c ) , 46.12 , ( 36.88 , 
 36.19 , 1 c ) , 32.72 , ( 29.77 , 29.53 , 1 c ) , 28.23 , 18.12 . ms ( esi ) m / z [ m + h ] : calcd , 440.2 ; 
 found , 440.04 .
compounds 72 and 73 were obtained as a 15:1 nonseparable mixture of isomers from reaction 
 of 49 with 4(5)-methylimidazole , which was carried to 
 the next step without further purification .
all isozymes of nos , rat and human nnos , 
 murine macrophage inos , and bovine enos , were recombinant enzymes , 
 overexpressed in escherichia coli and 
 purified following previously reported procedures .
the enzyme inhibition was determined by measuring the production 
 of nitric oxide from l - arginine using the hemoglobin capture 
 assay in the presence of different concentrations of inhibitors .
the assay was performed at 37 c in 100 
 mm hepes buffer with 10% glycerol ( ph 7.4 ) in the presence of 10 m l - arginine and tetrahydrobiopterin , 100 m nadph , 0.83 
 mm cacl2 , 320 units / ml of calmodulin , and 3 m 
 human oxyhemoglobin . for inos , cacl2 , and calmodulin
all assays were performed in 96-well 
 plates using a synergy h1 hybrid multimode microplate reader with 
 automated dispensing of nos enzyme and hemoglobin after 30 s ( maximum 
 delay ) , which initiated the assay .
the initial rates of no production 
 were determined by monitoring the formation of methemoglobin ( no mediated 
 conversion of oxyhemoglobin to methemoglobin ) by monitoring the absorbance 
 at 401 nm .
the entire kinetic readout was performed for 5 min with 
 measurements at every 22 s interval .
each compound was assayed at 
 least in duplicate , and nine concentrations ( 100 m to 10 nm 
 for nnos ; 500 m to 50 nm for inos and enos ) were used to construct 
 dose  response curves with slopes from initial 
 readouts .
ic50 values were calculated by nonlinear regression 
 using graphpad prism ( standard error values reported are from the 
 log ic50 calculations ) , and apparent ki values were determined using the cheng 
prusoff 
 equation [ ki = ic50/(1 + [ s]/km ) ] with the following km values for l - arginine : 1.3 ( rat nnos ) , 1.6 ( human 
 nnos ) , 8.2 ( murine macrophage inos ) , and 1.7 m ( bovine enos ) . 

the selectivity of an inhibitor was defined as the ratio of their 
 respective ki values .
the preparations of rat nnos , bovine 
 enos , and human nnos heme domains used for crystallographic studies 
 were carried out by the procedures described previously .
the heme domain samples of nnos ( at 9 mg / ml 
 containing 20 mm histidine ) , bovine enos ( 10 mg / ml containing 2 mm 
 imidazole ) , and human nnos ( 13 mg / ml ) were used for the sitting drop 
 vapor diffusion crystallization setup under conditions reported .
a new orthorhombic crystal form of human nnos 
 was obtained when the ph was raised to 6.2 from 5.0 and the protein 
 concentration dropped to 10 mg / ml .
the well solution compositions 
 were only slightly shifted from what were reported : 911% peg3350 , 
 40 mm citric acid , 60 mm bis - tris - propane , 10% glycerol , and 5 mm 
 tcep . from the sitting drop setup
, plate - like crystals grew to full 
 size at 4 c in 34 days without seeding .
fresh crystals 
 were first passed stepwise through cryoprotectant solutions and then 
 soaked with 10 mm inhibitor for 46 h at 4 c before being 
 flash cooled with liquid nitrogen .
the 
 cryogenic ( 100 k ) x - ray diffraction data were collected remotely at 
 the stanford synchrotron radiation light source ( ssrl ) or advanced 
 light source ( als ) through the data collection control software blu - ice and a crystal - mounting robot . when a q315r ccd 
 detector was used , 90100 of data were typically collected 
 with 0.5 per frame .
if a pilatus pixel array detector was used , 
 140160 of fine - sliced data were collected with 0.2 
 per frame .
raw ccd data frames were indexed , integrated , and scaled 
 using hkl2000 or mosflm , but the pixel array data were processed with xds and scaled with scala ( aimless ) . for rat nnos or bovine enos structures , the 
 binding of inhibitors was detected by the initial difference fourier 
 maps calculated with refmac . for human 
 nnos structures ,
one 
 homodimer in the known human nnos structure ( 4d1n ) was used as the 
 search model .
the new human nnos structure closely resembles that 
 of rat nnos to have only one homodimer in the asymmetric unit .
the 
 inhibitor molecules were then modeled in coot and refined using refmac or phenix .
the tls protocol was implemented 
 in the final stage of refinements with each subunit as one tls group . 
 the omit fo 
fc density maps were calculated by removing inhibitor coordinates 
 from the input pdb file before running one more round of tls refinement 
 in refmac or in phenix ( simulated annealling protocol with a 2000 
 k initial temperature ) .
the resulting map coefficients delfwt and 
 sigdelwt were used to generate maps that are displayed in figures . 

the refined structures were validated with the validation service 
 in the rcsb protein data bank . the crystallographic data collection 
 and structure refinement statistics are summarized in table s1 of the supporting information , with 
 the pdb accession codes included .
caco-2 permeability assays were performed by cyprotex ( watertown , 
 ma ) using the caco-2 epithelial monolayers .
caco-2 cells , grown in 
 tissue culture flasks , were trypsinized , suspended in media , and plated 
 in 96-well plates to be grown for 3 weeks ; the proper formation of 
 monolayer was determined by fluorescent measurement of transport of 
 an impermeable dye , lucifer yellow .
all assays were performed with 
 compounds at a concentration of 10 m for 2 h. for apical to 
 basolateral ( ab ) permeability , compounds were added on the 
 apical side ( a ) and permeation determined on the basolateral side 
 ( b ) , where the receiving buffer was removed for analysis by lc / ms / ms 
 using an agilent 6410 mass spectrometer ( esi , mrm mode ) coupled with 
 an agilent 1200 hplc .
the buffers used were 100 m lucifer yellow 
 in transport buffer ( 1.98 g / l glucose in 10 mm hepes , 1 hank s 
 balanced salt solution , ph 6.5 ) ( apical side ) and transport buffer , 
 ph 7.4 ( basolateral side ) .
the apparent permeability ( papp ) is expressed using the following equation : papp = ( dq / dt)/c0a , where dq / dt is the rate of permeation , c0 is initial concentration , and a is the monolayer area . for bidirectional permeability ,
the efflux 
 ratio was defined as papp(ba)/papp(ab ) ; high efflux ratio ( > 3 ) indicates 
 that a compound is a potential substrate for p - gp or other active 
 transport systems .
screening 
 of compound 9 for off - target receptor activity was performed 
 at the nimh psychoactive drug screening program at unc chapel hill . 
 in the primary radioligand
binding assays , the compound was tested 
 at a single concentration ( 10 m ) in quadruplicate in 96-well 
 plates . for receptors with which a compound displayed more than 50% 
 inhibition at 10 m concentration ,
the compound was subjected 
 to secondary radioligand binding assays to determine equilibrium binding 
 affinity at specific targets . in the secondary binding assays ,
compound 9 was tested at 11 concentrations ( 100.1 m ) 
 and in triplicate .
both primary and secondary radioligand binding 
 assays were carried out in a final volume of 125 l per well 
 in the appropriate binding buffer , and the radioligand concentration 
 was at a concentration close to the kd . in a typical assay , 25 l of radioligand was added to each 
 well of a 96-well plate , followed by addition of 25 l binding 
 buffer with or without compound .
the reaction started upon addition 
 of 75 l of fresh membrane protein ( typically 2550 g 
 per well ) , and the reaction was incubated in the dark at room temperature 
 for 90 min .
the reaction was stopped by vacuum filtration onto cold 
 0.3% polyethylenimine ( pei)-soaked 96-well filter mats using a 96-well 
 filtermate harvester , followed by three washes with cold wash buffers . 

scintillation cocktail was then melted onto the microwave - dried filters 
 on a hot plate , and radioactivity was counted in a microbeta counter . 
 for primary binding assay analysis ,
nonspecific binding in the presence 
 of 10 m of an appropriate reference compound was set as 100% 
 inhibition ; total binding in the absence of test compound or reference 
 compound was set as 0% inhibition .
the radioactivity in the presence 
 of test compound was calculated with the equation : % inhibition = 
 ( sample cpm 
nonspecific 
 cpm )  100 , where the radioactivity was measured in counts per 
 minute ( cpm / well ) . for secondary binding results , counts ( cpm / well ) 

were pooled and fitted to a three - parameter logistic function for 
 competition binding in prism to determine ic50 values : y = bottom + ( top  bottom)/1 + 1050 , where y is the 
 total binding in the presence of a corresponding concentration of 
 compound ( x , in this case , concentration of 9 ) , and top and bottom are the total and nonspecific binding 
 in the absence and presence of 10 m reference compound .
the cyp inhibition assay of 9 was performed 
 against the five major liver microsomal cyp enzymes : cyp1a2 , cy2c9 , 
 cyp2c19 , cyp2d6 , and cyp3a4 ( by sai life sciences ) . in the assay , 
 a 25 l aliquot of microsomes diluted in kphos buffer ( 0.4 mg / ml ) 
 was added to individual wells of the reaction plate .
fluvoxamine , 
 sulfaphenazole , quinidine , ticlopidine , and ketoconazole ( positive 
 control inhibitors for cyp1a2 , cyp2c9 , cyp2d6 , cyp2c19 , and cyp3a4 , 
 respectively ) , diluted in buffer ( 25 l ) , were added separately 
 to the respective wells . compound 9 , diluted in dmso 
 to a concentration of 10 m , was directly spiked into microsomal 
 mix ( 2 ) , and 50 l was aliquoted into individual wells . 

an aliquot of 25 l of phenacetin , diclofenac , bufuralol , s - mephenytoin , and midazolam ( 4 ) for cyp1a2 , cyp2c9 , 
 cyp2d6 , cyp2c19 , and cyp3a4 , respectively , was added separately to 
 wells and incubated for 5 min at 37 c .
the reactions were initiated 
 using 25 l of nadph ( 4 ) and further incubated for 5 min 
 for cyp3a4 ( midazolam ) , 10 min for cyp1a2 , cyp2c9 , and cyp2d6 , and 
 20 min for cyp2c19 .
all reactions were terminated using 100 l 
 of ice - cold acetonitrile containing internal standard ( imipramine 
 and glipizide at 1 m ) .
the plates were centrifuged at 4000 
 rpm for 15 min , 100 l aliquots were subjected to lc - m / ms on 
 a shimadzu api 4000 system ( mrm mode ) , and the metabolites were detected . 

the respective peak area ratios ( pa ) of metabolites and internal standard 
 was used to determine % inhibition , where % activity = ( pa ratios 
 in the presence of compound / pa ratios in dmso control )  100 , 
 and % inhibition = 100  % activity . the inhibitory potency of 9 , (
r , r)-12 , 13 , and 20 on the 7-benzyloxy-4-(trifluoromethyl)coumarin ( bfc ) debenzylase 
 activity of human cyp3a4 was evaluated fluorimetrically in a reconstituted 
 system with cytochrome p450 reductase ( cpr ) .
the reaction was carried 
 out at room temperature in 100 mm phosphate buffer , ph 7.4 , containing 
 catalase and superoxide dismutase ( 2 u / ml each ) .
a mixture of 1 m 
 cyp3a4 and 1 m cpr was preincubated for 1 h at room temperature 
 and diluted by 20-fold before measurements .
bfc ( 50 m ) and 
 various concentrations of inhibitors were added 2 min prior to initiation 
 of the reaction with 100 m nadph .
formation of 7-hydroxy-4-trifluoromethylcoumarin 
 ( ex = 430 nm ; em = 500 nm ) was 
 followed in a hitachi f100 fluorimeter .
ic50 values were 
 derived from the [ % activity ] vs [ inhibitor ] plots .
 selective 
 inhibition of neuronal nitric oxide synthase ( nnos ) is an important 
 therapeutic approach to target neurodegenerative disorders . 
 however , 
 the majority of the nnos inhibitors developed are arginine mimetics 
 and , therefore , suffer from poor bioavailability . 
 we designed a novel 
 strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine 
 head with promising structural components from previous inhibitors . 
 in conjunction with extensive structure  activity studies , several 
 highly potent and selective inhibitors of nnos were discovered . 
 x - ray 
 crystallographic analysis reveals that these type ii inhibitors utilize 
 the same hydrophobic pocket to gain strong inhibitory potency ( 13 ) , as well as high isoform selectivity . 
 interestingly , select 
 compounds from this series ( 9 ) showed good permeability 
 and low efflux in a caco-2 assay , suggesting potential oral bioavailability , 
 and exhibited minimal off - target binding to 50 central nervous system 
 receptors . furthermore , even with heme - coordinating groups in the 
 molecule , modifying other pharmacophoric fragments minimized undesirable 
 inhibition of cytochrome p450s from human liver microsomes . 
Introduction
Chemistry
Results and Discussion
Conclusions
Experimental Section