the term neurodenegerative 
 disorder is used to 
 describe diseases characterized by the progressive breakdown of neuronal 
 function and structure . this term encompasses disorders such as alzheimer s , 
 parkinson s , and huntington s diseases , as well as amyotrophic 
 lateral sclerosis ( als ) , among others , although neuronal damage is 
 also associated with stroke and ischemic events , cerebral palsy , and 
 head trauma . although the human and economic cost of neurodegeneration 
 continues to be astronomical , treatment is largely limited to palliative 
 care and prevention of symptom progression . therefore , there is a 
 constant demand for novel and effective approaches to slow or prevent 
 the progression of these diseases . nitric oxide ( no ) is an important second messenger in the 
 human body , and dysregulation of its production is implicated in many 
 pathologies . no is produced by the nitric oxide synthase enzymes , 
 of which there are three isoforms : endothelial nitric oxide synthase 
 ( enos ) , which regulates blood pressure and flow , inducible nitric 
 oxide synthase ( inos ) , involved in immune system activation , and nnos , 
 which is required for normal neuronal signaling . nonetheless , overexpression of nnos in neural tissue and 
 increased levels of no can result in protein nitration and oxidative 
 damage to neurons , especially if peroxynitrite is formed from excess 
 no . indeed , overexpression of nnos or excess no has been 
 implicated in or associated with many neurodegenerative disorders . the inhibition of nnos is , therefore , a viable therapeutic strategy 
 for preventing or treating neuronal damage . each monomer consists 
 of both a reductase domain with fad , fmn , and nadph binding sites , 
 and a heme - containing oxygenase domain , where the substrate ( l - arginine ) and cofactor ( 6r)-5,6,7,8-tetrahydrobiopterin 
 ( h4b ) bind . activated and regulated by calmodulin binding , 
 electron flow proceeds from one monomer s reductase domain 
 to the other s oxygenase domain , catalyzing the oxidation of arginine to citrulline with concomitant 
 production of no . not unexpectedly , 
 most investigated nnos inhibitors are mimetics 
 of arginine and act as competitive inhibitors . one major challenge 
 in designing nnos inhibitors is that enos and inos share high sequence 
 similarity and an identical overall architecture with nnos , especially in their substrate - binding sites . lack of isoform selectivity 
 could have deleterious effects ; inhibition of enos can cause severe 
 hypertension , and inos inhibition could impair immune system activation . previously , in our laboratories , fragment hopping and subsequent structure - based optimization afforded compounds 1 and 2 ( representative nnos inhibitors are shown in figure 1 ) . these compounds are highly potent and selective 
 nnos inhibitors , and compound 1 reverses a hypoxic - ischemic 
 brain damage phenotype in newborn rabbit kits when administered intravenously 
 to the dam . representative nnos inhibitors 
 discussed in this study . although effective , compounds 1 and 2 suffer from several drawbacks . like most arginine mimics , they are 
 very polar and hydrophilic and contain numerous basic moieties and 
 hydrogen - bond donors , as well as many rotatable bonds and a high total 
 polar surface area ( tpsa ) , all properties that hamper both gi absorption 
 and blood  brain barrier permeation . many attempts to improve the bioavailability of these compounds 
 have been made , including alkylation , fluorination , introduction of lipophilic 
 tails , and replacement of the amines ; most of these strategies either diminished potency 
 or selectivity or were synthetically challenging . the chiral scaffolds 
 of 1 and 2 are also difficult ( > 12 steps ) 
 to prepare , making them less desirable than simpler scaffolds , such 
 as 3(26 ) and the astrazeneca 
 candidate 4 ( ar - r17477 ; potencies and selectivities are 
 given in figure 1(27 ) ) from a clinical standpoint . nonetheless , these simplified molecules 
 are not without fault ; their isoform selectivities are lower , 3 suffers from poor caco-2 permeability , and 4 is much less potent in cell - based assays than against isolated enzymes , both likely the result , in part , 
 of the amidine moiety , which will be charged at physiological ph . one avenue that we wished to explore for the generation of simpler 
 nnos inhibitor scaffolds was the replacement of the amidine group 
 of a molecule such as 3 with another arginine isostere . such a group should be stable , weakly basic ( pka between 6 and 8) , and possess as few hydrogen - bond donors 
 as possible . one such moiety is the 2-aminoquinoline group ( pka of 7.3 ) , which 
 is desirable because of both its resemblance to the aminopyridines 
 of 1 and 2 ( in both structure and pka , which is 7.1 for 2-aminopyridine ) and its considerably higher clogp . we were 
 also encouraged by the reported use of dihydroaminoquinolines as nnos 
 inhibitors by jaroch et al . additionally , a recent report 
 indicates that 2-aminoquinoline - based bace-1 inhibitors have high 
 cellular activity and good blood brain barrier permeability 
 in a rat model . to this end , 2-aminoquinoline 
 and the structure of 3 were effectively hybridized  
 to produce compound 5 , a simple compound with good calculated 
 physicochemical properties . there is a hydrophobic 
 pocket at the far end of the substrate access channel of nnos ; contact 
 between an inhibitor and the residues lining this pocket is implicated 
 in high selectivity for nnos over the other two isoforms . preliminary docking studies and crystallography indicated that elongation 
 of the chain between the aminoquinoline system and the distal fluorophenyl 
 ring of 5 , moving the position of the secondary amine , 
 or a combination of both , might provide the right length and orientation 
 to reach this hydrophobic pocket , and a series of analogues investigating 
 chain length ( 69 ) and nitrogen position 
 was , therefore , prepared ( figure 2 ) . additionally , 
 on the basis of computer modeling , we hypothesized that placement 
 of the  tail  of the inhibitor at position 6 of the 
 aminoquinoline system ( instead of position 7 ) could also be effective ; 
 to this end , compounds 1013 were 
 prepared ( figure 2 ) . finally , several literature 
 studies indicate that the use of other halogens 
 and substitution patterns on the noncoordinating aryl ring could be 
 beneficial for enhancing potency and selectivity , so a small series 
 of 7-substituted compounds ( 1416 ) with different halogens and substitution patterns was prepared 
 ( figure 2 ) . all compounds were assayed against 
 purified rat nnos , and select compounds were assayed against enos , 
 inos , and human nnos , and for cellular permeability in a caco-2 model . 6- and 
 7-substituted 2-aminoquinolines were prepared by methods 
 originally reported by manimaran et al . and johnson et al . in the present study , 7-substituted aminoquinolines ( 59 and 1416 ) were prepared by a versatile , 
 late - divergent route that began with the preparation of 3-methylcinnamanilide 
 ( 17 ) by literature procedures . compound 17 was subsequently treated with an excess 
 of aluminum chloride in chlorobenzene to affect cyclization and concomitant 
 cleavage of the c - aryl bond to yield the carbostyril 18 as a mixture of the 7-isomer 18a ( major ) and 5-isomer 18b ( minor ) . the isomers were not separated at this stage but 
 were converted into the 2-chloroquinolines 19a and 19b ; the unwanted 5-isomer 19b was removed by 
 fractional crystallization . pure 19a was converted into 2-acetamidoquinoline 20 by the method of krdi , and free - radical bromination afforded 
 versatile intermediate 21 ( scheme 1 ) . to prepare aminoquinoline analogues 
 with one methylene unit between 
 the quinoline system and the secondary amine ( 5 and 9 , schemes 2 and 3 ) , compound 21 was treated with 3-fluorophenethylamine 
 ( 22 , scheme 2 ) or 3-fluoro-1-phenylpropanamine ( 25 , scheme 3 , prepared by hydrogenation of 3-fluorophenethyl cyanide [ 24 , prepared from 23 ] ) to afford amines 26 and 27 , respectively . deacetylation afforded the final compounds 
 as free - bases , which were readily converted to the water - soluble dihydrochloride 
 salts 5 and 9 . aminoquinolines possessing 
 two methylene units between the quinoline 
 system and secondary amine ( 6 , 7 , 8 , and 1416 , scheme 4 ) were likewise prepared from bromide 21 by homologation with cyanide ion to afford nitrile 28 . this compound was reduced to the polar quinolinyl - ethanamine ( 29 ) using hydrogen and raney nickel ; 29 was used 
 crude in the next step . from this amine indirect  reductive amination , 
 where 29 was treated with 3-fluorobenzaldehyde ( 30 ) under mildly acidic , dehydrating conditions . when the 
 aldehyde was consumed ( as measured by tlc ) , subsequent deacetylation , workup , and acidification afforded 6 . to prepare phenethyl analogues 7 , 14 , 15 , and 16 ( see scheme 4 ) , requisite phenylacetaldehydes 3538 were prepared by dess - martin oxidation of commercially available 
 phenethyl alcohols 3134 , respectively . a  direct  reductive amination 
 using 29 and the desired aldehyde was used to assemble 
 the cores of the final analogues . yields were low because of dialkylation 
 and aldehyde condensation byproducts ; the use of other solvents , dehydrating 
 agents , and reductants failed to alleviate these problems ; the aldehydes 
 may be light - and acid - sensitive as well . for these analogues , the 
 intermediate acetamides were immediately deprotected after isolation 
 ( because of some concerns about their stability ) to yield 7 , 14 , 15 , and 16 and converted 
 into dihydrochloride salts , which could be easily purified by crystallization , 
 trituration , or preparative hplc . finally , the preparation of propyl 
 analogue 8 began with 3-fluorophenylpropionic acid ( 39 ) . reduction to phenylpropanol 40 , followed by swern oxidation , afforded sensitive 
 aldehyde 41 . reductive amination using amine 29 , deacetylation , workup , and acidification afforded 8 , as also shown in scheme 4 . 6-substituted 
 2-aminoquinolines were prepared similarly to those 
 described above , beginning instead with 4-methylcinnamanilide 
 ( 42 , scheme 5 ) . using the cyclization compound 46 was treated with 22 , and the resulting acetamide 
 was deacetylated , isolated , and acidified as before to yield 10 . likewise , 
 as shown in scheme 6 , homologation 
 of 46 with cyanide ion afforded 47 , which 
 was readily reduced to ethanamine 48 . the indirect reductive 
 amination procedure ( using 30 ) afforded 11 after deacetylation , isolation , and acidification . the direct reductive 
 amination employing aldehyde 35 similarly afforded 12 after deacetylation / acidification , while the same reductive 
 amination procedure using 41 instead yielded 13 after deprotection and salt formation . compounds 516 were assayed against 
 purified rat nnos , bovine enos , and murine macrophage inos using the 
 hemoglobin capture assay , as previously described . the apparent ki values and isoform selectivities 
 are summarized in table 1 , and values for compounds 1 , 2 , and 3 are included for comparative 
 purposes ; the ic50 values and selectivities for 4 are given in figure 1 . the compounds 
 were assayed for in 
 vitro inhibition against three purified nos isoforms : rat nnos , bovine 
 enos , and murine inos , using known literature methods ( see experimental section for details ) , and ki values are calculated directly from ic50 values . 
 ic50 values are the average of at least two replicates 
 from nine data points ; all experimental standard error values are 
 less than 14% , and all correlation coefficients are > 0.81 . the lead 7-substituted 2-aminoquinoline , compound 5 , has potent nnos inhibitory activity ( 74 nm ) and high n / i 
 selectivity 
 ( 124-fold ) , yet it is only weakly selective for nnos over enos ( around 
 6-fold ) . the crystal structures of 5 bound to both nnos 
 and enos ( figure 3a and b , respectively ) indicate 
 that the bound conformation of 5 is virtually identical 
 in both isoforms . in both cases , the aminoquinoline acts as an arginine 
 mimic and interacts with the active site glutamate as arginine does 
 ( glu592 in nnos ( 1om4 ) ; glu363 in enos ( 2nse ) ) , while the secondary amine sits between the heme propionates , 
 making h - bonds to both . to simultaneously establish hydrogen bonds 
 between the aminoquinoline and glu592 as well as between the secondary 
 amine and both heme propionates , the rigid quinoline plane must tilt 
 significantly from the heme plane . the fluorophenethyl moiety , as 
 predicted from the short linker length , does not quite reach the hydrophobic 
 pocket ( figure 3 ) consisting of tyr706 , leu337 , 
 met336 , and trp306 in nnos ( tyr477 , leu107 , val106 , and trp76 in enos ) . these contacts are absent in 5 , thus resulting in poor selectivity for nnos over enos ; 
 this observation is similar to that of the crystal structure of 3 . active site structures 
 of lead 5 bound to rat nnos 
 ( a ) and bovine enos ( b ) . the omit fo fc density map for the inhibitor is shown at 
 the 2.5  contour level . major hydrogen bonds are shown as dashed 
 lines . in each panel , the four residues that line a hydrophobic pocket 
 are highlighted by a dot surface representation . while residues in 
 chain a of nnos ( a ) and enos ( b ) are colored green and cyan , respectively , 
 the residue from chain b ( second monomer in the homodimeric structure ) 
 is distinguished by a different color . we sought to improve potency and isoform selectivity by elongating 
 the chain between the aminoquinoline and the noncoordinating aryl 
 ring ( figure 2 ) . to this end , extra methylene 
 groups were inserted between the secondary amine and fluorophenyl 
 group ( 9 ) or between the quinoline and the secondary 
 amine ( 7 , 8) . we reasoned that moving the 
 amine farther from the quinoline could also have the advantage of 
 relaxing the constraints on the quinoline ring orientation but still 
 allow the amine to interact with the heme propionates , thus , in turn , 
 anchoring the tail in a favorable orientation to make hydrophobic 
 contacts . there are two factors that affect the comparative 
 inhibitor potency 
 in this series of aminoquinoline compounds : the linker length and 
 the position of the amine group . contrary to our prediction regarding 
 amine position , the structure of nnos with 6 bound ( figure 4a ) reveals that placing two carbons between the 
 quinoline and the amine actually diminishes the interaction with the 
 heme propionates ( more than 3.6  distance ) , leading to increased 
 flexibility as evidenced by the disordered fluorophenethyl tail in 
 the structure of 6 and decreased potency relative to 5 . superimposition of these two nnos structures ( 5 and 6 , figure 4b ) reveals that 
 the loose interaction between the amine of 6 and the 
 heme propionates ( lacking of h - bonds ) allows the quinoline to assume 
 a more parallel orientation ( relative to the heme ) than observed in 
 the structure of 5 . however , as the 4-atom linker ( of 5 and 6 ) is not long enough to bring the fluorophenyl 
 ring in contact with the aforementioned hydrophobic pocket ( as in 
 figure 3 ) , the majority of the stabilization 
 results from the hydrogen bonds from the aminoquinoline and the linker 
 amine . therefore , 5 , with an extra hydrogen bond , is 
 a stronger inhibitor than 6 . fc density map for the inhibitor is shown at the 2.5  
 contour level . ( b ) overlay of 5 ( cyan ) and 6 ( yellow ) in nnos . the different tilt angles of the aminoquinoline 
 ring relative to the heme plane is in part related to whether hydrogen 
 bonds ( dashed lines ) from the heme propionates to the linker amine 
 are present ( compound 5 ) or absent ( compound 6 ) . in general , compounds with shorter 
 linkers ( 5 and 6 ) have lower nnos inhibitory 
 activity than compounds with 
 longer linkers ( 7 and 9 , table 1 ) . the ideal chain length appears to be five atoms 
 between the quinoline and fluorophenyl groups . nnos inhibitory activity 
 is very similar between 7 and 9 ( table 1 ) with the nitrogen placement not drastically affecting 
 potency ; it appears that the influence of the amine position is weakened 
 in these inhibitors with longer linker lengths . the omit electron 
 density map reveals that 7 ( figure 5a ) , which lacks a strong secondary amine  heme propionate interaction , 
 is more flexible / disordered in the fluorophenyl tail region relative 
 to the structure of 9 ( which does show the amine nonetheless , the similar potencies of 7 and 9 indicate that the nitrogen position is not crucial for these compounds 
 with longer linkers , likely because additional stabilization results 
 from contact with the hydrophobic pocket . indeed , the structure of 9 ( figure 5b ) shows numerous favorable 
 hydrophobic contacts between the fluorophenyl group and the nonpolar 
 residues at the far end of the substrate access channel , tyr706 , leu337 , 
 met336 , and trp306 ( of the other monomer of the nnos homodimer ) . although 
 the tail of 7 ( figure 5a ) is more 
 disordered in structure than that of 9 ( figure 5b ) , these hydrophobic contacts exist with 7 as well . therefore , when the linker is long enough to allow 
 contact between the fluorophenyl ring and the hydrophobic pocket , 
 the strong combined stabilization from both the hydrophobic interactions 
 and the aminoquinoline glu592 interaction may effectively outweigh 
 any lack of interaction between the secondary amine and heme propionates . active 
 site structure of 7 ( a ) or 9 ( b ) 
 bound to nnos . the omit fo  fc density map for the inhibitor is shown at 
 the 2.5  contour level . chain lengths that are longer than the ideal ( e.g. , compound 8 , with six atoms between the quinoline and fluorophenyl group ) 
 result in a drop in potency when compared with 7 or 9 . the crystal structure with 8 ( figure 6a ) shows that the fluorophenyl ring of 8 makes hydrophobic contacts similar to those of 7 and 9 . nonetheless , to make these contacts , the flexible chain 
 has to assume a kinked conformation , in contrast to the fully extended 
 linker conformation seen in 9 ( figure 5b ) . the kinked conformation of 8 may result in 
 unfavorable torsional strain in the linker region upon binding . active site 
 structure of 8 ( a ) or 15 ( b ) 
 bound to nnos . the omit fo  fc density map for the inhibitor is shown at 
 the 2.5  contour level . it is worth noting that 7-substituted 2-aminoquinolines are , 
 as 
 a whole , very poor inos inhibitors ; an earlier report also describes 
 the parent compound , 2-aminoquinoline , as having only weak inos inhibitory 
 activity ( 1.7 m ) . compounds 7 , 9 , and 15 have ki values of 44 m , 32.3 m , and 28.4 m , 
 respectively , and 7 has nearly 900-fold selectivity for 
 nnos over inos , a value which is significantly higher than those of 14 , and is among the highest selectivity 
 reported for nnos over inos for nonpeptidic inhibitors . contact with 
 the substrate - channel hydrophobic pocket ( vide supra ) could be in 
 part responsible for this high n / i selectivity . murine inos contains 
 a polar asparagine residue ( asn115 ) in 
 this pocket ( at the position of leu337 of nnos ) that would strongly 
 disfavor binding by a hydrophobic group . nonetheless , even the short - chain 
 inhibitors ( 5 and 6 ) still possess good n / i selectivity , despite not reaching this distal pocket , indicating 
 that interactions with residues at this end of the binding site are 
 not the full determinant of this poor inos inhibition . it is reported 
 that the heme - binding sites themselves differ between inos and nnos 
 isoforms , with the former possessing 
 a smaller and more rigid active site that may not tolerate the bulky 
 and inflexible aminoquinoline as well . interestingly , the selectivity 
 patterns ( higher n / i selectivity ) contrast with many aminopyridine - based 
 inhibitors , which have higher n / e selectivity . in some cases ( such 
 as the r , r - enantiomer of 1 ) , this high n / e selectivity can be explained by water - mediated contacts 
 made between the center pyrrolidine ring and asp597 , a residue that exists in both nnos and inos but is asn369 
 in enos . this aspartate residue can provide considerable electrostatic 
 or hydrogen - bonding stabilization in nnos versus enos ; this stabilization 
 also manifests itself in the high n / e selectivity of dipeptide - based 
 inhibitors , but no contacts with asp597 are 
 observed in the aminoquinoline crystal structures . in other cases , 
 high n / e selectivity is rationalized by the tighter -stacking 
 with tyr706 of nnos than with the analogous tyr477 of enos , leading 
 to greater nonbonded contacts and better desolvation . while no clear -stacking interactions are visible 
 in the nnos crystal structures of 6 , 7 , 8 , or 9 , hydrophobic contacts and desolvation 
 may still play a substantial role in n / e selectivity for aminoquinolines . the binding mode of the aminoquinoline portion is identical in the 
 structure of 7 bound to nnos ( figure 5a ) or enos ( figure 7 ) and does not 
 contribute to isoform selectivity . however , the length of the linker 
 in 7 enables the fluorophenyl ring to make good hydrophobic 
 contacts with the residues met336 , leu337 , tyr706 , and trp306 ( from 
 the other monomer of the nnos homodimer ) . the bulky and flexible met336 
 side chain makes extensive contacts with the fluorophenyl group of 7 , whereas the analogous residue , val106 in enos , with a smaller 
 surface area , can not make that many contacts . additionally , the side 
 chain of tyr706 in nnos rotates by about 60 in order to make 
 better contacts with the tail of 7 , while in the enos 
 structure ( figure 7 ) , tyr477 remains in its 
 original side chain orientation . overall , these differences are fairly 
 subtle but still contribute to the slightly tighter binding of 7 to nnos over enos . small changes in these hydrophobic contacts 
 could also explain why 7 is more selective than 9 . the omit fo  fc density 
 map for the inhibitor is shown at the 2.5  contour level . major hydrogen bonds are shown as dashed lines . while 7-substituted aminoquinolines 
 ( 59 and 1416 ) are all highly 
 potent against nnos , the analogous 6-substituted aminoquinolines 1013 have low potency , regardless of 
 chain length or nitrogen position . this disparity is also explained 
 by the crystal structures of the bound 2-aminoquinolines . in the heme - binding 
 pocket , the aminoquinoline system does not stack parallel to the heme 
 but rather tilts down slightly toward the back wall  
 of this pocket ( figure 4a and b ) . in cases 
 where an h - bond is formed between the secondary amine and heme propionates , 
 the angle between the planes of the aminoquinoline and heme can be 
 as large as 45 , held in this conformation by the h - bond . even 
 when no hydrogen bond is present , the aminoquinoline still tilts to 
 avoid unfavorable contact with val567 and phe584 , bulky residues that 
 project downward from the roof of this pocket . a large or flexible 
 substituent located at position 6 , in any case , would clash directly 
 with these bulky residues or the heme propionates , or force the rigid 
 aminoquinoline system into a position where it can no longer be accommodated 
 in the heme - binding pocket . this also explains why 11 has low nnos inhibitory activity despite sharing a similar overall 
 structure with 4 ( the same number of atoms and a similar 
 orientation between the arginine - mimic portions and the noncoordinating 
 aryl rings ) . the flexibility ( more rotatable bonds ) of 4 allows it to be accommodated more easily in the heme - binding pocket , 
 whereas 11 is too rigid to fit . it also was reported 
 in the literature that rigid fused 2-aminodihydroquinoline - based nnos 
 inhibitors show a similar sar regarding substituent placement ; large 
 amine - containing tails can be easily placed in the region analogous 
 to the 7-position , whereas the area occupied by the 6-position can 
 only fit small substituents , such as fluorine . interestingly , 
 the replacement of the fluorine in the 3-fluorophenyl 
 group of 7 with a bulkier chlorine ( compound 15 ) does not significantly decrease the nnos inhibitory potency of 15 and is only modestly detrimental to isoform selectivity , 
 which remains 431-fold and 110-fold for inos and enos , respectively . as shown in figure 6b , 15 binds 
 to nnos in a manner very similar to that of 7 ( figure 5a ) . without a strong interaction between the amine 
 and the heme propionates , the chlorophenethyl tail is partially disordered 
 but can still be located based on the partial density contoured at 
 0.5 . in this model , the chlorine atom is not pointing directly 
 into the hydrophobic pocket , so the switch between chlorine and fluorine 
 should not significantly alter contacts with the enzyme . placement 
 of the fluorine ( or chlorine ) at the 4-position , however , is a disfavored 
 modification ( compare 7 to 14 or 15 to 16 ) . this drop in potency could arise from unfavorable 
 steric clashes between the 4-position substituent ( which would face 
 directly toward the back of the hydrophobic pocket ) and any hydrophobic 
 pocket residue , especially met336 and leu337 . encouraged by 
 the high potency and selectivity of 7 and 15 , we assayed these compounds ( and lead 5 ) against purified 
 human nnos ( table 2 ) . the human isoform has 
 an active site that is nearly identical 
 to that in the rat enzyme , with the exception 
 of the hydrophobic pocket , where leu337 is replaced by a histidine 
 ( his341 ) . this pocket is smaller and more polar , and may prefer to 
 bind inhibitors with less bulky and more hydrophilic tails . previously , 
 aminopyridine - based inhibitors showed lower potency against the human 
 enzyme when compared to the rat enzyme , and the same trend is observed for the aminoquinolines , although 5 , 7 , and 15 still display good 
 human nnos inhibition . because of the very similar selectivities ( ki - human / ki - rat ) 
 among these three compounds , it can be concluded that the modifications 
 that are well tolerated by the rat isoform ( chain elongation and replacement 
 of fluorine with chlorine ) are likewise tolerated similarly by human 
 nnos , including the introduction of the bulkier chlorine . ic50 values are the average 
 of at least two replicates from nine data points ; all experimental 
 standard error values are less than 10% , and all correlation coefficients 
 are > 0.90 . finally , compounds 7 and 15 were assayed 
 in a caco-2 monolayer permeability assay ( table 3 ) . this assay is an approximation of both a compound s ability 
 to penetrate the epithelium of the gi tract as well as the blood brain 
 barrier ; ideally , an orally bioavailable nnos inhibitor 
 should show high permeability in this assay . an efflux ratio ( ratio 
 of membrane permeability ( ab ) to efflux ( ba ) ) < 
 3 is considered favorable . pleasingly , both 7 and 15 display good membrane permeability in the apical to basolateral 
 ( ab ) direction and high compound recovery values . compound 15 even shows improved membrane permeation relative to compound 4 , and both 7 and 15 display relatively 
 low efflux ratios , diminishing the possibility that p - gp or other 
 active transport mechanisms are significantly acting on these compounds 
 ( especially on 15 ) . interestingly , compound 15 is more membrane - permeable than 7 despite their nearly 
 identical structures ; this could be the result of the higher clogp 
 of 15 ( 3.8 ) relative to 7 ( 3.2 ) or to variability 
 in the assay . in summary , we have prepared a series of novel simplified 2-aminoquinolines 
 based on the rationale that they might bind to and inhibit nnos in 
 a manner similar to that of aminopyridines , while being less polar , 
 less basic , more lipophilic , and , therefore , more bioavailable . compounds 
 were assayed with purified nos enzymes , and it was revealed that 7-substituted 
 2-aminoquinolines are highly potent inhibitors of nnos and that subtle 
 modifications ( such as increasing the chain length between the aminoquinoline 
 and a noncoordinating aryl ring ) can enhance potency and greatly improve 
 isoform selectivity to > 200-fold over both inos and enos . crystal 
 structures indicate that these compounds act as competitive arginine 
 mimics , where the aminoquinoline moiety makes hydrogen bonds with 
 the active - site glutamate residue , and that the noncoordinating aryl 
 rings are stabilized in a hydrophobic pocket on the far end of the 
 substrate access channel . enhanced hydrophobic contacts with 7 in this pocket in nnos , relative to that of enos , may also , 
 in part , dictate the high isoform selectivity . most promisingly , two 
 of these highly effective compounds , 7 and 15 , show good permeability in a caco-2 assay . these results indicate 
 that these compounds have high potential for oral bioavailability 
 and brain penetration and that the 7-substituted 2-aminoquinoline 
 cores offer very promising leads for further nnos inhibitor development . anhydrous solvents ( thf , ch2cl2 , and dmf ) were 
 distilled prior to use . the 
 remaining solvents , reactants , and reagents were purchased from commercial 
 vendors and were used without further purification , with the exception 
 of acetamide , which was heated to 80 c and dried under vacuum 
 before use . melting points were determined in capillary tubes using 
 a buchi melting point b-540 apparatus and are uncorrected . h nmr spectra were recorded at 500 mhz , using a bruker avance iii 
 500 ( direct cryoprobe ) , and c nmr spectra were obtained 
 at 126 mhz using the same instrument . 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 . flash 
 column chromatography was performed using an agilent 971-fp automated 
 flash purification system , using a varian column station with silicycle 
 cartridges ( 880 g ) , or manually in glass columns using silicycle 
 siliaflash p60 4063 m silica gel . analytical hplc was 
 performed either using a beckman system gold 125 solvent module and 
 166 detector or an agilent infinity 1260 system and an injection volume 
 of 10 l . a phenomenex gemini c18 5 m , 110  reverse - phase 
 column , gemini nx 5 m , 100  column ( both with dimensions 
 of 250 mm  4.6 mm ) , or phenomenex synergi 5 m , polar 
 rp column ( 4.6  50 mm ) was used for all hplc experiments . the 
 purity of all final target compounds was found to be 95% by 
 hplc , using either isocratic elution at 70% meoh in h2o 
 ( with 0.1% tfa ) or a gradient of 6595% meoh in h2o ( with 0.1% tfa ) at 0.8 ml / min . when the polar rp column was used , 
 elution was isocratic at either 50% acetonitrile in h2o 
 or 35% acetonitrile in h2o at 1.5 ml / min . 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  
 21.2 mm ) . analytical thin - layer chromatography was performed on silicycle 
 extra hard 250 m tlc plates . compounds were visualized with 
 short - wavelength uv light , ninhydrin , and kmno4 stain , 
 where relevant . compounds 17 , 35 , 36 , and 42 were prepared by literature procedures , 
 and their spectral data are consistent with those data reported for 
 the same . compound 26 ( 0.187 g , 0.554 
 mmol ) was diluted in meoh ( 8 ml ) , and k2co3 ( 0.077 
 g , 0.554 mmol ) was added . the mixture was heated at 50 c for 
 2 h and then at reflux for an additional 1 h. the mixture was cooled 
 and concentrated , and the residue was diluted in etoac ( 50 ml ) , washed 
 with h2o ( 2  50 ml ) , and dried over anhydrous sodium 
 sulfate . the solution was concentrated , the residue was diluted in 
 methanolic hcl ( 1.4 m , 12 ml ) , and the mixture was heated 
 for 3 h at 50 c , upon which a white crystalline precipitate 
 formed . the mixture was cooled and filtered , and additional product 
 was obtained upon concentration of the filtrate and recrystallization 
 of the residue from meoh . a total of 0.140 g of product ( 69% ) was 
 obtained : mp 283285 c ( dec . ) . h nmr ( 500 
 mhz ; dmso - d6 ) :  14.47 ( s , 1 h ) , 
 9.65 ( br s , 2 h ) , 9.31 ( br m , 1 h ) , 8.39 ( d , j = 
 9.3 hz , 1 h ) , 8.30 ( br s , 1 h ) , 7.99 ( d , j = 8.2 
 hz , 1 h ) , 7.87 ( s , 1 h ) , 7.68 ( d , j = 8.5 hz , 1 h ) , 
 7.40 ( td , j = 7.8 , 6.4 hz , 1 h ) , 7.167.09 
 ( m , 4 h ) , 4.364.35 ( m , 2 h ) , 3.233.22 ( m , 2 h ) , 3.06 
 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; 
 dmso - d6 ) :  ( 163.2 + 161.3 , 1 c ) , 
 154.7 ( 1 c ) , 142.6 ( 1 c ) , ( 140.09 + 140.03 , 1 c ) , 136.5 ( 1 c ) , 135.5 
 ( 1 c ) , ( 130.60 + 130.53 , 1 c ) , 129.1 ( 1 c ) , 126.5 ( 1 c ) , ( 124.82 + 
 124.81 , 1 c ) , 120.9 ( 1 c ) , 118.8 ( 1 c ) , ( 115.53 + 115.36 , 1 c ) , 114.5 
 ( 1 c ) , ( 113.70 + 113.54 , 1 c ) , 49.5 ( 1 c ) , 47.3 ( 1 c ) , 31.0 ( 1 c ) . hrms calcd for c18h18fn3 , 295.1485 ; found , 295.1487 . to a solution of 29 ( 0.062 
 g , 0.266 mmol ) in 5:1 chcl3/meoh ( 6 ml ) was added aldehyde 30 ( 0.033 g , 0.319 mmol ) and anhydrous sodium sulfate ( approximately 
 0.5 g ) . the mixture was stirred rapidly for 90 min , and additional 
 na2so4 ( 0.3 g ) and a catalytic amount 
 of glacial acoh ( approximately 10 l ) were added . after a total 
 of 3 h , extra na2so4 ( 0.3 g ) was added . after 4 h , tlc indicated the consumption of amine 29 , 
 the mixture was filtered to remove the na2so4 , and the filter cake was washed with 10 ml of chcl3 . the mixture was concentrated , the oily residue was diluted in meoh 
 ( 5 ml ) , then nabh4 ( 0.015 g , 0.4 mmol ) was added . 
 after being stirred for 20 min at room temperature , the solution was 
 concentrated , and the residue was partitioned between etoac and h2o ( 20 ml each ) . the layers were separated , and the aqueous 
 layer was extracted with etoac ( 20 ml ) . concentration afforded an oily residue that was purified by flash 
 column chromatography ( sio2 ) , eluting with a gradient of 
 etoac to 10% meoh in etoac to yield the intermediate acetamide ( 0.055 
 g , 75% , confirmed by ms ) , which was immediately dissolved in meoh 
 ( 6 ml ) . k2co3 ( 0.023 g , 0.167 mmol ) was added , 
 and the mixture was heated to vigorous reflux for 1 h 45 min . the 
 mixture was cooled and concentrated , and the residue was partitioned 
 between etoac and 1:1 h2o / sat . the layers were separated , and the aqueous layer was extracted with 
 etoac ( 5 ml ) . the combined organic layers were dried over anhydrous 
 sodium sulfate and concentrated to yield a sticky residue that was 
 diluted with ch2cl2 ( 5 ml ) and filtered to remove 
 particulate matter . methanolic hcl ( 1.4 m , 2 ml ) was added , 
 the mixture was stirred for 10 min , and ether ( 25 ml ) was added slowly 
 until a whitish precipitate formed . this solid was collected and dried 
 to afford the title compound as a cream - colored amorphous solid ( 0.052 
 g , 65% based on 29 ) : mp 278279 c . h nmr ( 500 mhz ; dmso - d6 ) :  
 14.36 ( s , 1 h ) , 9.65 ( s , 2 h ) , 9.20 ( br s , 1 h ) , 8.36 ( d , j = 9.3 hz , 1 h ) , 8.25 ( br s , 1 h ) , 7.91 ( d , j = 8.2 hz , 1 h ) , 7.59 ( s , 1 h ) , 7.51 ( m , j = 5.0 
 hz , 2 h ) , 7.447.39 ( m , 2 h ) , 7.307.26 ( m , 1 h ) , 7.09 
 ( d , j = 9.3 hz , 1 h ) , 4.22 ( s , 2 h ) , 3.22 ( br s , 
 4 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 162.9 + 160.9 , 1 c ) , 154.3 ( 1 c ) , 142.8 ( 1 c ) , 142.4 
 ( 1 c ) , 135.9 ( 1 c ) , ( 134.63 + 134.57 , 1 c ) , ( 130.76 + 130.70 , 1 c ) , 
 129.1 ( 1 c ) , ( 126.24 + 126.22 , 1 c ) , 125.8 ( 1 c ) , 119.8 ( 1 c ) , 117 
 ( 1 c ) , ( 116.96 + 116.79 , 1 c ) , ( 115.90 + 115.73 , 1 c ) , 113.4 ( 1 c ) , 
 49.2 ( 1 c ) , 47.1 ( 1 c ) , 31.6 ( 1 c ) . hrms calcd 
 for c18h18fn3 , 295.1485 ; found , 295.1487 . to a solution of 29 ( 0.74 g , 
 0.321 mmol ) in 7:1 chcl3/meoh ( 8 ml ) , aldehyde 35 ( 0.052 g , 0.375 mmol ) was added , followed by glacial acoh ( 7 l ) 
 and anhydrous mgso4 ( approximately 0.5 g ) . the mixture 
 was stirred at room temperature for 30 min and then cooled to 0 c . sodium triacetoxyborohydride ( 0.079 g , 0.375 mmol ) was added in one 
 portion , and the mixture was slowly warmed to room temperature over 
 45 min , stirred 15 min at room temperature , and diluted with chcl3 ( 30 ml ) . nahco3 ( 10 ml ) , and the aqueous layer was 
 extracted with chcl3 ( 5 ml ) . the solution was concentrated , and the residue was purified 
 by flash column chromatography ( sio2 ) , eluting with a gradient 
 of etoac to 18% meoh in etoac to yield the intermediate acetamide 
 as a sticky syrup ( 0.030 g , 25% ) , which was immediately dissolved 
 in meoh ( 4 ml ) . k2co3 ( 0.023 g , 0.163 mmol ) 
 was added , and the mixture was heated to vigorous reflux for 2 h. 
 the mixture was cooled and concentrated , and the residue was partitioned 
 between etoac and 3:2 h2o / sat . the 
 layers were separated , and the aqueous layer was extracted with etoac 
 ( 2 ml ) . nacl ( 4 
 ml ) , dried over anhydrous sodium sulfate , concentrated to yield a 
 sticky residue that was diluted with ch2cl2 ( 4 
 ml ) , and filtered to remove particulate matter . ( 1.4 
 m , 2 ml ) was added , the mixture was stirred for 10 min , ether ( 20 
 ml ) was added slowly , and the mixture was sonicated until a whitish 
 precipitate formed . this solid was collected and dried to afford the 
 title compound as a hygroscopic , cream - colored amorphous solid ( 0.023 
 g , 18% based on 29 ) : mp 247249 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) :  
 14.34 ( s , 1 h ) , 9.21 ( br s , 3 h ) , 8.37 ( d , j = 9.3 
 hz , 1 h ) , 8.27 ( br s , 1 h ) , 7.92 ( d , j = 8.2 hz , 
 1 h ) , 7.60 ( s , 1 h ) , 7.437.38 ( m , 2 h ) , 7.187.08 ( m , 
 4 h ) , 3.253.16 ( m , 6 h ) , 3.02 ( t , j = 8.1 
 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 163.7 + 161.8 , 1 c ) , 154.8 ( 1 c ) , 143.3 ( 1 c ) , 
 142.9 ( 1 c ) , ( 140.55 + 140.49 , 1 c ) , 136.4 ( 1 c ) , ( 131.07 + 131.00 , 
 1 c ) , 129.6 ( 1 c ) , 126.3 ( 1 c ) , ( 125.35 + 125.33 , 1 c ) , 120.2 ( 1 c ) , 
 117.5 ( 1 c ) , ( 116.03 + 115.86 1 c ) , ( 114.19 + 114.02 , 1 c ) , 113.86 
 ( 1 c ) , 47.7 ( 1 c ) , 32.1 ( 1 c ) , 31.6 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.064 g , 0.280 mmol ) was diluted in 7:1 chcl3/meoh ( 7 
 ml ) , and aldehyde 41 ( 0.049 g , 0.322 mmol ) was added , 
 followed by glacial acetic acid ( 6 l ) and anhydrous mgso4 ( 0.5 g ) . the mixture was stirred for 30 min , cooled 
 to 0 c , and sodium triacetoxyborohydride ( 0.070 g , 0.333 
 mmol ) was added in one portion . the mixture was allowed to warm to 
 room temperature slowly over 50 min and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 8 ml ) , and the 
 aqueous layer was extracted with chcl3 ( 5 ml ) . the organic 
 phase was washed with sat . nacl ( 10 ml ) , dried over anhydrous 
 sodium sulfate , and concentrated . the resulting residue was purified 
 by flash column chromatography ( sio2 ) eluting with a gradient 
 of etoac to 17% meoh in etoac to yield the intermediate acetamide 
 ( 0.045 g , 44% ) as a sticky semisolid . this substance was immediately 
 diluted with anhydrous meoh ( 6 ml ) , and k2co3 ( 0.034 g , 0.246 mmol ) was added . the mixture was heated at reflux 
 for 1 h 50 min , cooled , and concentrated . the residue was diluted 
 with etoac ( 10 ml ) , and the solution was washed with h2o / sat . nacl ( 5 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting syrup was diluted in ch2cl2 ( 3 
 ml ) and filtered to remove particulate matter , and methanolic hcl 
 was added ( 1.4 m , 3 ml ) and the mixture stirred at room temperature 
 for 10 min . ether ( 30 ml ) was added , the mixture was sonicated , concentrated , 
 and the residue was washed twice with ether ( 2 ml each ) to afford 
 the product as a cream - colored hygroscopic solid ( 0.042 g , 37% from 29 ) : mp 8183 c ( softens ) , 210 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) :  
 14.31 ( s , 1 h ) , 9.219.13 ( m , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.25 ( br s , 1 h ) , 7.91 ( d , j = 
 8.2 hz , 1 h ) , 7.59 ( s , 1 h ) , 7.42 ( dd , j = 8.2 , 1.3 
 hz , 1 h ) , 7.387.34 ( m , 1 h ) , 7.127.03 ( m , 4 h ) , 3.213.14 
 ( m , 4 h ) , 2.952.90 ( m , 2 h ) , 2.71 ( t , j = 
 7.6 hz , 2 h ) , 1.96 ( dt , j = 15.3 , 7.7 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  
 ( 163.2 + 161.3 , 1 c ) , 154.3 ( 1 c ) , ( 143.71 + 143.65 , 1 c ) , 142.8 ( 1 
 c ) , 142.5 ( 1 c ) , 135.8 ( 1 c ) , ( 130.32 + 130.25 , 1 c ) , 129.1 ( 1 c ) , 
 125.8 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 119.7 ( 1 c ) , 117.0 ( 1 c ) , ( 115.09 
 + 114.92 , 1 c ) , 113.4 ( 1 c ) , ( 112.95 + 112.79 , 1 c ) , 47.1 ( 1 c ) , 46.1 
 ( 1 c ) , 31.73 ( 1 c ) , 31.54 ( 1 c ) , 26.8 ( 1 c ) . anhydrous cs2co3 ( 0.105 g , 0.322 mmol ) was diluted with anhydrous dmf 
 ( 3 ml ) , 25 ( 0.050 g , 0.322 mmol ) was added as a solution 
 in dmf ( 2 ml ) , and the mixture was stirred for 30 min at room 
 temperature . compound 21 ( 0.075 g , 0.269 mmol ) was then 
 added as a solution in dmf ( 1.3 ml ) over several minutes . the 
 residue was partitioned between etoac and h2o ( 10 ml each ) , 
 the layers were separated , and the aqueous layer was saturated with 
 nacl and extracted with etoac ( 2  5 ml ) . nacl ( 10 ml ) , dried over anhydrous 
 sodium sulfate , and concentrated . the residue was purified by flash 
 column chromatography ( sio2 ) , eluting with a gradient of 
 etoac to 15% meoh in etoac to yield 27 as a yellow syrup 
 ( 0.039 g , 41% ) , which was used without further characterization . this 
 compound was diluted with anhydrous meoh ( 6 ml ) , and anhydrous k2co3 ( 0.031 g , 0.022 mmol ) was added . the mixture 
 was heated at reflux for 2 h 15 min , cooled , and concentrated . the 
 residue was diluted with etoac ( 10 ml ) , and 3 ml each of h2o and sat . the layers were separated , the aqueous 
 layer was extracted with etoac ( 3  4 ml ) , and the combined organic 
 layers were washed with sat . nacl ( 4 ml ) , dried over anhydrous 
 sodium sulfate , and concentrated . the residue was diluted with ch2cl2 ( 5 ml ) , filtered to remove particulate matter , 
 and reconcentrated . methanolic hcl ( 1.4 m , 3 ml ) was added , 
 the mixture was stirred for 5 min , and ether ( 30 ml ) was added slowly 
 until a white precipitate formed . this solid was collected and dried 
 to afford the title compound as a white microcrystalline solid ( 0.029 
 g , 28% based on 21 ) after drying in vacuo : mp 250252 
 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) :  14.44 ( s , 1 h ) , 9.50 ( s , 2 h ) , 9.30 ( br s , 
 1 h ) , 8.39 ( d , j = 9.2 hz , 1 h ) , 8.30 ( br s , 1 h ) , 
 7.98 ( d , j = 8.2 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.66 ( d , j = 8.3 hz , 1 h ) , 7.35 ( td , j = 8.0 , 6.4 
 hz , 1 h ) , 7.15 ( d , j = 9.3 hz , 1 h ) , 7.107.02 
 ( m , 3 h ) , 4.32 ( t , j = 5.5 hz , 2 h ) , 2.952.90 
 ( m , 2 h ) , 2.70 ( t , j = 7.6 hz , 2 h ) , 2.00 ( quintet , j = 7.7 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 163.2 + 161.3 , 1 c ) , 154.6 ( 1 c ) , 
 ( 143.67 + 143.61 , 1 c ) , 142.6 ( 1 c ) , 136.6 ( 1 c ) , ( 130.32 + 130.25 , 
 1 c ) , 129.0 ( 1 c ) , 126.4 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 120.9 ( 1 c ) , 
 118.7 ( 1 c ) , ( 115.08 + 114.91 , 1 c ) , 114.5 ( 1 c ) , ( 112.95 + 112.78 , 
 1 c ) , 49.3 ( 1 c ) , 46.0 ( 1 c ) , 31.5 ( 1 c ) , 26.7 ( 1 c ) ; one of the aminoquinoline 
 carbons is not visible due to baseline broadening ; esims m / z ( rel . anhydrous cs2co3 ( 0.090 
 g , 0.288 mmol ) was diluted in anhydrous dmf ( 5 ml ) , and amine 22 ( 0.040 g , 0.288 mmol ) was added . the mixture was stirred 
 for 30 min at room temperature before compound 46 ( 0.070 
 g , 0.250 mmol ) was added dropwise as a solution in anhydrous dmf ( 2 
 ml ) . the resultant suspension was stirred for 16 h at room temperature 
 and concentrated , the residue was partitioned between etoac and h2o ( 5 ml each ) , and the layers were separated . the aqueous 
 layer was extracted with etoac ( 2  5 ml ) , and the organic layers 
 were washed with sat . nacl ( 5 ml ) , dried over anhydrous sodium 
 sulfate , and concentrated . the residue was purified by flash column 
 chromatography ( sio2 ) , eluting with a gradient of etoac 
 to 10% meoh in etoac to yield the intermediate acetamide as a yellow 
 syrup ( 0.040 g , 47% , confirmed by ms ) . this syrup was dissolved in 
 meoh ( 5 ml ) , and k2co3 ( 0.026 g , 0.148 mmol ) 
 was added . the mixture was heated at reflux for 2 h , cooled to room 
 temperature , and concentrated . the layers 
 were separated , and the aqueous layer was extracted with etoac ( 2 
  5 ml ) . the combined organic phase was washed with sat aq . nacl 
 ( 4 ml ) , dried over anhydrous sodium sulfate , and concentrated . the 
 resulting residue was diluted in ch2cl2 ( 5 ml ) 
 and filtered to remove particulate matter , and methanolic hcl ( 1.4 
 m , 3 ml ) was added . after 10 min , ether ( 20 ml ) was added , and a precipitate 
 formed . this was collected and dried to yield the title compound as 
 a cream - colored powder ( 0.036 g , 38% from 46 : mp 277278 
 c ) . h nmr ( 500 mhz ; dmso - d6 ) :  14.35 ( s , 1 h ) , 9.58 ( s , 2 h ) , 9.28 ( br s , 1 h ) , 
 8.37 ( d , j = 9.5 hz , 1 h ) , 8.32 ( br s , 1 h ) , 8.05 
 ( s , 1 h ) , 7.96 ( d , j = 8.6 hz , 1 h ) , 7.78 ( d , j = 8.5 hz , 1 h ) , 7.39 ( td , j = 7.8 , 6.4 
 hz , 1 h ) , 7.167.09 ( m , 4 h ) , 4.29 ( s , 2 h ) , 3.203.19 
 ( m , 2 h ) , 3.05 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 163.2 
 + 161.2 , 1 c ) , 154.4 ( 1 c ) , 142.8 ( 1 c ) , ( 140.11 + 140.05 , 1 c ) , 135.9 
 ( 1 c ) , 134.2 ( 1 c ) , 130.59 ( 1 c ) , ( 130.58 + 130.52 , 1 c ) , 128.6 ( 1 
 c ) , ( 124.83 + 124.81 , 1 c ) , 120.5 ( 1 c ) , 117.6 ( 1 c ) , ( 115.53 + 115.36 , 
 1 c ) 114.5 ( 1 c ) , ( 113.69 + 113.52 , 1 c ) , 49.2 ( 1 c ) , 47.0 ( 1 c ) , 
 31.0 ( 1 c ) . amine 48 ( 0.050 g , 0.218 mmol ) 
 was dissolved in anhydrous chcl3 ( 3 ml ) , and aldehyde 30 ( 0.034 g , 0.274 mmol ) was added , followed by 3 ml of a 
 2:1 mixture of chcl3/meoh and anhydrous sodium sulfate 
 ( 0.5 g ) . the mixture was stirred rapidly at room temperature 
 for 90 min , after which glacial acetic acid ( 10 l ) was added . 
 after a total of 4 h , the amine appeared to be consumed by tlc , and 
 the mixture was filtered to remove the sodium sulfate . the filtrate 
 was concentrated , and the oily residue was diluted in meoh ( 5 ml ) . nabh4 ( 0.020 g , 0.523 mmol ) was added , and the mixture 
 was stirred for 40 min at room temperature . the mixture was concentrated 
 and partitioned between etoac and h2o ( 10 ml of each ) . the layers were separated , and the aqueous layer was extracted with 
 etoac ( 10 ml ) . the organic layer was washed with h2o and 
 sat aq . nacl ( 20 ml each ) , dried over anhydrous sodium sulfate , and 
 concentrated . the residue was purified by flash column chromatography 
 ( sio2 ) , eluting with a gradient of etoac to 20% meoh in 
 etoac to yield the intermediate acetamide as a yellow syrup ( 0.052 
 g , 72% , confirmed by ms ) . this compound was immediately diluted in 
 meoh ( 5 ml ) , and k2co3 ( 0.021 g , 0.154 mmol ) 
 was added . the mixture was heated at vigorous reflux for 2 h , cooled , 
 and concentrated , and the resulting residue was diluted with etoac 
 ( 20 ml ) and washed with h2o ( 10 ml ) . the aqueous layer 
 was extracted with etoac ( 2  10 ml ) , and the combined organic 
 layers were washed with sat . concentration afforded a white solid , which was diluted 
 with methanolic hcl ( 1.4 m , 3 ml ) and stirred for 10 min . the addition of ether ( 50 ml ) resulted in the precipitation of a solid 
 that was collected , washed with ether ( 20 ml ) and dried in vacuo to 
 yield the title compound as a white solid ( 0.043 g , 54% from 48 ) : mp 282284 c . h nmr ( 500 mhz ; 
 dmso - d6 ) :  14.24 ( s , 1 h ) , 9.62 
 ( s , 2 h ) , 9.16 ( br s , 1 h ) , 8.35 ( d , j = 9.4 hz , 
 1 h ) , 8.19 ( br s , 1 h ) , 7.80 ( d , j = 0.7 hz , 1 h ) , 
 7.717.67 ( m , 2 h ) , 7.527.48 ( m , 2 h ) , 7.42 ( d , j = 7.7 hz , 1 h ) , 7.307.26 ( m , 1 h ) , 7.12 ( d , j = 9.3 hz , 1 h ) , 4.22 ( s , 2 h ) , 3.173.14 ( m , 4 
 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 162.9 + 160.9 , 1 c ) , 154.0 ( 1 c ) , 142.8 ( 1 c ) , ( 134.62 
 + 134.56 , 1 c ) , 134.0 ( 1 c ) , 133.3 ( 1 c ) , ( 130.76 + 130.70 , 1 c ) , 
 128.3 ( 1 c ) , ( 126.24 + 126.22 , 1 c ) , 121.0 ( 1 c ) , 117.7 ( 1 c ) , ( 116.96 
 + 116.79 , 1 c ) , ( 115.90 + 115.74 , 1 c ) , 114.0 ( 1 c ) , 49.2 ( 1 c ) , 47.2 
 ( 1 c ) , 30.8 ( 1 c ) ; one of the aminoquinoline carbons is not visible 
 because of baseline broadening ; esims m / z ( rel . hrms calcd for c18h18fn3 , 295.1485 ; found , 295.1486 . to a solution of 48 ( 0.074 
 g , 0.321 mmol ) in 7:1 chcl3/meoh ( 8 ml ) , aldehyde 35 ( 0.049 g , 0.353 mmol ) was added , followed by glacial acoh 
 ( 7 l ) and anhydrous mgso4 ( approx 0.5 g ) . the mixture 
 was stirred at room temperature for 20 min and then cooled to 0 c . sodium triacetoxyborohydride ( 0.082 g , 0.385 mmol ) was added in one 
 portion , and the mixture was slowly warmed to room temperature over 
 50 min , then diluted with ch2cl2 ( 10 ml ) . nahco3 ( 2  20 ml ) , and the aqueous layer was extracted with 
 ch2cl2 ( 2  10 ml ) . the solution was concentrated , and the residue was 
 purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of etoac to 20% meoh in etoac to yield the intermediate 
 acetamide as a sticky syrup ( 0.039 g , 34% ) that was immediately dissolved 
 in meoh ( 3 ml ) . k2co3 ( 0.023 g , 0.167 mmol ) 
 was added , and the mixture was heated to vigorous reflux for 1 h 50 
 min . the mixture was cooled and concentrated , and the residue was 
 partitioned between etoac ( 10 ml ) and 1:1 h2o / sat . the layers were separated , and the aqueous layer was 
 extracted with etoac ( 2  4 ml ) . nacl ( 4 ml ) , dried over anhydrous sodium 
 sulfate , and concentrated to yield a sticky residue that was diluted 
 with ch2cl2 ( 3 ml ) and filtered to remove particulate 
 matter . methanolic hcl ( 1.4 m , 2 ml ) was added , the mixture 
 was stirred for 10 min and concentrated , and the residue was recrystallized 
 from 1:1 meoh / ether ( 1 ml ) to yield the product as a pale tan hygroscopic 
 solid ( 0.025 g , 21% based on 48 ) : mp 223226 c 
 ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) :  14.37 ( s , 1 h ) , 9.359.23 ( m , 3 h ) , 8.36 ( d , j = 9.4 hz , 1 h ) , 8.30 ( br s , 1 h ) , 7.83 ( s , 1 h ) , 7.737.69 
 ( m , 2 h ) , 7.427.38 ( m , 1 h ) , 7.187.09 ( m , 4 h ) , 3.223.19 
 ( m , 4 h ) , 3.14 ( t , j = 7.9 hz , 2 h ) , 3.04 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 163.2 + 161.3 , 1 c ) , 154.0 ( 1 c ) , 
 142.8 ( 1 c ) , ( 140.14 + 140.08 , 1 c ) , 134.6 ( 1 c ) , 134.1 ( 1 c ) , 133.3 
 ( 1 c ) , ( 130.58 + 130.51 , 1 c ) , 128.3 ( 1 c ) , ( 124.86 + 124.84 , 1 c ) , 
 120.9 ( 1 c ) , 117.5 ( 1 c ) , ( 115.54 + 115.37 , 1 c ) , 114.0 ( 1 c ) , ( 113.68 
 + 113.52 , 1 c ) , 47.35 ( 1 c ) , 47.20 ( 1 c ) , 31.0 ( 1 c ) , 30.8 ( 1 c ) . 
 esims m / z ( rel . hrms calcd for c19h20fn3 , 309.1641 ; found , 309.1647 . to a solution of 48 ( 0.060 g , 0.261 mmol ) in 10:1 chcl3/meoh ( 5 
 ml ) , aldehyde 41 ( 0.047 g , 0.313 mmol ) was added , followed 
 by glacial acoh ( 6 l ) and anhydrous mgso4 ( approx 
 0.5 g ) . the mixture was stirred at room temperature for 25 min and 
 then cooled to 0 c . sodium triacetoxyborohydride ( 0.070 g , 0.332 
 mmol ) was added in one portion , and the mixture was slowly warmed 
 to room temperature over 30 min , filtered , and concentrated . the residue 
 was purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of etoac to 20% meoh in etoac to yield a sticky yellow 
 solid ( 0.036 g , 27% ) . this substance was immediately dissolved in 
 meoh ( 3 ml ) , k2co3 ( 0.030 g , 0.217 mmol ) was 
 added , and the mixture was heated to vigorous reflux for 2 h. the 
 mixture was cooled and concentrated , and the residue was partitioned 
 between etoac ( 6 ml ) and 1:1 h2o / sat . the layers were separated , and the aqueous layer was extracted with 
 etoac ( 2  3 ml ) . nacl ( 3 ml ) , dried over anhydrous sodium sulfate , and concentrated 
 to yield a sticky residue that was diluted with ch2cl2 ( 3 ml ) and filtered to remove particulate matter . methanolic 
 hcl ( 1.4 m , 2 ml ) was added , the mixture was stirred for 10 
 min , concentrated , and the residue was washed with 1:1 ch2cl2/ether ( 3 ml ) to yield the product as a yellow - green 
 hygroscopic solid ( 0.033 g , 32% based on 48 ) : mp 70 c 
 ( softens ) , 211213 c ( dec ) . h nmr ( 500 mhz ; 
 dmso - d6 ) :  14.31 ( s , 1 h ) , 9.20 
 ( br s , 3 h ) , 8.34 ( d , j = 9.4 hz , 1 h ) , 8.22 ( br 
 s , 1 h ) , 7.82 ( s , 1 h ) , 7.70 ( s , 2 h ) , 7.387.33 ( m , 1 h ) , 
 7.147.03 ( m , 4 h ) , 3.233.17 ( m , 2 h ) , 3.11 ( t , j = 7.8 hz , 2 h ) , 2.942.89 ( m , 2 h ) , 2.71 ( t , j = 7.6 hz , 2 h ) , 1.97 ( quintet , j = 7.6 
 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 163.2 + 161.3 , 1 c ) , 154.0 ( 1 c ) , ( 143.72 + 143.66 , 
 1 c ) , 142.8 ( 1 c ) , 134.6 ( 1 c ) , 134.1 ( 1 c ) , 133.3 ( 1 c ) , ( 130.31 
 + 130.25 , 1 c ) , 128.2 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 120.9 ( 1 c ) , 
 117.5 ( 1 c ) , ( 115.09 + 114.93 , 1 c ) , 114.0 , ( 112.95 + 112.78 , 1 c ) , 
 47.3 ( 1 c ) , 46.1 ( 1 c ) , 31.5 ( 1 c ) , 30.9 ( 1 c ) , 26.8 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.076 g , 0.333 
 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 36 ( 0.045 g , 0.327 mmol ) was added as a solution in 1 ml of 
 chcl3 , followed by glacial acetic acid ( 7 l ) and 
 anhydrous mgso4 ( 0.5 g ) . the flask was sheathed 
 with aluminum foil , and the mixture was stirred for 45 min and cooled 
 to 0 c , and sodium triacetoxyborohydride ( 0.085 g , 0.401 mmol ) 
 was added in one portion . the mixture was allowed to warm to room 
 temperature slowly over 1 h and 15 min and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 10 ml ) , and the 
 aqueous layer was extracted with chcl3 ( 2  5 ml ) . nacl ( 20 ml ) , dried over 
 anhydrous sodium sulfate , and concentrated . the resulting residue 
 was purified by flash column chromatography ( sio2 ) eluting 
 with a gradient of etoac to 14% meoh in etoac to yield the intermediate 
 acetamide ( 0.051 g , 44% ) as an oil that began to solidify on standing . this substance was immediately diluted with anhydrous meoh ( 8 ml ) , 
 and k2co3 ( 0.030 g , 0.217 mmol ) was added . the 
 mixture was heated at reflux for 2 h , cooled , and concentrated . the 
 residue was diluted with etoac ( 10 ml ) , and the solution was washed 
 with h2o / sat . the aqueous layer was 
 extracted with etoac ( 3  6 ml ) , and the combined organic layers 
 were washed with sat . nacl ( 6 ml ) , dried over anhydrous sodium 
 sulfate , and concentrated . the resulting syrup was diluted in ch2cl2 ( 5 ml ) , filtered to remove particulate matter , 
 and reconcentrated . to the residue was added methanolic hcl ( 1.4 
 m , 1 ml ) , and the mixture was stirred at room temperature for 1 h , 
 upon which a white crystalline solid formed . the mixture was cooled 
 to 30 c and filtered to yield the title compound as 
 white flocculent crystals ( 0.021 g , 16% from 29 ) : mp 
 279281 c . h nmr ( 500 mhz ; dmso - d6 ) :  14.34 ( s , 1 h ) , 9.20 ( s , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.268.24 ( br s , 1 h ) , 7.92 ( d , j = 8.2 hz , 1 h ) , 7.60 ( s , 1 h ) , 7.42 ( dd , j = 8.2 , 1.2 hz , 1 h ) , 7.33 ( td , j = 6.1 , 2.5 hz , 
 2 h ) , 7.217.17 ( m , 2 h ) , 7.09 ( d , j = 9.3 
 hz , 1 h ) , 3.263.16 ( m , 6 h ) , 2.98 ( t , j = 
 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  ( 162.1 + 160.2 1 c ) , 154.3 ( 1 c ) , 142.8 ( 1 
 c ) , 142.4 ( 1c ) , 135.9 ( 1 c ) , ( 133.34 + 133.32 , 1 c ) , ( 130.59 + 130.53 , 
 1 c ) , 129.1 ( 1 c ) , 125.8 ( 1 c ) , 119.7 ( 1 c ) , 117.0 ( 1 c ) , ( 115.45 
 + 115.28 , 1 c ) , 113.4 ( 1 c ) , 47.7 ( 1 c ) , 47.2 ( 1 c ) , 31.7 ( 1 c ) , 30.7 
 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.076 g , 0.333 
 mmol ) was diluted in 8:1 chcl3/meoh ( 7 ml ) , and aldehyde 37 ( 0.051 g , 0.330 mmol ) was added as a solution in 1 ml of 
 chcl3 , followed by glacial acetic acid ( 7 l ) and 
 anhydrous mgso4 ( 0.5 g ) . the mixture was stirred 
 for 30 min , and cooled to 0 c , and sodium triacetoxyborohydride 
 ( 0.085 g , 0.401 mmol ) was added in one portion . the mixture was allowed 
 to warm to room temperature slowly over 1 h and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 10 ml ) , and the 
 aqueous layer was extracted with chcl3 ( 10 ml ) . nacl ( 20 ml ) , dried over anhydrous 
 sodium sulfate , and concentrated . the resulting residue was purified 
 by flash column chromatography ( sio2 ) eluting with a gradient 
 of etoac to 13% meoh in etoac to yield the intermediate acetamide 
 ( 0.039 g , 32% ) as a semisolid . this substance was diluted with anhydrous 
 meoh ( 6 ml ) , and k2co3 ( 0.029 g , 0.210 mmol ) 
 was added . the mixture was heated at reflux for 2 h , cooled , and concentrated . the residue was immediately diluted with etoac ( 10 ml ) , and the solution 
 was washed with h2o / sat . nacl ( 5 ml ) , dried over anhydrous 
 sodium sulfate , and concentrated . the resulting syrup was diluted 
 in ch2cl2 ( 5 ml ) , filtered to remove particulate 
 matter , and reconcentrated . to the residue was added methanolic hcl 
 ( 1.4 m , 3 ml ) , the mixture was stirred at room temperature 
 for 5 min , and ether ( 20 ml ) was added , upon which an off - white solid 
 ( 0.030 g , 23% ) was collected . an analytically pure sample for assay was prepared by preparative lc - ms , using the instrument and column 
 detailed in the general procedures section , 
 eluting with a gradient of 95% h2o + 0.1%/formic acid 5% 
 mecn + 0.1% formic acid for 2 min , to 70% h2o at 27 min , 
 then to 0% h2o at 32 min . evaporation and retreatment of 
 the residue with methanolic hcl ( 1 ml ) and ether ( 1 ml ) afforded the 
 pure compound as a white flocculent solid ( 0.014 g , 11% from 29 ) : mp 281282 c . h nmr ( 500 mhz ; 
 dmso - d6 ) :  14.29 ( s , 1 h ) , 9.17 
 ( br s , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.25 ( br 
 s , 1 h ) , 7.92 ( d , j = 8.2 hz , 1 h ) , 7.60 ( s , 1 h ) , 
 7.447.34 ( m , 4 h ) , 7.27 ( d , j = 7.4 hz , 1 
 h ) , 7.09 ( d , j = 9.3 hz , 1 h ) , 3.253.20 ( m , 
 4 h ) , 3.203.16 ( m , 2 h ) , 3.00 ( t , j = 8.1 
 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  154.3 ( 1 c ) , 142.8 ( 1 c ) , 142.5 ( 1 c ) , 139.8 ( 1 
 c ) , 135.8 ( 1 c ) , 133.2 ( 1 c ) , 130.5 ( 1 c ) , 129.1 ( 1 c ) , 128.6 ( 1 c ) , 
 127.5 ( 1 c ) , 126.8 ( 1 c ) , 125.8 ( 1 c ) , 119.7 ( 1 c ) , 116.9 ( 1 c ) , 113.4 
 ( 1 c ) , 47.2 ( 1 c ) , 31.6 ( 1 c ) , 31.0 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.076 g , 0.333 
 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 38 ( 0.051 g , 0.330 mmol ) was added as a solution in 1 ml of 
 chcl3 , followed by glacial acetic acid ( 7 l ) and 
 anhydrous mgso4 ( 0.5 g ) . the mixture was stirred 
 for 30 min and cooled to 0 c , and sodium triacetoxyborohydride 
 ( 0.085 g , 0.401 mmol ) was added in one portion . the mixture was allowed 
 to warm to room temperature slowly over 1 h 15 min and was diluted 
 with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 10 
 ml ) , and the aqueous layer was extracted with chcl3 ( 2 
  5 ml ) . the organic phase was washed with sat . nacl ( 20 
 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting 
 residue was purified by flash column chromatography ( sio2 ) eluting with a gradient of etoac to 13% meoh in etoac ) to yield 
 the intermediate acetamide ( 0.032 g , 26% ) as a white semisolid . this 
 substance was immediately diluted with anhydrous meoh ( 7 ml ) , and 
 k2co3 ( 0.024 g , 0.174 mmol ) was added . the mixture 
 was heated at reflux for 2 h , cooled , and concentrated . the residue 
 was diluted with etoac ( 10 ml ) , and the solution was washed with h2o / sat . nacl ( 5 ml ) , dried over anhydrous sodium sulfate , and 
 concentrated . the resulting syrup was diluted in ch2cl2 ( 5 ml ) , filtered to remove particulate matter , and reconcentrated . 
 to the residue was added methanolic hcl ( 1.4 m , 3 ml ) , the 
 mixture was stirred at room temperature for 5 min , and ether ( 20 ml ) 
 was added , upon which an off - white solid ( 0.027 g , 20% ) was collected . an analytically pure sample for assay was prepared by preparative 
 lc - ms , using the instrument and column detailed in the general procedures section , eluting with a gradient 
 of 95% h2o + 0.1% formic acid/5% mecn + 0.1% formic acid 
 for 5 min , to 93% h2o in 30 min , then to 0% h2o at 32 min . evaporation and retreatment of the residue with methanolic 
 hcl ( 1 ml ) and ether ( 1 ml ) afforded the pure compound as a white 
 flocculent solid ( 0.0094 g , 7.4% from 29 ) : mp 288290 
 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) :  14.20 ( s , 1 h ) , 9.07 ( s , 3 h ) , 8.36 ( dd , j = 9.2 , 0.5 hz , 1 h ) , 8.22 ( br s , 1 h ) , 7.91 ( d , j = 8.1 hz , 1 h ) , 7.58 ( s , 1 h ) , 7.447.41 ( m , 3 
 h ) , 7.32 ( d , j = 8.4 hz , 2 h ) , 7.07 ( d , j = 9.2 hz , 1 h ) , 3.283.14 ( m , 6 h ) , 2.97 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) :  154.4 ( 1 c ) , 142.8 ( 1 c ) , 142.4 ( 1 c ) , 136.2 
 ( 1 c ) , 131.4 ( 1 c ) , 130.6 ( 1 c ) , 129.1 ( 1 c ) , 128.6 ( 1 c ) , 125.8 ( 1 
 c ) , 119.8 ( 1 c ) , 117.0 ( 1 c ) , 113.4 ( 1 c ) , 47.40 ( 1 c ) , 47.22 ( 1 c ) , 
 31.7 ( 1 c ) , 30.8 ( 1 c ) ; one of the aminoquinoline carbons is not visible 
 due to line - broadening . compound 17 ( 4.26 
 g , 18.0 mmol ) was diluted in chlorobenzene ( 45 ml ) , and anhydrous 
 alcl3 ( 12.0 g , 5.00 mmol ) was added . the mixture was heated 
 to 90 c for 2 h , upon which the solution became black . h2o ( 300 ml ) , which 
 was extracted with etoac ( 2  300 ml ) . the organic phase was 
 washed with h2o ( 200 ml ) , and the aqueous layer was extracted 
 with etoac ( 100 ml ) . the 
 orange solution was filtered through celite and concentrated to afford 
 the mixture of products as a beige solid ( 2.53 g , 88% ) after washing 
 with hexanes and drying . h nmr spectra indicated that 18a and 18b were present as a 70:30 mixture ( consistent 
 with prior reports ) , which was used without any 
 further purification . a mixture 
 of 18a and 18b ( 2.53 g , 15.9 mmol ) was diluted 
 in pocl3 ( 35 ml ) , and the mixture was heated at 
 reflux for 70 min , before the clear orange solution was cooled to 
 room temperature and poured into ice the beaker was immersed in ice and cooled to 0 
 c with stirring , and solid naoh was added until the ph of the 
 mixture was approximately 7 . the resultant cloudy suspension was extracted 
 with etoac ( 300 ml ) , and the organic layers were washed with h2o ( 100 ml ) and sat . the organic layer was 
 dried over anhydrous sodium sulfate and concentrated , and the residue 
 was purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of hexanes to 12% ethyl acetate in hexanes to afford 
 orange crystals . fractional crystallization from hot isopropanol yielded 
 pure 19a ( 0.850 g , 30% ) as light orange iridescent crystals ; 
 the analytical data for this compound are identical to those in prior 
 literature reports.h nmr ( 500 mhz ; cdcl3 ) :  8.05 ( d , j = 8.5 hz , 1 h ) , 8.00 
 ( m , 1 h ) , 7.71 ( d , j = 8.3 hz , 1 h ) , 7.39 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.32 ( d , j = 8.5 
 hz , 1 h ) , 2.56 ( s , 3 h ) . chloride 19a ( 0.300 g , 1.69 mmol ) was diluted 
 with molten anhydrous 
 acetamide ( 8 g , 135 mmol ) , and k2co3 ( 1.17 g , 
 8.45 mmol ) was added . the mixture was heated in a sand bath , at reflux 
 ( 230 c ) for 17 h. the mixture was cooled , poured into 
 h2o ( 120 ml ) , and extracted with etoac ( 4  30 ml ) . the organic layers were washed with h2o ( 3  100 ml ) 
 and sat . nacl ( 50 ml ) , dried over anhydrous sodium sulfate , and 
 concentrated . purification of the residue by flash column chromatography 
 ( sio2 , 15% etoac in ch2cl2 ) afforded 
 the desired compound as a white solid ( 0.265 g , 78% ) . h nmr chemical shifts for this compound are consistent with those 
 reported by inglis et al . for the 7-isomer.h nmr ( 500 mhz ; cdcl3 ) :  9.899.88 
 ( br s , 1 h ) , 8.40 ( d , j = 8.9 hz , 1 h ) , 8.15 ( d , j = 9.0 hz , 1 h ) , 7.67 ( d , j = 8.3 hz , 
 1 h ) , 7.58 ( d , j = 0.6 hz , 1 h ) , 7.29 ( dd , j = 8.3 , 1.4 hz , 1 h ) , 2.54 ( s , 3 h ) , 2.27 ( s , 3 h ) . compound 20 ( 0.265 g , 1.32 
 mmol ) was diluted in anhydrous 
 benzene ( 10 ml ) . n - bromosuccinimide ( 0.247 g , 1.39 
 mmol ) and a catalytic amount ( 0.020 g ) of benzoyl peroxide 
 were added , and the mixture was heated to reflux under argon until 
 an orange tint was no longer visible in the solution refluxing in 
 the condenser ( typically 4 h ) . the mixture was cooled , concentrated , 
 and purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of 7% to 14% etoac in ch2cl2 , to yield the product ( 0.236 g , 64% ) as a flocculent yellow solid . h nmr chemical shifts for this compound are consistent with 
 those reported by inglis et al . for the 7-isomer.h nmr ( 500 mhz ; cdcl3 ) :  
 8.438.41 ( m , 2 h ) , 8.16 ( d , j = 8.9 hz , 1 
 h ) , 7.797.77 ( m , 2 h ) , 7.49 ( dd , j = 8.4 , 
 1.7 hz , 1 h ) , 4.65 ( s , 2 h ) , 2.27 ( s , 3 h ) . 3-fluorophenethyl 
 bromide ( 23 , 1.00 g , 12.3 mmol ) was diluted in dry dmf 
 ( 25 ml ) . sodium cyanide ( 1.06 g , 61.6 mmol ) was added in one portion , 
 and the mixture was heated to 60 c under argon for 16 h. the 
 mixture was cooled and concentrated , and the residue was partitioned 
 between etoac and h2o ( 50 ml each ) . the layers were separated , 
 and the aqueous phase was extracted with etoac ( 2  20 ml ) . nacl ( 50 
 ml each ) , dried over anhydrous sodium sulfate , and concentrated . the 
 resulting oil was purified by flash column chromatography ( sio2 ) , eluting with a gradient of 5% etoac in hexanes to 30% etoac 
 in hexanes to yield the desired product as a colorless oil ( 0.638 
 g , 87% ) . h nmr ( 500 mhz ; cdcl3 ) :  7.31 
 ( td , j = 7.9 , 6.0 hz , 1 h ) , 7.036.93 ( m , 
 3 h ) , 2.96 ( t , j = 7.4 hz , 2 h ) , 2.63 ( t , j = 7.4 hz , 2 h ) . compound 24 ( 0.180 g , 1.21 mmol ) 
 was diluted in etoh 
 ( 3 ml ) , and methanolic ammonia ( 7 m , 6 ml ) and raney nickel ( 1 
 g ) were added . the mixture was filtered through 
 a pall 0.2 m syringe filter and concentrated to yield a sticky 
 green syrup ( 0.083 g , 45% ) . the presence of amine was confirmed by h nmr spectrometry , tlc , and ninhydrin staining , and this 
 material was used crude without any further purification . anhydrous cs2co3 ( 0.295 
 g , 0.906 mmol ) was diluted in anhydrous dmf ( 10 ml ) . 3-fluorophenethylamine 
 ( 22 , 0.126 g , 0.906 mmol ) was added , and the mixture 
 was stirred at room temperature for 30 min before a solution of 21 ( 0.220 g , 0.788 mmol ) in dmf ( 4 ml ) was added slowly over 
 5 min . the cloudy yellow mixture was stirred at room temperature for 
 16 h and concentrated . the residue was diluted with etoac ( 50 ml ) 
 and washed with h2o ( 2  50 ml ) and sat . the organic phase was dried over anhydrous sodium sulfate , 
 concentrated , and purified by flash column chromatography ( sio2 ) eluting with 10% meoh in etoac to yield the product as a 
 clear yellow oil ( 0.187 g , 70% ) . h nmr ( 500 mhz ; cdcl3 ) :  8.95 ( s , 1 h ) , 8.40 ( br d , j = 
 8.2 hz , 1 h ) , 8.15 ( d , j = 8.9 hz , 1 h ) , 7.74 ( d , j = 8.3 hz , 1 h ) , 7.72 ( s , 1 h ) , 7.41 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.267.22 ( m , 1 h ) , 6.98 ( d , j = 7.7 hz , 1 h ) , 6.936.88 ( m , 2 h ) , 4.00 ( s , 2 
 h ) , 2.95 ( t , j = 7.0 hz , 2 h ) , 2.85 ( t , j = 7.0 hz , 2 h ) , 2.23 ( s , 3 h ) . c nmr ( 126 mhz ; cdcl3 ) :  169.3 ( 1 c ) , ( 163.9 + 162.0 , 1 c ) , 151.3 ( 1 c ) , 
 146.5 ( 1 c ) , ( 142.47 + 142.41 , 1 c ) , 142.37 ( 1 c ) , 138.5 ( 1c ) , ( 129.95 
 + 129.88 , 1 c ) , 127.8 ( 1 c ) , 125.76 ( 1 c ) , 125.71 ( 1 c ) , 125.4 ( 1 
 c ) , ( 124.42 + 124.40 , 1 c ) , ( 115.63 + 115.46 , 1 c ) , 114.0 ( 1 c ) , ( 113.23 
 + 113.06 , 1 c ) , 53.7 ( 1 c ) , 50.1 ( 1 c ) , 36.1 ( 1 c ) , 24.9 ( 1 c ) . esims m / z ( rel . compound 21 ( 0.216 g , 0.773 mmol ) was diluted with anhydrous 
 dmf ( 10 ml ) , and nacn ( 0.190 g , 3.87 mmol ) was added . the orange mixture 
 was stirred at room temperature for 17 h. the mixture was concentrated 
 and partitioned between etoac and h2o ( 50 ml each ) , and 
 the layers were separated . the aqueous phase was extracted with etoac 
 ( 2  50 ml ) , and the combined organic layers were washed with 
 h2o ( 2  80 ml ) and sat . the residue was purified 
 by flash column chromatography ( sio2 ) , eluting with a gradient 
 of 15% etoac in ch2cl2 to 25% etoac in ch2cl2 to yield the title compound as a white solid 
 ( 0.109 g , 63% ) : mp 180182 c . h nmr ( 500 
 mhz ; cdcl3 ) :  8.44 ( dd , j = 8.3 , 
 0.4 hz , 1 h ) , 8.278.22 ( m , 1 h ) , 8.18 ( d , j = 9.0 hz , 1 h ) , 7.81 ( d , j = 8.4 hz , 1 h ) , 7.79 
 ( s , 1 h ) , 7.40 ( dd , j = 8.3 , 1.7 hz , 1 h ) , 3.94 ( s , 
 2 h ) , 2.28 ( s , 3 h ) . c nmr ( 126 mhz ; cdcl3 ) :  169.2 ( 1 c ) , 151.5 ( 1 c ) , 138.8 ( 1 c ) , 132.1 ( 1 c ) , 128.7 
 ( 1 c ) , 126.2 ( 1 c ) , 125.6 ( 1 c ) , 124.9 ( 1 c ) , 117.3 ( 1 c ) , 114.7 ( 1 
 c ) , 25.1 ( 1 c ) , 24.0 ( 1 c ) . esims m / z ( rel . compound 28 ( 0.060 g , 0.266 
 mmol ) was diluted in absolute 
 etoh ( 7 ml ) , and methanolic ammonia ( 7 n , 7 ml ) was added . raney nickel 
 ( 1.5 g , washed with h2o and meoh ) was added , and 
 the mixture was degassed and hydrogenated with a h2-filled 
 balloon at room temperature for 30 min while stirring rapidly . the 
 clear solution was decanted from the nickel and was filtered through 
 a pall 0.2 m syringe filter to remove fine particulate matter . the solution was concentrated and dried in vacuo to yield an off - white 
 semisolid ( 0.062 g , 100% ) . conversion to this amine was confirmed 
 by tlc and ninhydrin staining , and it was used without any further 
 characterization or purification . dess - martin 
 periodinane ( 1.02 g , 2.4 mmol ) was 
 diluted in anhydrous ch2cl2 ( 25 ml ) under argon , 
 and when solution was affected , 3-chlorophenethyl alcohol ( 33 , 0.313 g , 2.00 mmol ) was added dropwise . the mixture was stirred 
 for 2 h and 15 min at room temperature and was then quenched by the 
 addition of 20 ml of sat . after stirring at room temperature for 15 min , the layers were 
 separated , and the aqueous layer was extracted with ch2cl2 ( 2  50 ml ) . the organic layer was washed with 
 h2o and sat . nacl ( 50 ml each ) and was dried over anhydrous 
 sodium sulfate and concentrated . the resulting semisolid residue was 
 triturated with 10% etoac in hexanes , and the solid was filtered out 
 and discarded . the filtrate was concentrated , and the oily residue 
 was purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of hexanes to 10% etoac in hexanes to afford the title 
 compound as a clear yellow volatile oil ( 0.241 g , 78% ) . h nmr ( 500 mhz ; cdcl3 ) :  9.75 ( t , j = 2.1 hz , 1 h ) , 7.317.23 ( m , 3 h ) , 7.117.09 ( m , 
 1 h ) , 3.69 ( d , j = 2.1 hz , 2 h ) . dess - martin 
 periodinane ( 1.02 g , 2.4 mmol ) was 
 diluted in anhydrous ch2cl2 ( 25 ml ) under argon , 
 and when the solution was affected , 4-chlorophenethyl alcohol ( 33 , 0.313 g , 2 mmol ) was added dropwise . the mixture was stirred 
 for 2 h and 15 min at room temperature and was then quenched by the 
 addition of 20 ml of sat . after stirring at room temperature for 15 min , the layers were 
 separated , and the aqueous layer was extracted with ch2cl2 ( 2  50 ml ) . the organic layer was washed with 
 h2o and sat . nacl ( 50 ml each ) and was dried over anhydrous 
 sodium sulfate and concentrated . the resulting semisolid residue was 
 triturated with 10% etoac in hexanes , and the solid was filtered and 
 discarded . the filtrate was concentrated , and the oily residue was 
 purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of hexanes to 15% etoac in hexanes to afford the title 
 compound as a clear yellow volatile oil ( 0.211 g , 88% ) . h nmr ( 500 mhz ; cdcl3 ) :  9.75 ( t , j = 2.1 hz , 1 h ) , 7.34 ( d , j = 8.3 hz , 2 h ) , 7.15 
 ( d , j = 8.2 hz , 2 h ) , 3.69 ( d , j = 2.0 hz , 2 h ) . 3-fluorophenylpropionic acid 
 ( 39 , 
 0.500 g , 2.97 mmol ) was diluted in anhydrous thf ( 2 ml ) under argon 
 and cooled to 0 c . borane - thf ( 1 m , 4.16 ml , 4.16 mmol ) was 
 added dropwise , and the mixture was allowed to warm to room temperature 
 and stirred for 18 h. the reaction was quenched by the addition of 
 1:1 thf / h2o ( 5 ml ) . when gas evolution ceased , solid k2co3 was added until the mixture separated into 
 two layers , which were separated . the aqueous layer was extracted 
 with etoac ( 2  5 ml ) , and the combined organic layers were washed 
 with h2o ( 15 ml ) and sat . nacl ( 15 ml ) , dried over 
 anhydrous sodium sulfate , and concentrated to yield the product as 
 a clear oil ( 0.446 g , 97% ) after drying in vacuo . h nmr 
 ( 500 mhz ; cdcl3 ) :  7.297.25 ( m , 1 h ) , 7.00 
 ( d , j = 7.6 hz , 1 h ) , 6.956.89 ( m , 2 h ) , 
 3.71 ( t , j = 6.4 hz , 2 h ) , 2.74 ( t , j = 7.7 hz , 2 h ) , 1.951.89 ( m , 2 h ) , 1.39 ( s , 1 h ) . anhydrous 
 ch2cl2 ( 10 ml ) 
 was cooled to 78 c , and anhydrous dmso ( 0.546 g , 7.00 
 mmol ) was added , followed , dropwise , by oxalyl chloride ( 0.380 g , 
 3.00 mmol ) . once gas evolution ceased , compound 40 ( 0.308 
 g , 2 mmol ) was added dropwise , and the resulting milky solution was 
 stirred for 15 min . et3n ( 1.17 ml , 8.4 mmol ) was added 
 slowly , and the mixture was stirred for 15 min at 78 c 
 and then warmed to room temperature and stirred for 1 h. the yellow 
 mixture was diluted with h2o ( 30 ml ) , and the layers were 
 separated . the aqueous layer was extracted with ch2cl2 ( 2  15 ml ) , and the organic layers were washed with 
 h2o and sat . the solution was dried 
 over anhydrous sodium sulfate , concentrated , and the resulting residue 
 was purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of hexanes to 10% etoac in hexanes to yield the title 
 aldehyde as a colorless volatile oil ( 0.220 g , 72% ) . h 
 nmr ( 500 mhz ; cdcl3 ) :  9.82 ( s , 1 h ) , 7.277.23 
 ( m , 1 h ) , 6.97 ( d , j = 7.6 hz , 1 h ) , 6.926.89 
 ( m , 2 h ) , 2.96 ( t , j = 7.5 hz , 2 h ) , 2.812.78 
 ( m , 2 h ) . compound 43 ( 3.75 g , 15.8 mmol ) was diluted 
 in chlorobenzene ( 40 ml ) , 
 and aluminum chloride ( 10.5 g , 75.0 mmol ) was added . the mixture was 
 heated to 90 c under nitrogen for 2 h , upon which the mixture 
 became black , was subsequently cooled , and poured into ice the resulting suspension was extracted with etoac 
 ( 700 ml ) , and the organic layer was washed with h2o ( 300 
 ml ) and dried over anhydrous sodium sulfate . concentration afforded 
 an orange solid that was recrystallized from hot meoh ( 60 ml ) to yield 
 an orange iridescent solid ( 1.95 g , 77% ) . this was not characterized 
 but was instead diluted in pocl3 ( 30 ml ) and heated at 
 reflux for 70 min before cooling and pouring into ice the beaker was immersed in a 
 cooler of ice , with stirring , and solid naoh was added until the ph 
 was approximately 7 . the oily suspension was extracted with etoac 
 ( 400 ml ) , washed with sat . nacl ( 300 ml ) , and the organic layer 
 was dried over anhydrous sodium sulfate . the solution was concentrated 
 to yield a solid that was purified by flash column chromatography 
 ( sio2 ) , eluting with a gradient of hexanes to 40% etoac 
 in hexanes to yield the product as an orange crystalline solid ( 1.79 
 g , 64% from 42 ) . the h nmr chemical shifts 
 for this compound are identical to those previously reported by inglis 
 et al.h nmr ( 500 mhz ; cdcl3 ) :  8.01 ( d , j = 8.6 hz , 1 h ) , 7.91 
 ( d , j = 9.2 hz , 1 h ) , 7.577.55 ( m , 2 h ) , 
 7.34 ( d , j = 8.6 hz , 1 h ) , 2.53 ( s , 3 h ) . chloride 44 ( 0.300 g , 1.69 
 mmol ) was diluted with molten anhydrous 
 acetamide ( 8 g , 135 mmol ) , and k2co3 ( 1.17 g , 
 8.45 mmol ) was added . the mixture was heated in a sand bath , at reflux 
 ( 230 c ) for 16 h. the mixture was cooled , poured into 
 h2o ( 120 ml ) , and extracted with etoac ( 4  30 ml ) . the organic layers were washed with h2o ( 3  100 ml ) 
 and sat . purification of the residue by flash column chromatography 
 ( sio2 ) , eluting with a gradient of 10% etoac in ch2cl2 to 30% etoac in ch2cl2 , afforded the desired compound as a white solid ( 0.250 g , 74% ) . h nmr chemical shifts for this compound are consistent with 
 those reported by inglis et al.h nmr ( 500 mhz ; cdcl3 ) :  8.36 ( br d , j = 8.6 hz , 1 h ) , 8.27 ( br s , 1 h ) , 8.09 ( d , j = 8.9 hz , 1 h ) , 7.70 ( d , j = 8.6 hz , 1 h ) , 7.55 
 ( s , 1 h ) , 7.50 ( dd , j = 8.6 , 1.9 hz , 1 h ) , 2.51 ( s , 
 3 h ) , 2.24 ( s , 3 h ) . compound 45 ( 0.300 g , 1.50 mmol ) 
 was diluted in anhydrous 
 benzene ( 10 ml ) . n - bromosuccinimide ( 0.280 g , 1.57 
 mmol ) and a catalytic amount ( 0.020 g ) of benzoyl peroxide 
 were added , and the mixture was heated to reflux under nitrogen until 
 an orange tint was no longer visible in the solution refluxing in 
 the condenser ( around 2 h 40 min ) . the mixture was cooled , concentrated , 
 and purified by flash column chromatography ( sio2 ) , eluting 
 with a gradient of 10% to 12% etoac in ch2cl2 to yield the product ( 0.262 g , 63% ) as a flocculent yellow solid . the h nmr chemical shifts for this compound are identical 
 to those previously reported by inglis et al.h nmr ( 500 mhz ; cdcl3 ) :  8.44 ( br 
 d , j = 8.6 hz , 1 h ) , 8.30 ( br s , 1 h ) , 8.17 ( d , j = 9.0 hz , 1 h ) , 7.82 ( m , j = 9.1 hz , 
 2 h ) , 7.72 ( dd , j = 8.6 , 2.1 hz , 1 h ) , 4.67 ( s , 2 
 h ) , 2.29 ( s , 3 h ) . compound 46 ( 0.254 g , 0.91 mmol ) 
 was diluted with anhydrous 
 dmf ( 10 ml ) , and nacn ( 0.230 g , 4.55 mmol ) was added . the orange mixture 
 was stirred at room temperature for 17 h. the mixture was concentrated 
 and partitioned between etoac and h2o ( 50 ml each ) , and 
 the layers were separated . the aqueous phase was extracted with etoac 
 ( 2  50 ml ) , and the combined organic layers were washed with 
 h2o ( 2  80 ml ) and sat . the residue was purified 
 by flash column chromatography ( sio2 ) , eluting with a gradient 
 of 15% etoac in ch2cl2 to 25% etoac in ch2cl2 to yield the title compound as a white solid 
 ( 0.170 g , 83% ) : mp 154155 c . h nmr ( 500 
 mhz ; cdcl3 ) :  8.48 ( d , j = 8.6 
 hz , 1 h ) , 8.22 ( br s , 1 h ) , 8.20 ( d , j = 9.0 hz , 
 1 h ) , 7.85 ( d , j = 8.7 hz , 1 h ) , 7.81 ( d , j = 1.0 hz , 1 h ) , 7.61 ( dd , j = 8.7 , 2.1 
 hz , 1 h ) , 3.96 ( s , 2 h ) , 2.30 ( s , 3 h ) . c nmr ( 126 mhz ; 
 cdcl3 ) :  169.1 ( 1 c ) , 151.3 ( 1 c ) , 145.8 ( 1 c ) , 
 138.6 ( 1 c ) , 129.8 ( 1 c ) , 128.3 ( 1 c ) , 126.74 ( 1 c ) , 126.64 ( 1 c ) , 
 126.2 ( 1 c ) , 117.6 ( 1 c ) , 114.9 ( 1 c ) , 25.0 ( 1 c ) , 23.7 ( 1 c ) . esims m / z ( rel . compound 47 ( 0.060 g , 0.266 mmol ) was diluted in absolute 
 etoh ( 7 ml ) , and methanolic ammonia ( 7 n , 7 ml ) was added . raney nickel 
 ( 1.5 g , washed with h2o and meoh ) was added , and 
 the mixture was degassed and hydrogenated with a balloon at room temperature 
 for 30 min while stirring rapidly . the clear solution was decanted 
 away from the nickel and was filtered through a pall 0.2 m 
 syringe filter to remove fine particulate matter . the solution was 
 concentrated and dried in vacuo to yield a colorless gum that became 
 a white semisolid upon standing ( 0.050 g , 82% ) . conversion to this 
 amine was confirmed by h nmr spectrometry , tlc , ms , and 
 ninhydrin staining , and it was used crude without any further characterization 
 or purification . rat and 
 human nnos , murine 
 macrophage inos , and bovine enos were recombinant enzymes , expressed 
 in e. coli and purified as previously reported . to test for 
 enzyme inhibition , the assay was performed at 37 c in 
 hepes buffer ( 100 mm , with 10% glycerol , ph 7.4 ) in the presence of 
 10 m l - arginine . also included were 100 m nadph , 
 0.83 mm cacl2 , approximately 320 units / ml of calmodulin , 
 10 m tetrahydrobiopterin , and human oxyhemoglobin ( 3 m ) . 
 for inos , cacl2 and calmodulin were omitted and replaced 
 with hepes buffer ( as neither are required for activation of inos ) . this assay was performed in 96-well plates using a synergy 4 biotek 
 hybrid reader , and the dispensing of nos enzyme and hemoglobin were 
 automated ; after 30 s ( maximum delay ) , no production was read by monitoring 
 the absorbance at 401 nm ( resulting from the conversion of oxyhemoglobin 
 to methemoglobin ) . each compound was assayed at least in duplicate , and nine concentrations 
 ( 500 m50 nm or 100 m10 nm for enos and 
 inos ; 50 m to 5 nm for nnos ) were used to construct dose ic50 values were calculated by nonlinear regression 
 using graphpad prism software ( standard error values reported are 
 from the logic50 calculations ) , and ki values were obtained using the cheng prusoff equation 
 [ ki = ic50/(1 + [ s]/km ) ] using the following km values : 1.3 ( rat nnos ) , 1.6 ( human nnos ) , 
 8.2 ( murine macrophage inos ) , and 1.7 m ( bovine enos ) . the nnos or enos 
 heme domain proteins used for crystallographic studies were produced 
 by limited trypsin digest from the corresponding full length enzymes 
 and further purified through a superdex 200 gel filtration column 
 ( ge healthcare ) as described previously . the nnos heme domain ( at 9 mg / ml containing 20 mm histidine ) or 
 the enos heme domain ( at 12 mg / ml containing 2 mm imidazole ) was used 
 for the sitting drop vapor diffusion crystallization setup under conditions 
 previously reported . fresh crystals ( 12 days 
 old ) were first passed stepwise through cryoprotectant solutions and 
 then soaked with a 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 lightsource 
 ( 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 , 120130 of fine - sliced 
 data were collected with 0.2 per frame . raw ccd data frames 
 were indexed , integrated , and scaled using hkl2000 , but the pixel array data were processed with xds and scaled with scala . the binding of inhibitors was detected by the initial difference 
 fourier maps calculated with refmac . disordering in portions of inhibitors 
 bound in the nos active sites was often observed , sometimes resulting 
 in poor density quality . however , partial structural features usually 
 could still be visible if the contour level of the sigmaa weighted 
 2m|fo|  d|fc| map dropped to 0.5  , 
 which afforded the building of reasonable models into the disordered 
 regions . the tls protocol was implemented in the 
 final stage of refinements with each subunit as one tls group . fc density maps were calculated by repeating the last round 
 of tls refinement with the inhibitor coordinate removed from the input 
 pdb file to generate the map coefficients delfwt and phdelwt . the 
 refined structures were validated in coot before deposition 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 . ( watertown , ma ) using the following standard 
 procedure : caco-2 cells , grown in tissue culture flasks , were trypsinized , 
 resuspended , and grown and differentiated in 96-well plates for three 
 weeks ; monolayer formation was determined by measuring transport of 
 lucifer yellow , an impermeable dye . all assays were performed at a 
 concentration of 10 m for 2 h. for apical to basolateral ( ab ) 
 permeability , compounds were added on the apical side ( a ) , with permeation 
 determined at the receiving ( basolateral , b ) side , 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 . 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 tranport buffer , ph 7.4 ( basolateral 
 side ) . apparent permeability ( papp ) is 
 expressed using the following equation : papp = ( dq / dt)/c0a , where the numerator 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 values ( > 3 ) 
 indicate that a compound may be a substrate for p - gp or other active 
 transport systems .
since high levels of nitric oxide 
 ( no ) are implicated in neurodegenerative 
 disorders , inhibition of the neuronal isoform of nitric oxide synthase 
 ( nnos ) and reduction of no levels are therapeutically desirable . 
 nonetheless , 
 many nnos inhibitors mimic l - arginine and are poorly bioavailable . 
 2-aminoquinoline - based scaffolds were designed with the hope that 
 they could ( a ) mimic aminopyridines as potent , isoform - selective arginine 
 isosteres and ( b ) possess chemical properties more conducive to oral 
 bioavailability and cns penetration . 
 a series of these compounds was 
 synthesized and assayed against purified nnos enzymes , endothelial 
 nos ( enos ) , and inducible nos ( inos ) . 
 several compounds built on a 
 7-substituted 2-aminoquinoline core are potent and isoform - selective ; 
 x - ray crystallography indicates that aminoquinolines exert inhibitory 
 effects by mimicking substrate interactions with the conserved active 
 site glutamate residue . 
 the most potent and selective compounds , 7 and 15 , were tested in a caco-2 assay and showed 
 good permeability and low efflux , suggesting high potential for oral 
 bioavailability .