the centers for disease 
 control and prevention 
 ( cdc ) has declared vaccination and the control of infectious diseases 
 to be among the greatest public health achievements of the 20th century . vaccines afford protection by the induction of 
 immune responses , both humoral and cellular , specifically directed 
 against the pathogen . a significant trend in contemporary vaccinology 
 is the design of highly effective subunit vaccines , and the majority 
 of modern subunit vaccines that utilize highly purified , recombinantly 
 expressed protein immunogens are reliant on vaccine adjuvants ( substances that enhance immune responses ) to provide the initial , 
 innate immune - activating signals that determine the specificity , magnitude , 
 quality , and durability of downstream adaptive immune responses . with few exceptions , the majority of currently available vaccines 
 contain a single adjuvant :  alum  introduced by alexander 
 glenny in 1926 . alum  ( a 
 mixture of aluminum phosphate and aluminum hydroxide ) appears to promote 
 a t helper 2 ( th2 ) skewed antibody response . indeed , 
 alum - adjuvanted pertussis subunit vaccines , which supplanted killed whole - cell pertussis vaccines in the 1990s , 
 induce immunity that rapidly wanes ; the short - lived immunity 
 is thought to contribute to the recent re - emergence of pertussis in 
 the united states and elsewhere in the world . in experimental models of pertussis , alum - adjuvanted acellular pertussis 
 vaccines protected baboons in the short term from severe pertussis - like 
 symptoms but failed to prevent colonization of b. pertussis , allowing transmission of the pathogen to unvaccinated animals ; killed whole - cell pertussis vaccines , on the 
 other hand , elicited strong b. pertussis - specific 
 th17 and th1 memory , indicating that 
 both durability and quality of immune responses are pivotal in the 
 induction and maintenance of long - term sterilizing immunity . innate immune signals evoked by vaccine adjuvants include those originating 
 from toll - like receptors ( tlrs ) , as well as rig - i - like 
 receptors and nod - like receptors ( nlrs ) . there are 10 functional tlrs encoded in the human genome , which 
 are transmembrane proteins with an extracellular domain having leucine - rich 
 repeats ( lrr ) and a cytosolic domain called the toll / il-1 receptor 
 ( tir ) domain . the ligands for these receptors 
 are highly conserved molecules such as lipopolysaccharides ( lps ) ( recognized 
 by tlr4 ) , lipopeptides ( tlr2 in combination with tlr1 or tlr6 ) , flagellin 
 ( tlr5 ) , single stranded rna ( tlr7 and tlr8 ) , double stranded rna ( tlr3 ) , 
 cpg motif - containing dna ( recognized by tlr9 ) , and profilin present 
 on uropathogenic bacteria ( tlr11 ) . tlr1 , 
 -2 , -4 , -5 , and -6 recognize extracellular stimuli , while tlr3 , -7 , 
 -8 , and -9 function within the endolysosomal compartment . our 
 understanding of how the engagement of innate immune receptors by 
 vaccine adjuvants leads to the deployment and amplification of immunogen - specific 
 adaptive immune responses , and the maintenance of immunological memory is incomplete and may 
 involve multiple mechanisms and pathways ; these may include ( i ) enhanced 
 antigen uptake and presentation by professional antigen presenting 
 cells ( apcs ) , ( ii ) amplification of cross - talk between naive b lymphocytes 
 recognizing the immunogen and rare naive cd4 t cells expressing 
 t cell antigen receptors ( tcrs ) specific for antigen - derived peptide / major 
 histocompatibility complex class ii molecules ( mhcii ) displayed by 
 such naive b cells , ( iii ) accelerated differentiation of cd4 t cells into follicular helper t cells ( tfh ) , and ( iv ) subsequent b lymphocyte 
 differentiation events leading to immunologulin affinity maturation and the generation of antigen - specific memory b cells and plasma 
 cells . the need for the development of safe and effective 
 vaccine adjuvants has fueled our exploration of a variety of innate 
 immune stimuli , which include agonists of tlr2 , tlr7 , tlr8 , nucleotide oligomerization domain 1 ( nod1 ) , as well as c  c chemokine receptor type 1 ( ccr1 ) . activity relationship studies 
 have proven useful in providing tools with which to examine how these 
 different classes of innate immune signaling molecules affect and 
 modulate pathways linking the innate and adaptive immune systems described 
 above . tlr8 is expressed predominantly in myeloid dendritic 
 cells , monocytes , and monocyte - derived dendritic cells . engagement by tlr8 agonists evokes a dominant proinflammatory cytokine 
 profile , including tumor necrosis factor  ( tnf- ) , interleukin 
 ( il)-12 , and il-18 and appear uniquely potent in enhancing the production 
 of th1-polarizing cytokines tnf- and il-12 in apcs . our interest in small molecule agonists of tlr8 has 
 led to the exploration of the 2,3-diaminofuro[2,3-c]pyridines , 4-aminofuro[2,3-c]quinolines , 3-alkylquinoline-2-amines , and 1-alkyl-2-aminobenzimidazoles , all of which are pure tlr8 agonists with no 
 detectable activity at tlr7 . crystal structures of the ectodomain 
 of human tlr8 ( htlr8 ) cocrystallized with two regioisomers of dual 
 tlr7/8-agonistic n - aminomethylbenzyl - substituted 
 imidazoquinolines ( 1 , 2 ) showed subtle differences in their interactions in the 
 binding site of htlr8 ( figure 1 ) . the n - substituent of 1 was observed 
 to h - bond with a backbone carbonyl group , while in 2 , 
 a stronger salt - bridge was present , which fully explained the higher 
 tlr8 activity of 2 . we sought to apply these findings 
 and asked whether the tlr8-agonistic potency of the best - in - class 
 compound of the 3-alkylquinoline-2-amine series could be further enhanced by  designing in  
 functional groups which would mimic the ionic h - bond observed in the 
 htlr8/2 complex . we now report a focused and hypothesis - driven 
 exploration of introducing alkylamino groups at all possible positions 
 on the quinoline core . these studies led to the identification of 
 a novel tlr8 agonist which was 20-fold more potent than the 
 parent compound . left : structures of the dual tlr7/8-active n-4-aminomethylbenzyl ( 1 ) and n-3-aminomethylbenzyl ( 2 ) substituted 
 imidazoquinolines and pure tlr8-agonistic 3-pentylquinolin-2-amine 
 ( 3 ) . right : crystal structures of 1 and 2 bound to human tlr8 . dashed lines in yellow the dual tlr7/8-active regioisomeric imidazoquinolines 1 and 2 ( figure 1 ) , synthesized when we first began our investigations on tlr - active 
 compounds , showed substantially different 
 agonistic potencies in human tlr7 ( 1 , 50 nm ; 2 , 215 nm ) and tlr8 ( 1 , 55 nm ; 2 , 14 nm ) 
 primary screens ( figure s1 in supporting information ) . the crystal structures of these two congeners bound to the ectodomain 
 of human tlr8 reveal the structural basis of enhanced tlr8-agonistic 
 potency of 2 relative to 1 : the 3-aminomethylbenzyl 
 substituent in 2 forms a strong ionic h - bond ( salt bridge ) 
 with the side chain carboxylate of asp545 , while the 4-aminomethylbenzyl 
 substituent in 1 is observed to engage the backbone carbonyl 
 of gly351 in a weaker h - bond ( figure 1 ) . the stronger interaction of 2 in its 
 binding site resulted not only in enhancement of agonistic activity 
 in primary screens ( figure s1 ) but also 
 in higher proinflammatory cytokine induction in whole human blood 
 assays ( data not shown ) . the 3-pentylquinoline-2-amine 3 , derived from structure - based ligand design , had previously been 
 identified as a human tlr8-specific agonist ( ec50 = 200 
 nm ) . we asked whether grafting 
 the aminomethylbenzyl group on to the 3-pentylquinoline-2-amine moiety 
 would result in augmented activity . direct snar displacement 
 of the 4-chloro-3-(pent-1-yn-1-yl)quinoline intermediate4 with 3- or 4-cyanobenzylzinc 
 bromide as nucleophile afforded the 4-substituted 
 3-pentynylquinolines 5a and 5b ( scheme 1 ) ; reduction of the 
 nitriles with lialh4 and subsequent boc protection of the 
 resultant amines yielded the intermediates 6a and 6b . hydrogenation of the alkynyl 
 group and boc - deprotection furnished the desired target compounds 9a and 9b ( scheme 1 ) which retained specificity for tlr8 but with marginal 
 improvement in potency ( 150 and 120 nm , respectively ; table 1 ) . in order to examine relieving 
 possible steric bulk of the aminomethylbenzyl substituent at c4 , we 
 undertook the synthesis of the 4-aminobutyl ( 14a ) and 
 5-aminopentyl ( 14b ) analogues ( scheme 2 ) , the lengths of which were found to be 
 optimal in sar studies on several tlr8-active chemotypes . installation 
 of the 4-alkylnitrile groups of 10a , b was 
 carried out with cyanoalkylzinc bromides under negishi conditions 
 ( scheme 2 ) , and the 
 remainder of the sequence of reactions was similar to those described 
 in scheme 1 . the potencies 
 of 14a and 14b remained virtually unchanged 
 ( 190 and 250 nm , respectively ; table 1 ) . reagents : ( i ) 3-cyanobenzylzinc 
 bromide ( for 5a ) or 4-cyanobenzylzinc bromide ( for 5b ) , lici , dmf ; ( ii ) ( a ) liaih4 , thf , ( b ) boc2o , ch3oh ; ( iii ) m - cpba , chcl3 ; 
 ( iv ) ( a ) benzoyl isocyanate , ch2ci2 , ( b ) naoch3 , ch3oh ; ( v ) ( a ) pt / c , etoac , 30 psi , ( b ) hci , 
 4 m. reagents : ( i ) 3-cyanopropylzinc 
 bromide ( for 10a ) or 4-cyanobutylzinc bromide ( for 10b ) , pd(pph3)4 , thf ; ( ii ) ( a ) lialh4 , thf , ( b ) boc2o , ch3oh ; ( iii ) m - cpba , chcl3 ; ( iv ) ( a ) benzoyl isocyanate , ch2ci2 , ( b ) naoch3 , ch3oh ; ( v ) 
 ( a ) pt / c , etoac , 30 psi , ( b ) hci , 4 m. ec50 values represent the arithmetic 
 mean values obtained on quadruplicate samples . our first attempts at attaching 
 amine - bearing appendages on the quinoline core at c4 to allow for 
 additional salt - bridge interactions with asp545 appeared unfruitful , 
 and we set out to systematically examine substitutions at all other 
 positions . we desired an efficient synthetic strategy to access 5- , 
 6- , 7- , and 8-substituted 2-amino-3-pentylquinolines . a one - pot method 
 for the syntheses of 2-aminoquinoline-3-carboxamides has been reported 
 using 2-aminobenzaldehyde and active methylene - group - bearing cyanoacetamides . we envisioned that a modified friedlnder 
 synthesis of key bromo - substituted 2-amino-3-pentylquinolines could 
 be directly obtained via condensation our initial attempts 
 at model reactions with alkane nitriles and the unsubstituted 2-aminobenzaldehyde 
 proceeded very well in the presence of n - butyllithium . however , in order to preempt possible debromination , we sought alternatives 
 and successfully utilized potassium tert - butoxide 
 to generate the pivotal bromo - substituted 2-amino-3-pentylquinolines 
 ( schemes 37 ) . reagents : ( i ) heptanenitrile , t - buok , dmso ; ( ii ) 3-cyanobenzylzinc bromide ( for 17a ) or 4-cyanobenzylzinc bromide ( for 17b ) or 
 2-cyanobenzylzinc bromide ( for 17c ) or benzylzinc bromide 
 ( for 17d ) , pd(pph3)4 , thf ; ( iii ) 
 liaih4 , thf ; ( iv ) koh , t - buoh , 8 h. we targeted 2-amino-3-pentylquinolines substituted 
 at c5 with 3-aminomethylbenzyl ( 18a ) , 4-aminomethylbenzyl 
 ( 18b ) , and 2-aminomethylbenzyl ( 18c ) substituents , 
 which were obtained by negishi coupling of corresponding cyanobenzylzinc 
 bromides with 16 ( scheme 3 ) . substantially improved potencies were observed for 
 both 18a and 18b ( ec50 of 49 
 and 38 nm , respectively ; figure 2 , table 1 ) , whereas the 2-aminomethylbenzyl - substituted 18c was 
 significantly weaker ( ec50 = 1000 nm ; table 1 ) than the parent compound , 3 , suggesting that the placement of the amine on the benzyl 
 substituent was an important determinant of activity . in order to 
 formally test whether the amine was participating in the predicted 
 salt bridge , the nitrile 17a was hydrolyzed to the carboxamide 
 analogue 18d ( scheme 3 ) . compound 18d and the 5-benzyl analogue 17d were found to be inactive ( table 1 ) , lending support to our hypothesis . agonistic activities 
 of analogues 18a , 18b , 34a , 
 and 34b in human tlr8 reporter gene assays . mean values 
  sd on quadruplicates are shown . also included is 3 , used as a reference / comparator compound . we next explored the role of conformational flexibility of 
 the aminomethylbenzyl substituent at c5 , and we therefore synthesized 
 the aryl aryl coupled 5-(aminomethyl)phenyl analogues 20a and 20b via suzuki reaction of cyanophenylboronic 
 acids with 16 ( scheme 4 ) . compound 20a was entirely inactive , 
 and the activity of 20b was attenuated ( 699 nm ) , strongly 
 pointing to the indispensability of conformational freedom . these 
 findings prompted us to synthesize 5-aminoalkyl analogues ( schemes 5 and 6 ) . the aminobutyl ( 34a ) , aminopentyl ( 34b ) , and aminohexyl ( 34c ) derivatives could be accessed 
 via negishi couplings ( scheme 6 ) ; the reactivity of 2-cyanoethylzinc bromide with 16 , however , was very poor even under microwave conditions , and the 
 aminopropyl analogue 23 was accessed via heck reaction 
 of acrylonitrile with 16 ( scheme 5 ) . a clear dependence on the length of the 
 alkylamine substituent was observed in these homologues with progressive 
 increases in potency from the aminopropyl ( 23 , 91 nm ) , 
 aminobutyl ( 34a , 27 nm ; figure 2 ) and aminopentyl ( 34b , 9 nm ; figure 2 ) analogues ; a further 
 increase in 
 length ( 34c , aminohexyl ) led to decreased activity ( 56 
 nm , table 1 ) . conversion 
 of the nitrile precursor 30a to the carboxamide derivative 34d ( scheme 6 ) resulted in a dramatic decrease in potency ( 2181 nm , table 1 ) , once again highlighting the 
 importance of the presence of a free amino functional group . reagents:(i ) 3-cyanophenylboronic 
 acid ( for 19a ) or 4-cyanophenylboronic acid ( for 19b ) , pd(dppf)c12 , k2co3 , 
 1,4-dioxane ; ( ii ) liaih4 , thf , 5 h. reagents : ( i ) acrylonitrile , 
 pd(oac)2 , pph3 , k2co3 , 
 dmf ; ( ii ) h2 , pt / c , 30 psi , etoac ; ( iii ) liaih4 , thf . reagents : ( i ) heptanenitrile , t - buok , dmso ; ( ii ) 3-cyanopropylzinc bromide ( for 30a32a and 33 ) or 4-cyanobutylzinc 
 bromide ( for 30b32b ) or 5-cyanopentylzinc 
 bromide ( for 30c32c ) , pd(pph3)4 , thf ; ( iii ) liaih4 , thf ; ( iv ) koh , t - buoh , 8 h ; ( v ) 1h - pyrazole-1-carboxamidine 
 hci , et3n , meoh . the dramatic enhancement 
 of potency in 34b seemed to unambiguously support our 
 hypothesis of a salt - bridge between asp545 and the 5-aminopentyl group 
 of the lead compound . given that guanidine carboxylate interaction 
 in proteins are consequential and significant gains 
 in interaction energies are observed in drugs such as zanamivir and 
 peramivir whose crystal structures show strong salt - bridges between 
 their guanidinium functional groups and the asp / glu residues that 
 they interact with , we synthesized from 34a the guanidine derivative 34e ( scheme 6 ) , the length of the c5 substituent 
 of which was calculated to be comparable to that of 34b . we found , to our surprise , a precipitous fall in activity ( 2862 
 nm , table 1 ) , the reasons 
 for which are yet to be understood . we also explored aminoalkyl 
 substitutions at c6 ( 35a  c ) , c7 ( 36a  c ) , and c8 ( 37 ) ( scheme 6 ) . compounds 35a  c showed slight decreases in activity 
 while analogues 36a  c displayed modest 
 gains in potency , with the most active compound being the 7-(5-aminopentyl)-3-pentylquinolin-2-amine , 36b ( 50 nm ) . the c8-substituted analogue 37 was 
 entirely devoid of activity ( table 1 ) . noting that the most potent analogues possessed 
 an aminopentyl substituent either at c5 ( 34b , 9 nm ) or 
 c7 ( 36b , 50 nm ) , we wished to synthesize a dually substituted 
 analogue . the key precursor 2-amino-4,6-dibromobenzaldehyde was synthesized 
 from 2-amino-4,6-dibromobenzoic acid using conventional methods , and 
 alkylamino substituents at c5 and c7 installed via negishi reaction 
 of 41 with 4-cyanobutylzinc bromide ( scheme 7 ) . the disubstituted analogue 43 , however , was 
 found to be weaker ( 621 nm ) than the parent compound . reagents : ( i ) liaih4 , thf ; ( ii ) mno2 , dcm ; ( iii ) heptanenitrile , t - buok , dmso ; ( iv ) 4-cyanobutylzinc bromide , pd(pph3)4 , thf ; ( v ) liaih4 , thf , 4 h. the most potent analogue 34b was characterized further 
 in cytokine / chemokine induction profiles in a panel of secondary screens 
 employing human peripheral blood mononuclear cells as well as whole 
 human blood . consistent with its specificity and potency for tlr8 , 
 we observed not only the induction of a specific set of proinflammatory 
 cytokines , including tnf- , il-12 , and ifn- ( figure 3 ) , but also that 
 the potency of 34b was significantly higher than that 
 of both 3 ( tlr8-specific ) and 1 ( dual tlr7/8-active ) . 
 as observed in our previous studies , these agonists also induce responses 
 which are distinctly biphasic , with higher concentrations of ligand 
 leading to an apparent suppression of cytokine production ( figure 3 ) . none of the active 
 compounds displayed any detectable cytotoxicity at concentrations 
 up to 100 g / ml , and the origin of the apparent suppression 
 of responses is presumed to be due to large excesses of ligand disfavoring 
 dimerization of tlr8 . representative cytokine induction data ( excerpted from 
 a 63 cytokine panel ) in human pbmcs . we compared the adjuvantic 
 activity of 34b ( tlr8 ec50 = 9 nm ) with that 
 of 3 ( 200 nm ) , as well as a first - generation c2-butylfuro[2,3-c]quinoline ( 1600 nm ) in a 
 rabbit model of immunization , using the diphtheria toxin mutein crm197 as a model antigen . crm197 has served as a carrier 
 protein for conjugate vaccines against encapsulated bacteria such 
 as haemophilus influenzae , streptococcus 
 pneumoniae , and neisseria meningitidis . we observed a clear dependence between antigen - specific igg titers 
 and tlr8-agonistic potency ( figure 4 ) . cohorts of adult 
 female new zealand white rabbits ( n = 4 ) were immunized 
 intramuscularly in the flank region with ( a ) 10 g of crm197 
 in 0.2 ml of saline ( unadjuvanted control ) or ( b ) 10 g of crm197 
 in 0.2 ml of saline plus 100 g of lead tlr8 agonists ( 3 , 34b , and a tlr8-specific furoquinoline agonist ) . preimmune test - bleeds were obtained on day 
 0 , and animals were immunized on days 1 , 15 , and 28 . crm197-specific elisas were performed using 
 automated liquid handling methods and are depicted as log10 ( immune / preimmune ) titers . an aspect of our work on vaccine adjuvant discovery , in addition 
 to elucidating of structure  activity relationships in lead 
 candidate vaccine adjuvants , is to delineate specific mechanisms by 
 which these compounds elicit adjuvantic effects . as alluded to earlier , 
 our understanding of how efferent signals arising from activation 
 of the innate immune system engage particular pathways in downstream 
 adaptive immune responses culminating , for instance , in the generation 
 of antigen - specific humoral responses is nascent and fragmentary . one of the questions that we have begun to address is how various 
 chemotypes acting on different innate immune receptors with divergent 
 outcomes effect enhancement of immune responses . pure tlr8 agonists , 
 as discussed earlier , evoke the production of th1-biased cytokines 
 such as tnf- , il-1 , il-12 , il-18 , and ifn- from cells 
 of the monocytoid lineage ; pure tlr7-active compounds induce the copious 
 production of ifn- from low - abundance plasmacytoid cells , activate 
 natural killer ( nk ) , and induce mitogenicity 
 in b lymphocytes ( manuscript in preparation ) and are much weaker in 
 inducing tnf- and ifn- ; tlr2 agonists , in contrast , 
 activate neutrophils as evidenced by rapid upregulation of cd11b and 
 p38 map kinase activity . the observation that 
 all these chemotypes display adjuvantic activities may signify that 
 the disparate outcomes in different cell types may point to different 
 mechanisms mediating adjuvantic activities such as , as discussed earlier , 
 enhanced antigen uptake and presentation by apcs , enhanced cd4 t helper cell activation , or affinity maturation of antibodies . in an 
 attempt to understand how tlr8 agonism may modulate adaptive immune 
 functions , we used eight - color flow cytometry to interrogate activation 
 markers ( cd40 , cd80 ) in major cellular subsets ( granulocytes , monocytes 
 ( cd14 ) , t cells ( cd3 ) , b cells ( cd19 ) , nk cells ( cd3cd56 ) , and cytokine - induced 
 killer cells ( cd3cd56 ) in human whole blood 
 stimulated with 34b , the significantly weaker tlr8-specific 3 , as well as the potent , dual tlr7/8-active 1 ; we found that whereas both 34b and 1 upregulate 
 cd40 , specifically in cd14 monocytes ( and not in other 
 subsets ) , the tlr8 stimulation with 34b strongly induces 
 cd80 expression in the monocytes ( figure 5 ) , and in these assays , differences in potency 
 between 34b and 1 become readily evident 
 ( figure 5 ) . these results 
 hint at a possible specific role of tlr8 agonists at enhancing antigen 
 presentation and point a way forward to exploring this phenomenon 
 in greater detail . top : gating strategy for identification 
 of b , t , nk lymphocytes , monocytes , and granulocytes . lymphocytic 
 subsets were identified based on cd3 , cd19 , cd56 staining patterns 
 as described in the experimental section . bottom : stimulation of whole human blood tlr8-active compounds leading 
 to upregulation of cd40 and cd80 in cd14 monocytes , denoted 
 by increase in mean fluorescence intensity ( mfi ) . in conclusion , our hypothesis - driven approach of augmenting 
 potency by exploiting key interactions identified in crystallographic 
 studies of tlr8 has yielded novel analogues of extraordinary potency 
 and specificity which are proving useful in understanding the immunological 
 basis of adjuvanticity in this chemotype . all of the solvents and reagents used were 
 obtained commercially and used as such unless noted otherwise . moisture- 
 or air - sensitive reactions were conducted under nitrogen atmosphere 
 in oven - dried ( 120 c ) glass apparatus . flash column 
 chromatography was carried out using redisep rf  gold  
 high performance silica columns on combiflash rf instruments unless otherwise mentioned , while thin - layer chromatography 
 was carried out on silica gel ccm precoated aluminum sheets . purity 
 for all final compounds was confirmed to be greater than 98% by lc ms 
 using a zorbax eclipse plus 4.6 mm  150 mm , 5 m analytical 
 reverse phase c18 column with h2o  ch3cn and h2o  meoh gradients and an agilent 
 6520 esi - qtof accurate mass spectrometer ( mass accuracy of 5 ppm ) 
 operating in the positive ion acquisition mode . compound 4 was synthesized as published by us earlier . to a solution of compound 4 ( 230 
 mg , 1 mmol ) in dmf ( 5 ml ) were added 3-cyanobenzylzinc bromide ( 4 
 ml , 2 mmol , 0.5 m in thf ) and licl ( 85 mg , 2 mmol ) . the resulting 
 reaction mixture was stirred for 24 h at room temperature under nitrogen 
 atmosphere . the reaction mixture was diluted with water and extracted 
 with etoac ( 3  30 ml ) . the combined organic layer was dried 
 over na2so4 and concentrated under reduced pressure . the crude material was purified by flash chromatography ( 40% etoac / hexanes ) 
 to obtain the compound 5a as a pale yellow solid ( 179 
 mg , 58% ) . h nmr ( 500 mhz , cdcl3 )  8.93 
 ( s , 1h ) , 8.10 ( dd , j = 1.2 , 8.4 hz , 1h ) , 7.87 ( dd , j = 1.3 , 8.3 hz , 1h ) , 7.67 ( ddd , j = 1.3 , 
 6.9 , 8.4 hz , 1h ) , 7.557.50 ( m , 2h ) , 7.497.46 ( m , 1h ) , 
 7.447.41 ( m , 1h ) , 7.34 ( t , j = 7.7 hz , 1h ) , 
 4.65 ( s , 2h ) , 2.47 ( t , j = 7.0 hz , 2h ) , 1.731.52 
 ( m , 2h ) , 1.02 ( t , j = 7.4 hz , 3h ) . c 
 nmr ( 126 mhz , cdcl3 )  152.89 , 147.08 , 145.67 , 140.55 , 
 132.90 , 132.02 , 130.52 , 130.36 , 129.55 , 129.51 , 127.65 , 126.62 , 123.91 , 
 119.01 , 118.90 , 112.77 , 98.31 , 77.53 , 35.56 , 22.20 , 21.80 , 13.70 . ms ( esi - tof ) for c22h18n2 [ m + h ] calculated 311.1543 , found 311.1441 . h nmr 
 ( 500 mhz , cdcl3 )  8.93 ( s , 1h ) , 8.09 ( dd , j = 0.8 , 8.4 hz , 1h ) , 7.84 ( dd , j = 0.8 , 
 8.5 hz , 1h ) , 7.67 ( ddd , j = 1.4 , 6.9 , 8.4 hz , 1h ) , 
 7.567.48 ( m , 3h ) , 7.327.26 ( m , 2h ) , 4.69 ( s , 2h ) , 
 2.46 ( t , j = 7.0 hz , 2h ) , 1.741.51 ( m , 2h ) , 
 1.01 ( t , j = 7.4 hz , 3h ) . c nmr ( 126 
 mhz , cdcl3 )  152.87 , 147.07 , 145.59 , 144.64 , 132.54 , 
 130.50 , 129.55 , 129.18 , 127.61 , 126.68 , 123.97 , 119.08 , 118.94 , 110.49 , 
 98.24 , 77.50 , 36.17 , 22.19 , 21.79 , 13.69 . ms ( esi - tof ) for c22h18n2 [ m + h ] calculated 311.1543 , 
 found 311.1504 . a solution of compound 5a ( 155 mg , 0.5 mmol ) in thf ( 5 ml ) was added slowly to a 
 solution of lialh4 ( 2.5 ml , 2.5 mmol , 1.0 m in thf ) in 
 thf ( 5 ml ) at 0 c under nitrogen atmosphere . the reaction mixture 
 was stirred for 1 h at 25 c and 5 h at 75 c . the reaction 
 mixture was cooled to room temperature and quenched carefully with 
 ice - cold water . the resulting mixture was basified with 10% naoh ( to 
 ph = 8.0 ) and extracted with ch2cl2 ( 3  
 30 ml ) . the combined organic layer was dried over na2so4 and concentrated under reduced pressure to obtain the residue . the residue was dissolved in meoh , and di - tert - butyl 
 dicarbamate ( 109 mg , 0.5 mmol ) was added and stirred under nitrogen 
 for 1 h. the solvent was removed under vacuum . the resulting residue 
 was purified by flash chromatography ( 20% etoac / hexanes ) to obtain 
 the compound 6a as a pale yellow solid ( 134 mg , 65% ) . ms ( esi - tof ) for c27h30n2o2 [ m + h ] calculated 415.2380 , found 415.2266 . to a stirred 
 solution of substrate 6a ( 124 mg , 0.3 mmol ) in chcl3 was added m - cpba ( 134 mg , 0.6 mmol ) . the reaction mixture was diluted with water and extracted with ch2cl2 ( 3  20 ml ) . the combined organic layer 
 was dried over na2so4 , concentrated 
 under reduced pressure , and the crude material was purified by flash 
 chromatography ( 10% meoh / ch2cl2 ) to obtain 7a as a yellow solid ( 98 mg , 76% ) . ms ( esi - tof ) for c27h30n2o3 [ m + h ] calculated 431.2329 , found 431.2122 . to a stirred solution of 7a ( 86 mg , 0.2 mmol ) in ch2cl2 was added 
 benzoyl isocyanate ( 88 mg , 0.6 mmol ) . the resulting reaction mixture 
 was stirred at 55 c for 1 h. after completion of reaction ( monitored 
 by tlc ) , the solvent was removed under reduced pressure . the residue 
 was redissolved in meoh ( 5 ml ) , and naome ( 54 mg , 1 mmol ) was added 
 and refluxed for 2 h. the solvent was removed and the crude material 
 was purified by flash chromatography ( 10% meoh / ch2cl2 ) to obtain 8a as a off - white solid ( 67 mg , 78% ) . ms ( esi - tof ) for c27h31n3o2 [ m + h ] calculated 430.2489 , found 430.2303 . to a solution 
 of compound 8a ( 43 mg , 0.1 mmol ) in anhydrous etoac ( 10 
 ml ) was added a catalytic amount of pt / c , and the reaction mixture 
 was subjected to hydrogenation at 30 psi for 30 min . the crude material was purified by flash chromatography ( 10% meoh / ch2cl2 ) to obtain n - boc protected 
 benzylamine as a white solid ( 32 mg ) . ms ( esi - tof ) for c27h35n3o2 [ m + h ] calculated 
 434.2802 , found 434.2612 . to a stirred solution of n - boc protected benzylamine ( 32 mg ) in 1,4-dioxane ( 1 ml ) was added 
 hydrogen chloride ( 1 ml , 4 m in dioxane ) , and the reaction mixture 
 was stirred for 1 h at room temperature . excess solvent was removed 
 under reduced pressure and the resulting residue was thoroughly washed 
 with diethyl ether to obtain the desired compound 9a as 
 a white solid ( 28 mg , 69% ) . h nmr ( 500 mhz , meod )  
 7.977.91 ( m , 1h ) , 7.777.64 ( m , 2h ) , 7.43 ( ddd , j = 1.4 , 7.0 , 8.4 hz , 1h ) , 7.38 ( t , j = 
 7.6 hz , 1h ) , 7.367.31 ( m , 1h ) , 7.28 ( s , 1h ) , 7.18 ( d , j = 8.0 hz , 1h ) , 4.62 ( s , 2h ) , 4.06 ( s , 2h ) , 2.81 ( t , j = 8.5 hz , 2h ) , 1.581.49 ( m , 2h ) , 1.471.39 
 ( m , 2h ) , 1.361.25 ( m , 2h ) , 0.89 ( t , j = 7.3 
 hz , 3h ) . c nmr ( 126 mhz , meod )  155.07 , 151.34 , 
 140.27 , 136.23 , 135.28 , 133.06 , 130.87 , 129.87 , 129.62 , 128.43 , 127.13 , 
 126.67 , 126.32 , 123.05 , 118.59 , 44.08 , 34.87 , 32.75 , 29.22 , 28.11 , 
 23.67 , 14.34 . ms ( esi - tof ) for c22h27n3 [ m + h ] calculated 334.2278 , found 334.2238 . h nmr ( 500 mhz , meod )  7.91 ( dd , j = 1.2 , 8.4 hz , 1h ) , 7.797.64 ( m , 2h ) , 7.447.37 
 ( m , 3h ) , 7.22 ( d , j = 8.0 hz , 2h ) , 4.62 ( s , 2h ) , 
 4.07 ( s , 2h ) , 2.79 ( t , j = 8.2 hz , 2h ) , 1.571.47 
 ( m , 2h ) , 1.461.38 ( m , 2h ) , 1.361.27 ( m , 2h ) , 0.89 
 ( t , j = 7.3 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  155.04 , 151.56 , 140.37 , 136.15 , 133.09 , 133.07 , 130.67 , 
 129.85 , 127.14 , 126.60 , 126.20 , 123.00 , 118.56 , 43.87 , 34.72 , 32.71 , 
 29.19 , 27.99 , 23.62 , 14.33 . ms ( esi - tof ) for c22h27n3 [ m + h ] calculated 334.2278 , found 334.2257 . to a solution of compound 4 ( 229.7 mg , 1 
 mmol ) in thf ( 4 ml ) were added 3-cyanopropylzinc bromide ( 4 ml , 2 
 mmol , 0.5 m in thf ) and pd(pph3)4 ( 115.5 mg , 
 0.1 mmol ) . the resulting reaction mixture was stirred for 12 h at 
 65 c under nitrogen atmosphere . the reaction mixture was diluted 
 with water and extracted with etoac ( 3  30 ml ) . the combined 
 organic layer was dried over na2so4 and concentrated 
 under reduced pressure . the crude material was purified by flash chromatography 
 ( 20% etoac / hexanes ) to obtain the compound 10a as a pale 
 yellow oil ( 144 mg , 55% ) . h nmr ( 500 mhz , cdcl3 )  8.84 ( s , 1h ) , 8.118.06 ( m , 1h ) , 7.98 ( dd , j = 1.0 , 8.9 hz , 1h ) , 7.69 ( ddd , j = 1.3 , 
 6.8 , 8.3 hz , 1h ) , 7.59 ( ddd , j = 1.3 , 6.8 , 8.3 hz , 
 1h ) , 3.42 ( t , j = 7.7 hz , 2h ) , 2.53 ( t , j = 7.0 hz , 2h ) , 2.46 ( t , j = 7.0 hz , 2h ) , 2.142.03 
 ( m , 2h ) , 1.771.62 ( m , 2h ) , 1.10 ( t , j = 7.4 
 hz , 3h ) . c nmr ( 126 mhz , cdcl3 )  152.74 , 
 147.14 , 146.93 , 130.56 , 129.49 , 127.51 , 126.37 , 123.25 , 119.48 , 118.15 , 
 98.32 , 77.10 , 28.90 , 25.79 , 22.27 , 21.83 , 17.31 , 13.81 . ms ( esi - tof ) 
 for c18h18n2 [ m + h ] calculated 
 263.1543 , found 263.1437 . h nmr 
 ( 500 mhz , cdcl3 )  8.83 ( s , 1h ) , 8.07 ( dd , j = 0.8 , 8.5 hz , 1h ) , 7.97 ( dd , j = 1.0 , 
 8.5 hz , 1h ) , 7.67 ( ddd , j = 1.4 , 6.9 , 8.3 hz , 1h ) , 
 7.57 ( ddd , j = 1.3 , 6.9 , 8.3 hz , 1h ) , 3.30 ( t , j = 7.5 hz , 2h ) , 2.51 ( t , j = 7.0 hz , 2h ) , 
 2.40 ( t , j = 7.0 hz , 2h ) , 1.951.84 ( m , 2h ) , 
 1.851.75 ( m , 2h ) , 1.761.65 ( m , 2h ) , 1.10 ( t , j = 7.4 hz , 3h ) . c nmr ( 126 mhz , cdcl3 )  152.78 , 148.54 , 146.91 , 130.45 , 129.34 , 127.22 , 126.48 , 
 123.54 , 119.52 , 117.88 , 97.62 , 77.51 , 29.32 , 29.02 , 25.41 , 22.33 , 
 21.81 , 17.20 , 13.79 . ms ( esi - tof ) for c19h20n2 [ m + h ] calculated 277.1699 , found 277.1581 . h nmr ( 500 mhz , meod )  8.11 ( d , j = 8.1 hz , 
 1h ) , 7.76 ( ddd , j = 1.2 , 7.1 , 8.4 hz , 1h ) , 7.67 ( dd , j = 1.2 , 8.4 hz , 1h ) , 7.57 ( ddd , j = 1.2 , 
 7.1 , 8.3 hz , 1h ) , 3.19 ( t , j = 8.2 hz , 2h ) , 3.01 
 ( t , j = 7.6 hz , 2h ) , 2.80 ( t , j = 
 8.2 hz , 2h ) , 1.981.87 ( m , 2h ) , 1.801.70 ( m , 2h ) , 1.651.55 
 ( m , 2h ) , 1.561.46 ( m , 2h ) , 1.471.38 ( m , 2h ) , 0.96 
 ( t , j = 7.2 hz , 3h ) . nmr ( 126 mhz , 
 meod )  154.80 , 154.01 , 135.98 , 133.05 , 126.77 , 126.45 , 124.51 , 
 122.47 , 118.61 , 40.50 , 32.81 , 29.52 , 29.45 , 28.86 , 28.16 , 27.56 , 23.73 , 
 14.42 . ms ( esi - tof ) for c18h27n3 [ m 
 + h ] calculated 286.2278 , found 286.2240 . h nmr ( 500 mhz , meod )  8.09 ( d , j = 8.2 hz , 1h ) , 7.76 ( ddd , j = 1.2 , 7.0 , 
 8.3 hz , 1h ) , 7.66 ( d , j = 7.6 hz , 1h ) , 7.56 ( ddd , j = 1.2 , 7.0 , 8.3 hz , 1h ) , 3.16 ( t , j = 
 6.7 hz , 2h ) , 2.97 ( t , j = 7.5 hz , 2h ) , 2.79 ( t , j = 8.4 hz , 2h ) , 1.801.64 ( m , 6h ) , 1.641.56 
 ( m , 2h ) , 1.541.46 ( m , 2h ) , 1.481.38 ( m , 2h ) , 0.96 
 ( t , j = 7.3 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  154.78 , 154.54 , 135.97 , 133.00 , 126.70 , 126.41 , 124.30 , 
 122.51 , 118.60 , 40.61 , 32.81 , 30.92 , 29.79 , 29.52 , 28.48 , 27.85 , 27.54 , 
 23.73 , 14.41 . ms ( esi - tof ) for c19h29n3 [ m + h ] calculated 300.2434 , found 300.2397 . to a solution of compound 15 ( 200 mg , 1 mmol ) in dmso ( 3 ml ) were added heptanenitrile ( 275 l , 
 2 mmol ) and t - buok ( 224 mg , 2 mmol ) . the resulting 
 reaction mixture was stirred for 3 h at 60 c under nitrogen 
 atmosphere . the reaction mixture was diluted with water and extracted 
 with etoac ( 3  50 ml ) . the combined organic layer was dried 
 over na2so4 and concentrated under reduced pressure . the crude material was purified by flash chromatography ( 50% etoac / hexanes ) 
 to obtain the compound 16 as an off - white solid ( 220 
 mg , 75% ) . h nmr ( 500 mhz , dmso - d6 )  7.82 ( s , 1h ) , 7.497.40 ( m , 2h ) , 7.34 ( dd , j = 7.6 , 8.3 hz , 1h ) , 6.58 ( s , 2h ) , 2.62 ( t , j = 7.8 hz , 2h ) , 1.661.57 ( m , 2h ) , 1.441.29 ( m , 4h ) , 
 0.89 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 
 mhz , dmso - d6 )  157.75 , 147.59 , 
 132.61 , 128.85 , 126.08 , 124.89 , 124.73 , 121.98 , 120.40 , 30.96 , 30.29 , 
 27.42 , 22.06 , 14.01 . ms ( esi - tof ) for c14h17brn2 [ m + h ] calculated 293.0648 , found 293.0684 . to a solution of compound 16 ( 58.8 mg , 0.2 
 mmol ) in thf ( 2 ml ) were added 3-cyanobenzylzinc bromide ( 0.8 ml , 
 0.4 mmol , 0.5 m in thf ) and pd(pph3)4 ( 11.6 
 mg , 0.01 mmol ) . the resulting reaction mixture was stirred at 65 c 
 under nitrogen atmosphere for 12 h. the reaction mixture was diluted 
 with water and extracted with etoac ( 3  10 ml ) . the combined 
 organic layer was dried over na2so4 and concentrated 
 under reduced pressure . the crude material was purified by flash chromatography 
 ( 60% etoac / hexanes ) to obtain the compound 17a as a pale 
 yellow solid ( 54 mg , 82% ) . h nmr ( 500 mhz , cdcl3 )  7.67 ( s , 1h ) , 7.61 ( d , j = 8.5 hz , 1h ) , 
 7.517.44 ( m , 3h ) , 7.407.33 ( m , 2h ) , 7.07 ( d , j = 7.0 hz , 1h ) , 4.80 ( s , 2h ) , 4.37 ( s , 2h ) , 2.52 ( t , j = 7.6 hz , 2h ) , 1.661.56 ( m , 2h ) , 1.381.21 
 ( m , 4h ) , 0.88 ( t , j = 7.1 hz , 3h ) . c nmr ( 126 mhz , cdcl3 )  155.95 , 147.28 , 142.44 , 134.55 , 
 133.17 , 132.16 , 131.77 , 130.13 , 129.40 , 128.69 , 125.59 , 124.49 , 123.66 , 
 122.91 , 119.00 , 112.68 , 38.40 , 31.52 , 31.49 , 27.56 , 22.63 , 14.15 . ms ( esi - tof ) for c22h23n3 [ m + h ] calculated 330.1965 , found 330.1896 . h nmr ( 500 mhz , 
 cdcl3 )  7.66 ( s , 1h ) , 7.62 ( d , j = 8.5 hz , 1h ) , 7.56 ( d , j = 8.3 hz , 2h ) , 7.48 ( dd , j = 7.0 , 8.4 hz , 1h ) , 7.307.25 ( m , 2h ) , 7.09 ( d , j = 6.8 hz , 1h ) , 4.81 ( s , 2h ) , 4.41 ( s , 2h ) , 2.52 ( t , j = 7.8 hz , 2h ) , 1.661.53 ( m , 2h ) , 1.391.19 
 ( m , 4h ) , 0.89 ( t , j = 7.1 hz , 3h ) . c 
 nmr ( 126 mhz , cdcl3 )  155.95 , 147.26 , 146.62 , 134.50 , 
 132.44 , 131.82 , 129.42 , 128.68 , 125.56 , 124.54 , 123.63 , 122.96 , 119.07 , 
 110.20 , 39.00 , 31.51 , 31.45 , 27.53 , 22.63 , 14.15 . ms ( esi - tof ) for 
 c22h23n3 [ m + h ] calculated 
 330.1965 , found 330.1899 . h nmr ( 500 mhz , 
 cdcl3 )  7.71 ( s , 1h ) , 7.69 ( dd , j = 1.1 , 7.7 hz , 1h ) , 7.61 ( d , j = 8.5 hz , 1h ) , 7.47 
 ( dd , j = 7.1 , 8.5 hz , 1h ) , 7.39 ( td , j = 1.4 , 7.7 hz , 1h ) , 7.29 ( td , j = 1.2 , 7.6 hz , 
 1h ) , 7.07 ( dd , j = 1.1 , 7.1 hz , 1h ) , 7.00 ( dd , j = 0.6 , 7.9 hz , 1h ) , 4.81 ( s , 2h ) , 4.57 ( s , 2h ) , 2.53 ( t , j = 7.6 hz , 2h ) , 1.671.60 ( m , 2h ) , 1.381.22 
 ( m , 4h ) , 0.87 ( t , j = 7.1 hz , 3h ) . c 
 nmr ( 126 mhz , cdcl3 )  156.01 , 147.18 , 144.65 , 134.08 , 
 133.05 , 132.87 , 131.83 , 129.79 , 128.64 , 126.92 , 125.57 , 124.59 , 123.80 , 
 123.06 , 118.25 , 112.43 , 36.89 , 31.56 , 31.48 , 27.55 , 22.62 , 14.16 . ms ( esi - tof ) for c22h23n3 [ m + h ] calculated 330.1965 , found 330.2008 . h nmr ( 500 mhz , dmso - d6 )  7.82 ( s , 1h ) , 7.417.31 ( m , 
 2h ) , 7.287.18 ( m , 4h ) , 7.187.11 ( m , 1h ) , 7.057.00 
 ( m , 1h ) , 6.21 ( s , 2h ) , 4.28 ( s , 2h ) , 2.552.49 ( m , 2h ) , 1.571.47 
 ( m , 2h ) , 1.321.17 ( m , 4h ) , 0.83 ( t , j = 7.2 
 hz , 3h ) . c nmr ( 126 mhz , dmso - d6 )  156.73 , 147.10 , 141.09 , 136.57 , 131.20 , 128.43 , 
 128.28 , 127.75 , 125.84 , 123.78 , 123.50 , 122.48 , 121.73 , 37.77 , 30.72 , 
 30.31 , 27.23 , 22.06 , 13.96 . ms ( esi - tof ) for c21h24n2 [ m + h ] calculated 305.2012 , found 305.1951 . a solution of compound 17a ( 33 mg , 0.1 mmol ) 
 in thf ( 5 ml ) was added slowly to a solution of lialh4 ( 0.5 
 ml , 0.5 mmol , 1.0 m in thf ) in thf ( 3 ml ) at 0 c under nitrogen 
 atmosphere . the reaction mixture was stirred for 2 h at 25 c 
 and 2 h at 75 c . the reaction mixture was carefully quenched 
 with ice - cold water ( 1 ml ) at 0 c , and 10% naoh ( 1 ml ) was added . the resulting mixture was stirred for 10 min at room temperature , 
 filtered through celite , and washed with ch2cl2 ( 15 ml ) . the resulting filtrate was dried over na2so4 and concentrated under reduced pressure and the crude material 
 was purified by neutral - alumina column chromatography ( 20% meoh / ch2cl2 ) to obtain the compound 18a as 
 a white solid ( 24 mg , 72% ) . h nmr ( 500 mhz , meod )  
 7.88 ( s , 1h ) , 7.467.38 ( m , 2h ) , 7.21 ( t , j = 7.5 hz , 1h ) , 7.187.10 ( m , 3h ) , 7.06 ( d , j = 7.4 hz , 1h ) , 4.34 ( s , 2h ) , 3.70 ( s , 2h ) , 2.55 ( t , j = 7.5 hz , 2h ) , 1.60 ( p , j = 7.6 hz , 2h ) , 1.421.20 
 ( m , 4h ) , 0.89 ( t , j = 7.1 hz , 3h ) . c 
 nmr ( 126 mhz , meod )  158.00 , 147.60 , 143.84 , 142.65 , 138.04 , 
 133.82 , 129.68 , 129.58 , 128.69 , 128.24 , 126.21 , 125.12 , 125.06 , 124.29 , 
 123.67 , 46.67 , 39.64 , 32.47 , 31.77 , 28.68 , 23.64 , 14.41 . ms ( esi - tof ) 
 for c22h27n3 [ m + h ] calculated 
 334.2278 , found 334.2214 . h nmr ( 500 mhz , meod )  7.87 ( s , 1h ) , 
 7.517.37 ( m , 2h ) , 7.22 ( d , j = 8.0 hz , 2h ) , 
 7.14 ( d , j = 7.9 hz , 2h ) , 7.127.09 ( m , 1h ) , 
 4.32 ( s , 2h ) , 3.72 ( s , 2h ) , 2.54 ( t , j = 7.6 hz , 
 2h ) , 1.681.48 ( m , 2h ) , 1.401.21 ( m , 4h ) , 0.89 ( t , j = 7.1 hz , 3h ) . c nmr ( 126 mhz , meod )  
 158.00 , 147.60 , 141.33 , 141.04 , 138.13 , 133.81 , 129.74 , 129.56 , 128.59 , 
 125.10 , 124.98 , 124.27 , 123.63 , 46.35 , 39.28 , 32.48 , 31.78 , 28.66 , 
 23.64 , 14.41 . ms ( esi - tof ) for c22h27n3 [ m + h ] calculated 334.2278 , found 334.2191 . h nmr ( 500 mhz , meod ) 
  7.90 ( s , 1h ) , 7.477.33 ( m , 3h ) , 7.24 ( td , j = 1.3 , 7.5 hz , 1h ) , 7.15 ( td , j = 1.4 , 
 7.5 hz , 1h ) , 6.93 ( dd , j = 1.3 , 7.7 hz , 1h ) , 6.84 
 ( dd , j = 1.1 , 7.1 hz , 1h ) , 4.42 ( s , 2h ) , 3.84 ( s , 
 2h ) , 2.59 ( t , j = 7.8 hz , 2h ) , 1.681.57 ( m , 
 2h ) , 1.401.27 ( m , 4h ) , 0.90 ( t , j = 7.1 hz , 
 3h ) . c nmr ( 126 mhz , meod )  158.12 , 147.46 , 141.29 , 
 139.25 , 137.80 , 133.50 , 131.09 , 129.58 , 129.13 , 128.27 , 127.89 , 125.42 , 
 124.27 , 124.19 , 123.78 , 43.84 , 35.93 , 32.58 , 31.94 , 28.87 , 23.65 , 
 14.42 . ms ( esi - tof ) for c22h27n3 [ m 
 + h ] calculated 334.2278 , found 334.2195 . to a solution of compound 17a ( 33 mg , 0.1 mmol ) in t - buoh ( 2 ml ) was added potassium 
 hydroxide ( 84 mg , 1.5 mmol ) . the reaction was allowed to cool to room temperature , 
 the solvent was removed under reduced pressure and the crude solubilized 
 in ethyl acetate . the organic layer was washed with water and saturated 
 aqueous ammonium chloride and dried over na2so4 and evaporated under reduced pressure . the residue was purified 
 by silica gel flash - column chromatography ( 15% meoh / ch2cl2 ) to afford the compound ( 18d ) as a white 
 solid ( 18 mg , 52% ) . h nmr ( 500 mhz , meod )  7.88 
 ( s , 1h ) , 7.78 ( d , j = 0.8 hz , 1h ) , 7.68 ( dt , j = 1.9 , 7.0 hz , 1h ) , 7.497.43 ( m , 2h ) , 7.387.27 
 ( m , 2h ) , 7.16 ( dd , j = 3.2 , 5.0 hz , 1h ) , 4.42 ( s , 
 2h ) , 2.56 ( t , j = 7.1 hz , 2h ) , 1.58 ( p , j = 7.5 hz , 2h ) , 1.361.27 ( m , 2h ) , 1.281.19 ( m , 2h ) , 
 0.86 ( t , j = 7.2 hz , 3h ) . c nmr ( 126 
 mhz , meod )  172.26 , 157.87 , 147.04 , 142.96 , 137.61 , 135.13 , 
 134.03 , 133.11 , 129.84 , 129.66 , 128.98 , 126.42 , 125.39 , 125.32 , 124.08 , 
 123.44 , 39.47 , 32.39 , 31.68 , 28.61 , 23.59 , 14.39 . ms ( esi - tof ) for 
 c22h25n3o [ m + h ] calculated 
 348.2070 , found 348.2022 . to a stirred solution of compound 16 ( 59 mg , 0.2 mmol ) in 1,4-dioxane ( 2 ml ) were added 3-cyanophenylboronic 
 acid ( 44 mg , 0.3 mmol ) , pd(dppf)cl2 ( 14.6 mg , 0.02 mmol ) , 
 and k2co3 ( 83 mg , 0.6 mmol ) . the resulting reaction 
 mixture was stirred for 12 h at 90 c under nitrogen atmosphere . the reaction mixture was diluted with water and extracted with etoac 
 ( 3  20 ml ) . the combined organic layer was dried over na2so4 and concentrated under reduced pressure , and 
 crude material was purified by flash chromatography ( 60% etoac / hexanes ) 
 to obtain the compound 19a as a brownish solid ( 40 mg , 
 63% ) . h nmr ( 500 mhz , cdcl3 )  7.777.66 
 ( m , 4h ) , 7.647.52 ( m , 3h ) , 7.15 ( dd , j = 
 1.2 , 7.1 hz , 1h ) , 4.89 ( s , 2h ) , 2.53 ( t , j = 7.6 
 hz , 2h ) , 1.671.57 ( m , 2h ) , 1.391.31 ( m , 4h ) , 0.89 
 ( t , j = 6.9 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  156.25 , 146.96 , 141.74 , 137.22 , 134.46 , 
 133.42 , 132.79 , 131.10 , 129.35 , 128.42 , 126.31 , 124.37 , 123.92 , 122.25 , 
 118.90 , 112.78 , 31.69 , 31.59 , 27.74 , 22.57 , 14.15 . ms ( esi - tof ) for 
 c21h21n3 [ m + h ] calculated 
 316.1808 , found 316.1823 . h nmr 
 ( 500 mhz , cdcl3 )  7.837.76 ( m , 2h ) , 7.71 
 ( d , j = 8.4 hz , 1h ) , 7.61 ( s , 1h ) , 7.607.53 
 ( m , 3h ) , 7.17 ( dd , j = 1.2 , 7.2 hz , 1h ) , 4.89 ( s , 
 2h ) , 2.52 ( t , j = 7.7 hz , 2h ) , 1.671.55 ( m , 
 2h ) , 1.401.31 ( m , 4h ) , 0.89 ( t , j = 7.0 hz , 
 3h ) . c nmr ( 126 mhz , cdcl3 )  156.24 , 
 146.97 , 145.32 , 137.73 , 132.84 , 132.32 , 130.78 , 128.40 , 126.40 , 124.35 , 
 123.80 , 122.09 , 119.04 , 111.30 , 31.68 , 31.56 , 27.73 , 22.57 , 14.16 . ms ( esi - tof ) for c21h21n3 [ m + h ] calculated 316.1808 , found 316.1845 . h nmr ( 500 mhz , meod ) 
  7.73 ( s , 1h ) , 7.587.39 ( m , 5h ) , 7.30 ( d , j = 6.7 hz , 1h ) , 7.16 ( d , j = 6.6 hz , 1h ) , 3.88 ( s , 
 2h ) , 2.56 ( t , j = 7.6 hz , 2h ) , 1.681.53 ( m , 
 2h ) , 1.381.29 ( m , 4h ) , 0.90 ( t , j = 6.7 hz , 
 3h ) . c nmr ( 126 mhz , meod )  158.28 , 147.40 , 144.04 , 
 141.73 , 141.28 , 134.86 , 130.00 , 129.55 , 129.53 , 129.41 , 127.59 , 125.60 , 
 124.69 , 124.34 , 123.07 , 46.69 , 32.56 , 31.81 , 28.73 , 23.59 , 14.39 . 
 ms ( esi - tof ) for c21h25n3 [ m + h ] calculated 320.2121 , found 320.2072 . h nmr ( 500 mhz , meod ) 
  7.72 ( s , 1h ) , 7.577.45 ( m , 4h ) , 7.39 ( d , j = 8.1 hz , 2h ) , 7.13 ( dd , j = 1.7 , 6.6 hz , 1h ) , 
 3.89 ( s , 2h ) , 2.54 ( t , j = 7.6 hz , 2h ) , 1.671.51 
 ( m , 2h ) , 1.381.26 ( m , 4h ) , 0.89 ( t , j = 7.0 
 hz , 3h ) . c nmr ( 126 mhz , meod )  158.26 , 147.42 , 
 142.97 , 141.07 , 140.07 , 134.82 , 131.06 , 129.43 , 128.56 , 125.58 , 124.65 , 
 124.33 , 123.07 , 46.45 , 32.58 , 31.88 , 28.78 , 23.57 , 14.40 . ms ( esi - tof ) 
 for c21h25n3 [ m + h ] calculated 
 320.2121 , found 320.2073 . a solution of 16 ( 147 mg , 
 0.5 mmol ) and acrylonitrile ( 66 l , 1 mmol ) in dmf ( 4 ml ) was 
 treated with pd(oac)2 ( 11.2 mg , 0.05 mmol ) , pph3 ( 26.2 mg , 0.1 mmol ) , and k2co3 ( 138 mg , 1 
 mmol ) . the resulting reaction mixture was stirred for 12 h at 110 
 c under nitrogen atmosphere . the reaction mixture was diluted 
 with water and extracted with etoac ( 3  20 ml ) . the combined 
 organic layer was dried over na2so4 and concentrated 
 under reduced pressure . the crude material was purified by flash chromatography 
 ( 60% etoac / hexanes ) to obtain the compound 21 as a pale 
 yellow solid ( 73 mg , 55% ) . ms ( esi - tof ) for c17h19n3 [ m + h ] calculated 266.1652 , found 266.1663 . 
 to a solution of compound 21 ( 53 mg , 0.2 mmol ) in anhydrous 
 etoac ( 10 ml ) was added a catalytic amount of pt / c , and the reaction 
 mixture was subjected to hydrogenation at 30 psi for 3 h. the reaction 
 mixture was filtered , and the filtrate concentrated under reduced 
 pressure . the crude material was purified using silica gel column 
 chromatography ( 60% etoac / hexanes ) to obtain compound 22 as white solid ( 40 mg , 75% ) . ms ( esi - tof ) for c17h21n3 [ m + h ] calculated 268.1808 , found 
 268.1821 . a solution of compound 22 ( 27 mg , 0.1 mmol ) 
 in thf ( 5 ml ) was added slowly to a solution of lialh4 ( 0.5 
 ml , 0.5 mmol , 1.0 m in thf ) in thf ( 3 ml ) at 0 c under nitrogen 
 atmosphere . the reaction mixture was stirred for 2 h at 25 c 
 and 2 h at 60 c . the reaction mixture was carefully quenched 
 with ice - cold water ( 1 ml ) at 0 c , and 10% naoh ( 1 ml ) was added . the resulting mixture was stirred for 10 min at room temperature , 
 filtered through celite , and washed with ch2cl2 ( 15 ml ) . the resulting filtrate was dried over na2so4 and concentrated under reduced pressure and the crude material 
 was purified by flash neutral - alumina column chromatography ( 20% meoh / ch2cl2 ) to obtain the compound 23 as 
 a white solid ( 15 mg , 55% ) . h nmr ( 500 mhz , meod )  
 7.99 ( s , 1h ) , 7.407.37 ( m , 2h ) , 7.09 ( dd , j = 2.8 , 5.4 hz , 1h ) , 3.02 ( t , j = 7.5 hz , 2h ) , 2.73 
 ( t , j = 7.2 hz , 2h ) , 2.68 ( t , j = 
 7.6 hz , 2h ) , 1.921.80 ( m , 2h ) , 1.781.68 ( m , 2h ) , 1.481.40 
 ( m , 4h ) , 0.95 ( t , j = 7.1 hz , 3h ) . c 
 nmr ( 126 mhz , meod )  158.00 , 147.49 , 139.44 , 133.35 , 129.58 , 
 125.36 , 123.72 , 123.66 , 123.41 , 42.35 , 35.26 , 32.80 , 32.15 , 30.76 , 
 29.17 , 23.69 , 14.46 . ms ( esi - tof ) for c17h25n3 [ m + h ] calculated 272.2121 , found 272.2155 . h nmr ( 500 mhz , cdcl3 )  7.74 ( d , j = 2.2 hz , 1h ) , 7.58 ( s , 1h ) , 
 7.56 ( dd , j = 2.2 , 8.9 hz , 1h ) , 7.51 ( d , j = 8.8 hz , 1h ) , 4.87 ( s , 2h ) , 2.57 ( t , j = 7.7 hz , 2h ) , 1.781.66 ( m , 2h ) , 1.451.36 ( m , 4h ) , 
 0.93 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 
 mhz , cdcl3 )  156.49 , 145.21 , 134.44 , 132.05 , 129.08 , 
 127.43 , 125.84 , 124.79 , 115.57 , 31.76 , 31.24 , 27.56 , 22.67 , 14.17 . ms ( esi - tof ) for c14h17brn2 [ m + 
 h ] calculated 293.0648 , found 293.0654 . h nmr ( 500 mhz , cdcl3 )  7.81 ( d , j = 2.2 hz , 1h ) , 7.63 ( s , 1h ) , 
 7.45 ( d , j = 8.5 hz , 1h ) , 7.32 ( dd , j = 1.9 , 8.5 hz , 1h ) , 4.93 ( s , 2h ) , 2.55 ( t , j = 
 7.9 hz , 2h ) , 1.761.67 ( m , 2h ) , 1.441.36 ( m , 4h ) , 0.93 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  156.88 , 147.41 , 135.17 , 128.30 , 128.09 , 
 125.98 , 124.16 , 123.19 , 122.73 , 31.79 , 31.24 , 27.56 , 22.67 , 14.17 . ms ( esi - tof ) for c14h17brn2 [ m + 
 h ] calculated 293.0648 , found 293.0669 . h nmr ( 500 mhz , cdcl3 )  7.82 ( dd , j = 1.4 , 7.5 hz , 1h ) , 
 7.65 ( s , 1h ) , 7.55 ( dd , j = 1.4 , 7.9 hz , 1h ) , 7.08 
 ( t , j = 7.7 hz , 1h ) , 5.04 ( s , 2h ) , 2.58 ( t , j = 7.5 hz , 2h ) , 1.781.65 ( m , 2h ) , 1.481.30 
 ( m , 4h ) , 0.92 ( t , j = 7.0 hz , 3h ) . c 
 nmr ( 126 mhz , cdcl3 )  157.09 , 143.79 , 135.84 , 132.41 , 
 126.93 , 125.67 , 124.65 , 122.94 , 120.61 , 31.76 , 31.08 , 27.58 , 22.67 , 
 14.16 . ms ( esi - tof ) for c14h17brn2 [ m + h ] calculated 293.0648 , found 293.0675 . h nmr ( 500 mhz , cdcl3 )  7.86 ( s , 1h ) , 7.56 ( d , j = 8.4 hz , 1h ) , 7.44 ( dd , j = 7.1 , 8.4 
 hz , 1h ) , 7.10 ( dd , j = 1.1 , 7.1 hz , 1h ) , 4.83 ( s , 
 2h ) , 3.15 ( t , j = 7.4 hz , 2h ) , 2.62 ( t , j = 7.7 hz , 2h ) , 2.36 ( t , j = 7.0 hz , 2h ) , 2.122.01 
 ( m , 2h ) , 1.791.68 ( m , 2h ) , 1.451.37 ( m , 4h ) , 0.93 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  155.95 , 147.16 , 135.47 , 131.33 , 128.61 , 
 125.08 , 123.79 , 123.28 , 122.75 , 119.71 , 31.84 , 31.77 , 31.02 , 27.95 , 
 26.64 , 22.66 , 16.84 , 14.21 . ms ( esi - tof ) for c18h23n3 [ m + h ] calculated 282.1965 , found 282.1904 . h 
 nmr ( 500 mhz , cdcl3 )  7.84 ( s , 1h ) , 7.54 ( d , j = 8.4 hz , 1h ) , 7.43 ( dd , j = 7.1 , 8.4 
 hz , 1h ) , 7.07 ( dd , j = 1.1 , 7.1 hz , 1h ) , 4.80 ( s , 
 2h ) , 3.02 ( t , j = 7.5 hz , 2h ) , 2.62 ( t , j = 7.6 hz , 2h ) , 2.37 ( t , j = 7.1 hz , 2h ) , 1.931.83 
 ( m , 2h ) , 1.801.69 ( m , 4h ) , 1.471.36 ( m , 4h ) , 0.93 
 ( t , j = 7.1 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  155.86 , 147.12 , 136.99 , 131.59 , 128.56 , 
 124.66 , 123.49 , 123.01 , 122.83 , 119.68 , 31.82 , 31.77 , 31.69 , 29.93 , 
 27.97 , 25.25 , 22.67 , 17.27 , 14.22 . ms ( esi - tof ) for c19h25n3 [ m + h ] calculated 296.2121 , 
 found 296.2068 . h nmr ( 500 mhz , cdcl3 )  7.84 ( s , 1h ) , 7.53 ( d , j = 8.4 hz , 1h ) , 7.42 ( dd , j = 7.1 , 8.4 
 hz , 1h ) , 7.07 ( dd , j = 1.2 , 7.0 hz , 1h ) , 4.80 ( s , 
 2h ) , 2.99 ( t , j = 7.6 hz , 2h ) , 2.62 ( t , j = 7.7 hz , 2h ) , 2.34 ( t , j = 7.1 hz , 2h ) , 1.801.64 
 ( m , 6h ) , 1.621.53 ( m , 2h ) , 1.451.36 ( m , 4h ) , 0.93 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  155.81 , 147.02 , 137.81 , 131.78 , 128.58 , 
 124.38 , 123.33 , 122.91 , 122.86 , 119.84 , 32.30 , 31.79 , 31.75 , 30.36 , 
 28.78 , 27.98 , 25.48 , 22.68 , 17.29 , 14.22 . ms ( esi - tof ) for c20h27n3 [ m + h ] calculated 310.2278 , 
 found 310.2323 . h nmr ( 500 mhz , cdcl3 )  7.64 ( s , 1h ) , 7.60 ( d , j = 8.5 hz , 1h ) , 7.41 ( d , j = 2.0 hz , 1h ) , 
 7.34 ( dd , j = 2.1 , 8.5 hz , 1h ) , 4.80 ( s , 2h ) , 2.89 
 ( t , j = 7.3 hz , 2h ) , 2.58 ( t , j = 
 7.7 hz , 2h ) , 2.33 ( t , j = 7.1 hz , 2h ) , 2.131.95 
 ( m , 2h ) , 1.791.65 ( m , 2h ) , 1.451.36 ( m , 4h ) , 0.93 
 ( t , j = 7.1 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  156.14 , 145.35 , 135.19 , 133.81 , 129.65 , 
 126.26 , 126.06 , 124.58 , 124.15 , 119.73 , 34.18 , 31.80 , 31.31 , 27.71 , 
 27.02 , 22.69 , 16.47 , 14.19 . ms ( esi - tof ) for c18h23n3 [ m + h ] calculated 282.1965 , found 282.1832 . h nmr ( 500 mhz , cdcl3 )  7.63 ( s , 1h ) , 7.59 ( d , j = 8.5 hz , 1h ) , 7.37 ( d , j = 1.9 hz , 1h ) , 
 7.34 ( dd , j = 2.1 , 8.5 hz , 1h ) , 4.78 ( s , 2h ) , 2.76 
 ( t , j = 7.4 hz , 2h ) , 2.57 ( t , j = 
 7.8 hz , 2h ) , 2.36 ( t , j = 7.1 hz , 2h ) , 1.891.80 
 ( m , 2h ) , 1.761.69 ( m , 4h ) , 1.481.30 ( m , 4h ) , 0.93 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 cdcl3 )  155.97 , 145.13 , 135.46 , 135.22 , 129.91 , 
 125.82 , 125.76 , 124.56 , 123.98 , 119.80 , 34.84 , 31.80 , 31.32 , 30.41 , 
 27.74 , 24.96 , 22.70 , 17.26 , 14.19 . ms ( esi - tof ) for c19h25n3 [ m + h ] calculated 296.2121 , 
 found 296.2146 . h nmr ( 500 mhz , cdcl3 )  7.63 ( s , 1h ) , 7.58 
 ( d , j = 8.5 hz , 1h ) , 7.37 ( d , j = 
 1.9 hz , 1h ) , 7.34 ( dd , j = 2.1 , 8.5 hz , 1h ) , 4.77 
 ( s , 2h ) , 2.73 ( t , j = 7.6 hz , 2h ) , 2.57 ( t , j = 7.7 hz , 2h ) , 2.33 ( t , j = 7.1 hz , 2h ) , 
 1.781.61 ( m , 6h ) , 1.561.46 ( m , 2h ) , 1.441.36 
 ( m , 4h ) , 0.93 ( t , j = 7.0 hz , 3h ) . c 
 nmr ( 126 mhz , cdcl3 )  155.89 , 145.03 , 136.28 , 135.24 , 
 130.01 , 125.75 , 125.63 , 124.55 , 123.88 , 119.93 , 35.44 , 31.81 , 31.33 , 
 30.77 , 28.42 , 27.76 , 25.47 , 22.70 , 17.27 , 14.19 . ms ( esi - tof ) for 
 c20h27n3 [ m + h ] calculated 
 310.2278 , found 310.2309 . h nmr ( 500 mhz , cdcl3 )  7.66 ( s , 1h ) , 7.55 
 ( d , j = 8.1 hz , 1h ) , 7.45 ( s , 1h ) , 7.09 ( dd , j = 1.7 , 8.1 hz , 1h ) , 4.83 ( s , 2h ) , 2.91 ( t , j = 7.3 hz , 2h ) , 2.57 ( t , j = 7.6 hz , 2h ) , 2.34 ( t , j = 7.1 hz , 2h ) , 2.122.02 ( m , 2h ) , 1.781.68 
 ( m , 2h ) , 1.511.31 ( m , 4h ) , 0.93 ( t , j = 7.0 
 hz , 3h ) . c nmr ( 126 mhz , cdcl3 )  156.54 , 
 146.63 , 140.61 , 135.31 , 127.40 , 124.69 , 123.73 , 123.46 , 123.24 , 119.70 , 
 34.72 , 31.80 , 31.27 , 27.73 , 26.85 , 22.69 , 16.52 , 14.19 . ms ( esi - tof ) 
 for c18h23n3 [ m + h ] calculated 
 282.1965 , found 282.1978 . h nmr ( 500 mhz , cdcl3 )  7.66 ( s , 1h ) , 7.54 
 ( d , j = 8.1 hz , 1h ) , 7.43 ( s , 1h ) , 7.09 ( dd , j = 1.7 , 8.2 hz , 1h ) , 4.81 ( s , 2h ) , 2.79 ( t , j = 7.4 hz , 2h ) , 2.57 ( t , j = 7.6 hz , 2h ) , 2.34 ( t , j = 7.1 hz , 2h ) , 1.941.81 ( m , 2h ) , 1.771.63 
 ( m , 4h ) , 1.451.36 ( m , 4h ) , 0.93 ( t , 3h ) . c nmr 
 ( 126 mhz , cdcl3 )  156.44 , 146.56 , 142.18 , 135.36 , 
 127.15 , 124.52 , 123.82 , 123.20 , 123.00 , 119.77 , 35.26 , 31.80 , 31.26 , 
 30.06 , 27.75 , 24.87 , 22.69 , 17.22 , 14.19 . ms ( esi - tof ) for c19h25n3 ms 
 ( esi - tof ) for c20h27n3 [ m + h ] calculated 310.2278 , found 310.2309 . h nmr ( 500 mhz , cdcl3 )  7.66 ( s , 1h ) , 7.49 
 ( dd , j = 1.5 , 8.0 hz , 1h ) , 7.35 ( dd , j = 1.4 , 7.1 hz , 1h ) , 7.16 ( dd , j = 7.1 , 8.0 hz , 
 1h ) , 4.79 ( s , 2h ) , 3.23 ( t , j = 7.2 hz , 2h ) , 2.58 
 ( t , j = 7.6 hz , 2h ) , 2.32 ( t , j = 
 7.1 hz , 2h ) , 2.192.09 ( m , 2h ) , 1.791.66 ( m , 2h ) , 1.451.33 
 ( m , 4h ) , 0.93 ( t , j = 7.1 hz , 3h ) . c 
 nmr ( 126 mhz , cdcl3 )  155.58 , 145.03 , 135.76 , 135.33 , 
 128.76 , 125.91 , 124.72 , 123.64 , 122.30 , 120.54 , 31.83 , 31.28 , 30.49 , 
 27.78 , 26.53 , 22.70 , 16.95 , 14.19 . ms ( esi - tof ) for c18h23n3 [ m + h ] calculated 282.1965 , 
 found 282.2008 . h nmr ( 500 mhz , meod )  7.95 ( s , 1h ) , 
 7.427.35 ( m , 2h ) , 7.08 ( dd , j = 3.1 , 5.1 
 hz , 1h ) , 3.00 ( t , j = 7.6 hz , 2h ) , 2.692.63 
 ( m , 4h ) , 1.781.64 ( m , 4h ) , 1.611.50 ( m , 2h ) , 1.461.40 
 ( m , 4h ) , 0.95 ( t , j = 7.0 hz , 3h ) . c 
 nmr ( 126 mhz , meod )  157.97 , 147.47 , 139.74 , 133.37 , 129.56 , 
 125.18 , 123.77 , 123.64 , 123.41 , 42.51 , 33.75 , 33.21 , 32.72 , 32.07 , 
 29.82 , 29.05 , 23.68 , 14.47 . ms ( esi - tof ) for c18h27n3 [ m + h ] calculated 286.2278 , found 286.2218 . h nmr ( 500 mhz , meod )  7.94 ( s , 1h ) , 7.427.32 
 ( m , 2h ) , 7.06 ( dd , j = 2.9 , 5.4 hz , 1h ) , 2.98 ( t , j = 7.6 hz , 2h ) , 2.67 ( t , j = 7.6 hz , 2h ) , 
 2.62 ( t , j = 7.0 hz , 2h ) , 1.781.65 ( m , 4h ) , 
 1.571.47 ( m , 2h ) , 1.491.39 ( m , 6h ) , 0.95 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , meod )  
 157.96 , 147.46 , 139.91 , 133.37 , 129.57 , 125.12 , 123.73 , 123.58 , 123.39 , 
 42.53 , 33.77 , 33.35 , 32.69 , 32.39 , 32.04 , 29.03 , 27.94 , 23.68 , 14.47 . 
 ms ( esi - tof ) for c19h29n3 [ m + h ] calculated 300.2434 , found 300.2374 . h nmr ( 500 mhz , meod ) 
  7.95 ( s , 1h ) , 7.417.32 ( m , 2h ) , 7.06 ( dd , j = 3.0 , 5.3 hz , 1h ) , 2.98 ( t , j = 7.6 
 hz , 2h ) , 2.67 ( t , j = 7.4 hz , 2h ) , 2.63 ( t , j = 7.0 hz , 2h ) , 1.71 ( dq , j = 7.8 , 15.5 
 hz , 4h ) , 1.501.37 ( m , 10h ) , 0.95 ( t , j = 
 7.0 hz , 3h ) . c nmr ( 126 mhz , meod )  157.96 , 147.45 , 
 140.00 , 133.37 , 129.56 , 125.08 , 123.72 , 123.55 , 123.39 , 42.40 , 33.46 , 
 33.34 , 32.68 , 32.51 , 32.00 , 30.48 , 28.99 , 27.93 , 23.68 , 14.47 . ms 
 ( esi - tof ) for c20h31n3 [ m + h ] calculated 314.2591 , found 314.2537 . h nmr ( 500 
 mhz , meod )  8.02 ( s , 1h ) , 7.457.31 ( m , 2h ) , 7.08 ( dd , j = 2.9 , 5.4 hz , 1h ) , 3.01 ( t , j = 7.7 
 hz , 2h ) , 2.68 ( t , j = 7.7 hz , 2h ) , 2.30 ( t , j = 7.3 hz , 2h ) , 2.031.91 ( m , 2h ) , 1.801.66 
 ( m , 2h ) , 1.481.38 ( m , 4h ) , 0.94 ( t , j = 7.0 
 hz , 3h ) . c nmr ( 126 mhz , meod )  178.76 , 157.96 , 
 147.27 , 139.18 , 133.57 , 129.64 , 125.45 , 123.92 , 123.69 , 123.43 , 35.95 , 
 32.85 , 32.76 , 32.13 , 29.12 , 28.38 , 23.68 , 14.47 . ms ( esi - tof ) for 
 c18h25n3o [ m + h ] calculated 
 300.2070 , found 300.2025 . 1h - pyrazole-1-carboxamidine 
 hydrochloride ( 16 mg , 0.11 mmol ) and et3n ( 15.3 l , 
 0.11 mmol ) were added to a solution of compound 34a ( 28.5 
 mg , 0.1 mmol ) in meoh ( 2 ml ) , and the reaction mixture was stirred 
 for 3 h at room temperature . the solvent was removed under reduced 
 pressure and the crude material was purified by basic - alumina column 
 chromatography ( 30% meoh / ch2cl2 ) to obtain 34e as a white solid ( 16 mg , 49% ) . h nmr ( 500 
 mhz , dmso - d6 )  7.82 ( s , 1h ) , 7.60 
 ( t , j = 5.7 hz , 1h ) , 7.367.24 ( m , 3h ) , 6.98 
 ( dd , j = 3.1 , 5.3 hz , 1h ) , 6.84 ( s , 2h ) , 6.22 ( s , 
 2h ) , 3.13 ( q , j = 6.9 hz , 2h ) , 2.92 ( t , j = 7.5 hz , 2h ) , 2.60 ( t , j = 7.6 hz , 2h ) , 1.681.56 
 ( m , 4h ) , 1.581.46 ( m , 2h ) , 1.391.29 ( m , 4h ) , 0.88 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 dmso - d6 )  156.72 , 156.66 , 146.95 , 
 137.62 , 130.71 , 127.62 , 123.66 , 123.27 , 121.56 , 121.26 , 40.52 , 31.08 , 
 30.96 , 30.56 , 28.36 , 27.64 , 27.62 , 22.03 , 14.00 . ms ( esi - tof ) for 
 c19h29n5 [ m + h ] calculated 
 328.2496 , found 328.2444 . h nmr ( 500 mhz , meod ) 
  7.70 ( s , 1h ) , 7.487.40 ( m , 2h ) , 7.36 ( dd , j = 2.0 , 8.5 hz , 1h ) , 2.73 ( t , j = 7.5 
 hz , 2h ) , 2.66 ( t , j = 7.3 hz , 2h ) , 2.62 ( t , j = 7.5 hz , 2h ) , 1.761.66 ( m , 4h ) , 1.571.47 
 ( m , 2h ) , 1.461.39 ( m , 4h ) , 0.94 ( t , j = 7.1 
 hz , 3h ) . c nmr ( 126 mhz , meod )  158.06 , 145.47 , 
 137.66 , 136.72 , 131.08 , 126.94 , 125.75 , 125.30 , 124.97 , 42.38 , 36.34 , 
 33.20 , 32.78 , 31.81 , 29.96 , 28.96 , 23.68 , 14.43 . ms ( esi - tof ) for 
 c18h27n3 [ m + h ] calculated 
 286.2278 , found 286.2283 . h nmr ( 500 mhz , 
 meod )  7.70 ( s , 1h ) , 7.45 ( d , j = 8.5 hz , 
 1h ) , 7.41 ( s , 1h ) , 7.35 ( dd , j = 2.0 , 8.5 hz , 1h ) , 
 2.72 ( t , j = 7.6 hz , 2h ) , 2.662.58 ( m , 4h ) , 
 1.761.65 ( m , 4h ) , 1.551.48 ( m , 2h ) , 1.471.35 
 ( m , 6h ) , 0.95 ( t , j = 6.9 hz , 3h ) . c 
 nmr ( 126 mhz , meod )  158.03 , 145.42 , 137.86 , 136.72 , 131.09 , 
 126.88 , 125.73 , 125.30 , 124.92 , 42.50 , 36.48 , 33.66 , 32.80 , 32.61 , 
 31.82 , 28.97 , 27.61 , 23.68 , 14.43 . ms ( esi - tof ) for c19h29n3 [ m + h ] calculated 300.2434 , 
 found 300.2487 . h nmr ( 500 mhz , 
 meod )  7.70 ( s , 1h ) , 7.44 ( d , j = 8.5 hz , 
 1h ) , 7.40 ( s , 1h ) , 7.34 ( dd , j = 2.0 , 8.5 hz , 1h ) , 
 2.70 ( t , j = 7.6 hz , 2h ) , 2.652.57 ( m , 4h ) , 
 1.761.62 ( m , 4h ) , 1.491.33 ( m , 10h ) , 0.94 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , meod )  
 158.02 , 145.41 , 137.96 , 136.71 , 131.10 , 126.86 , 125.72 , 125.30 , 124.90 , 
 42.52 , 36.50 , 33.73 , 32.80 , 32.70 , 31.83 , 30.18 , 28.97 , 27.89 , 23.68 , 
 14.44 . ms ( esi - tof ) for c20h31n3 [ m 
 + h ] calculated 314.2591 , found 314.2649 . h nmr ( 500 mhz , 
 meod )  7.71 ( s , 1h ) , 7.53 ( d , j = 8.1 hz , 
 1h ) , 7.33 ( s , 1h ) , 7.10 ( dd , j = 1.7 , 8.2 hz , 1h ) , 
 2.75 ( t , j = 7.6 hz , 2h ) , 2.65 ( t , j = 7.2 hz , 2h ) , 2.61 ( t , j = 7.6 hz , 2h ) , 1.781.65 
 ( m , 4h ) , 1.581.48 ( m , 2h ) , 1.461.39 ( m , 4h ) , 0.94 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  158.52 , 147.11 , 144.79 , 136.77 , 128.05 , 124.90 , 124.62 , 
 123.96 , 123.62 , 42.44 , 36.97 , 33.45 , 32.77 , 31.75 , 29.78 , 28.97 , 23.68 , 
 14.42 . ms ( esi - tof ) for c18h27n3 [ m 
 + h ] calculated 286.2278 , found 286.2287 . h nmr ( 500 mhz , meod ) 
  7.71 ( s , 1h ) , 7.53 ( d , j = 8.1 hz , 1h ) , 7.32 
 ( s , 1h ) , 7.08 ( dd , j = 1.7 , 8.1 hz , 1h ) , 2.74 ( t , j = 7.6 hz , 2h ) , 2.652.58 ( m , 4h ) , 1.761.66 
 ( m , 4h ) , 1.561.47 ( m , 2h ) , 1.461.36 ( m , 6h ) , 0.94 
 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  158.52 , 147.10 , 144.96 , 136.78 , 128.02 , 124.88 , 124.64 , 
 123.90 , 123.58 , 42.47 , 37.08 , 33.62 , 32.78 , 32.38 , 31.75 , 28.97 , 27.62 , 
 23.68 , 14.43 . ms ( esi - tof ) for c19h29n3 [ m + h ] calculated 300.2434 , found 300.2486 . h nmr ( 500 mhz , meod ) 
  7.71 ( d , j = 0.9 hz , 1h ) , 7.53 ( d , j = 8.2 hz , 1h ) , 7.32 ( s , 1h ) , 7.08 ( dd , j = 1.7 , 8.1 hz , 1h ) , 2.73 ( t , j = 7.6 hz , 2h ) , 2.662.56 
 ( m , 4h ) , 1.781.65 ( m , 4h ) , 1.551.35 ( m , 10h ) , 0.94 
 ( t , j = 7.1 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  158.50 , 147.09 , 145.03 , 136.78 , 128.00 , 124.86 , 124.65 , 
 123.89 , 123.56 , 42.40 , 37.07 , 33.42 , 32.79 , 32.43 , 31.75 , 30.14 , 28.98 , 
 27.85 , 23.69 , 14.43 . ms ( esi - tof ) for c20h31n3 h nmr ( 500 mhz , meod )  7.68 ( s , 1h ) , 7.44 
 ( dd , j = 1.5 , 8.0 hz , 1h ) , 7.32 ( dd , j = 1.4 , 7.1 hz , 1h ) , 7.10 ( dd , j = 7.1 , 8.0 hz , 
 1h ) , 3.06 ( t , j = 7.7 hz , 2h ) , 2.69 ( t , j = 7.1 hz , 2h ) , 2.62 ( t , j = 7.7 hz , 2h ) , 1.811.68 
 ( m , 4h ) , 1.611.51 ( m , 2h ) , 1.461.39 ( m , 4h ) , 0.94 
 ( t , j = 6.9 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  157.96 , 145.92 , 137.83 , 137.02 , 129.40 , 126.33 , 125.61 , 
 125.14 , 122.79 , 42.40 , 33.63 , 32.83 , 32.10 , 31.86 , 28.96 , 28.93 , 23.69 , 
 14.43 . ms ( esi - tof ) for c18h27n3 [ m 
 + h ] calculated 286.2278 , found 286.2283 . a solution of compound 38 ( 737 
 mg , 2.5 mmol ) in thf ( 20 ml ) was added slowly to a solution of lialh4 ( 10 ml , 10 mmol , 1.0 m in thf ) in thf ( 10 ml ) at 0 c 
 under nitrogen atmosphere . the reaction mixture was carefully quenched with 
 ice - cold water ( 1 ml ) at 0 c , and 10% naoh ( 1 ml ) was added . the resulting mixture was stirred for 10 min at room temperature , 
 filtered through celite and washed with ch2cl2 ( 50 ml ) . the resulting filtrate was dried over na2so4 and concentrated under reduced pressure and the crude material 
 was purified by flash column chromatography ( 30% etoac / hexanes ) to 
 obtain the compound 39 as a off - white solid ( 386 mg , 
 55% ) . ms ( esi - tof ) for c7h7br2no 
 [ m + h ] calculated 279.8967 , found 279.8975 . to a solution 
 of compound 39 ( 351 mg , 1.25 mmol ) in ch2cl2 ( 10 ml ) was added mno2 ( 326 mg , 3.75 mmol , activated ) . the 
 mixture was concentrated and purified by flash chromatography ( 20% 
 etoac / hexanes ) to give compound 40 as a yellow solid 
 ( 286 mg , 82% ) . h nmr ( 400 mhz , cdcl3 )  
 10.33 ( s , 1h ) , 7.07 ( d , j = 1.8 hz , 1h ) , 6.81 ( d , j = 1.1 hz , 1h ) , 6.56 ( s , 2h ) . c nmr ( 126 mhz , 
 cdcl3 )  194.69 , 152.20 , 130.08 , 129.98 , 124.03 , 
 118.97 , 113.96 . ms ( esi - tof ) for c7h5br2no [ m + h ] calculated 277.8811 , found 277.8811 . h nmr ( 400 mhz , cdcl3 )  7.96 
 ( s , 1h ) , 7.77 ( dd , j = 0.8 , 1.9 hz , 1h ) , 7.61 ( d , j = 1.9 hz , 1h ) , 4.98 ( s , 2h ) , 2.58 ( t , j = 7.6 hz , 2h ) , 1.811.68 ( m , 2h ) , 1.481.36 ( m , 4h ) , 
 0.94 ( t , j = 7.0 hz , 3h ) . c nmr ( 126 
 mhz , cdcl3 )  157.24 , 147.77 , 134.54 , 128.98 , 128.00 , 
 125.45 , 122.50 , 121.99 , 121.86 , 31.78 , 31.35 , 27.55 , 22.65 , 14.18 . ms ( esi - tof ) for c14h16br2n2 [ m + h ] calculated 370.9753 , found 370.9747 . to a solution of compound 41 ( 74 mg , 0.2 mmol ) in thf ( 2 ml ) were added 4-cyanobutylzinc bromide 
 ( 1.6 ml , 0.8 mmol , 0.5 m in thf ) and pd(pph3)4 ( 23 mg , 0.02 mmol ) . the resulting reaction mixture was stirred at 
 65 c under nitrogen atmosphere for 24 h. the reaction mixture 
 was diluted with water and extracted with etoac ( 3  10 ml ) . the crude material was 
 purified by flash chromatography ( 20% meoh / ch2cl2 ) to obtain the compound 42 as a pale yellow solid ( 19 
 mg , 25% ) . ms ( esi - tof ) for c24h32n4 [ m + h ] calculated 377.2700 , found 377.2691 . a solution 
 of compound 42 ( 19 mg , 0.05 mmol ) in thf ( 5 ml ) was added 
 slowly to a solution of lialh4 ( 0.5 ml , 0.5 mmol , 1.0 m 
 in thf ) in thf ( 3 ml ) at 0 c under nitrogen atmosphere . the 
 reaction mixture was stirred for 2 h at 25 c and 2 h at 60 c . the reaction mixture was carefully quenched with ice - cold water ( 1 
 ml ) at 0 c , and 10% naoh ( 1 ml ) was added . the resulting mixture 
 was stirred for 10 min at room temperature , filtered through celite , 
 and washed with ch2cl2 ( 25 ml ) . the resulting 
 filtrate was dried over na2so4 and concentrated 
 under reduced pressure and the crude material was purified by semipreparative 
 reverse phase hplc to obtain the compound 43 as a white 
 solid ( 5 mg , 26% ) . h nmr ( 400 mhz , meod )  8.33 
 ( s , 1h ) , 7.39 ( s , 1h ) , 7.28 ( s , 1h ) , 3.07 ( t , j = 
 7.7 hz , 2h ) , 2.94 ( t , j = 7.6 hz , 4h ) , 2.862.73 
 ( m , 4h ) , 1.821.66 ( m , 10h ) , 1.581.40 ( m , 8h ) , 0.95 
 ( t , j = 6.8 hz , 3h ) . c nmr ( 126 mhz , 
 meod )  154.80 , 148.71 , 141.75 , 138.97 , 136.98 , 128.10 , 125.79 , 
 119.63 , 115.24 , 40.68 , 40.68 , 36.73 , 32.74 , 32.45 , 32.12 , 31.61 , 30.84 , 
 29.02 , 28.52 , 28.42 , 27.37 , 27.14 , 23.63 , 14.44 . ms ( esi - tof ) for 
 c24h40n4 [ m + h ] calculated 
 385.3326 , found 385.3330 . the extracellular domain of human tlr8 ( htlr8 , residues 27827 ) 
 was prepared as described previously and 
 was concentrated to 16 mg / ml in 10 mm mes ( ph 5.5 ) , 50 mm nacl . the 
 protein solutions for the crystallization of htlr8/compound complexes 
 contained htlr8 ( 8.5 mg / ml ) and compound ( protein / compound molar ratio 
 of 1:10 ) in a crystallization buffer containing 7 mm mes ( ph 5.5 ) , 
 35 mm nacl . crystallization experiments were performed with sitting - drop 
 vapor - diffusion methods at 293 k. crystals of htlr8/compound were 
 obtained with reservoir solutions containing 912% ( w / v ) peg3350 , 
 0.3 m potassium formate , and 0.1 m sodium citrate ( ph 4.85.2 ) . diffraction 
 data sets were collected on beamlines pf - ar ne3a ( ibaraki , japan ) 
 and spring-8 bl41xu under cryogenic conditions at 100 k. crystals 
 of htlr8/compound were soaked into a cryoprotectant solution containing 
 15% ( w / v ) peg3350 , 0.23 m potassium formate , 75 mm sodium citrate , 
 ph 4.85.2 , 7.5 mm mes ph 5.5 , 38 mm nacl , and 25% glycerol . htlr8/compound 
 structures were determined by the molecular replacement method using 
 the molrep program with the htlr8/cl097 
 structure ( pdb code 3w3j ) as a search model . the model was further refined with stepwise 
 cycles of manual model building using the coot program and restrained refinement using refmac until the r factor was converged . compound molecule , n - glycans , and water molecules 
 were modeled into the electron density maps at the latter cycles of 
 the refinement . the statistics of the data 
 collection and refinement are also summarized in table s1 . the figures representing structures were prepared 
 with pymol ( schrdinger , new york , ny ) . coordinates have been 
 deposited in the protein data bank of the research collaboratory for 
 structural bioinformatics ; pdb codes for compounds 1 and 2 are , respectively , 5awd and 5awb . the induction of nf-b was quantified using human tlr-2 , -3 , 
 -4 , -5 , -7 , -8 , -9 , and nod-1/nod-2-specific , rapid - throughput , liquid 
 handler - assisted reporter gene assays as previously described by us . co - transfected with the appropriate htlr ( or 
 nod ) and secreted alkaline phosphatase ( sap ) were maintained in hek - blue 
 selection medium . stable expression of secreted alkaline phosphatase 
 ( sap ) under control of nf-b / ap-1 promoters is inducible by 
 appropriate tlr / nod agonists , and extracellular sap in the supernatant 
 is proportional to nf-b induction . reporter cells were incubated 
 at a density of 10 cells / ml in a volume of 80 
 l / well , in 384-well , flat - bottomed , cell culture - treated microtiter 
 plates in the presence of graded concentrations of stimuli . sap was 
 assayed spectrophotometrically using an alkaline phosphatase - specific 
 chromogen ( present in hek - detection medium as supplied by invivogen ) 
 at 620 nm . none of the compounds were active in the counterscreens 
 ( data not shown ) , confirming specificity for human tlr8 . fresh human peripheral blood mononuclear cells 
 ( hpbmc ) were isolated from human blood obtained by venipuncture with 
 informed consent and as per institutional guidelines on ficoll aliquots of pbmcs ( 10 cells in 100 l / well ) 
 were stimulated for 12 h with graded concentrations of test compounds . supernatants were isolated by centrifugation and were assayed in duplicates 
 using analyte - specific multiplexed cytokine / chemokine bead array assays 
 as reported by us previously . all experiments were performed at harlan laboratories ( indianapolis , 
 in ) in accordance with institutional guidelines . all antigen / adjuvant 
 preparations were entirely aqueous ; no liposomal or emulsifying agents 
 were used . cohorts of adult female new zealand wwhite rabbits ( n = 4 ) were immunized intramuscularly in the flank region 
 with ( a ) 10 g of crm197 in 0.2 
 ml of saline ( unadjuvanted control ) or ( b ) 10 g of crm197 in 
 0.2 ml of saline plus 100 g of lead tlr8 agonists . preimmune 
 test - bleeds were first obtained via venipuncture of the marginal vein 
 of the ear . a final 
 test - bleed was performed via the marginal vein of the ear on day 38 . crm197-specific 
 elisas were performed in 384-well format using automated liquid handling 
 methods as described by us elsewhere . a precision 2000 liquid handler ( bio - tek , winooski , vt ) was used 
 for all serial dilution and reagent addition steps , and a bio - tek 
 elx405 384-well plate washer was employed for plate washes ; 100 mm 
 phosphate - buffered saline ( pbs ) , ph 7.4 , containing 0.1% tween-20 
 was used as wash buffer . nunc - immuno maxisorp ( 384-well ) plates were 
 coated with 30 ml of crm197 ( 10 g / ml ) in 100 mm carbonate buffer , 
 ph 9.0 , overnight at 4 c . after 3 washes , the plates were blocked 
 with 3% bovine serum albumin ( in pbs , ph 7.4 ) for 1 h at rt . serum 
 samples ( in quadruplicate ) were serially diluted in a separate 384-well 
 plate using the liquid handler , and an amount of 30 l of the 
 serum dilutions was transferred using the liquid handler , and the 
 plate was incubated at 37 c for 2 h. the assay plate was washed 
 three times , and 30 l of 1:10  000 diluted appropriate 
 anti - rabbit immunoglobulin ( igg ,  chain ) conjugated with horseradish 
 peroxidase was added to all wells . following an incubation step at 
 37 c for 1 h and three washes , tetramethylbenzidine substrate 
 was added at concentrations recommended by vendor ( sigma ) . the chromogenic 
 reaction was terminated at 30 min by the addition of 2 m h2so4 . plates were then read at 450 nm using a spectramax 
 m4 device ( molecular devices , sunnyvale , ca ) . cell surface 
 marker upregulation was determined by flow cytometry using protocols 
 published by us previously and modified 
 for rapid throughput . briefly , heparin - anticoagulated whole blood 
 samples were obtained by venipuncture from healthy human volunteers 
 with informed consent and as per guidelines approved by the university 
 of kansas human subjects experimentation committee . serial dilutions 
 of selected compounds were performed using a bio - tek precision 2000 
 xs liquid handler in sterile 96-well polypropylene plates , to which 
 were added 100 ml aliquots of anticoagulated whole human blood . the 
 plates were incubated at 37 c for 16 h. negative ( endotoxin 
 free water ) controls were included in each experiment . the following 
 fluorochrome - conjugated antibodies were used : cd3-pe , cd19-fitc , cd56-apc 
 ( ebioscience , san diego , ca ) , cd14-v500 , cd28 pe - cy7 , cd40 v450 , cd80 
 apc - h7 , cd86 percp - cy5.5 ( becton - dickinson biosciences , san jose , 
 ca ) . following incubation , 2.5 g of each antibody was added 
 to wells with a liquid handler and incubated at 4 c in the dark 
 for 60 min . following staining , erythrocytes were lysed and leukocytes 
 fixed by mixing 200 ml of the samples in 800 ml prewarmed whole blood 
 lyse / fix buffer ( becton - dickinson biosciences , san jose , ca ) in 96 
 deep - well plates . after washing the cells twice at 300 g for 10 min 
 in rpmi , the cells were transferred to a 96-well plate . flow cytometry 
 was performed using a bd facsverse instrument for acquisition on 100  000 
 gated events . compensation for spillover was computed for each experiment 
 on singly stained comp beads ( becton - dickinson biosciences , san jose , 
 ca ) . cd28 , cd40 , cd80 , and cd86 activation in the major leukocyte 
 populations , viz . , natural killer lymphocytes ( nk cells cd3cd56 ) , cytokine - induced killer phenotype ( cik cells cd3cd56 ) , b lymphocytes ( cd19cd3 ) , t lymphocytes ( cd3cd56 ) , monocytes ( cd14 ) , polymorphonuclear cells ( cd14 ) were quantified using flowjo , version 7.0 , software 
 ( treestar , ashland , or ) .
human 
 toll - like receptor 8 ( htlr8 ) is expressed in myeloid dendritic cells , 
 monocytes , and monocyte - derived dendritic cells . 
 engagement by tlr8 
 agonists evokes a distinct cytokine profile which favors the development 
 of type 1 helper t cells . 
 crystal structures of the ectodomain of 
 htlr8 cocrystallized with two regioisomers of a dual tlr7/8-agonistic 
 n1-substituted imidazoquinolines showed subtle differences in their 
 interactions in the binding site of htlr8 . 
 we hypothesized that the 
 potency of a previously reported best - in - class pure tlr8 agonist , 
 3-pentylquinoline-2-amine , could be further enhanced by  designing 
 in  functional groups that would mimic key intermolecular interactions 
 that we had observed in the crystal structures . 
 we performed a focused 
 exploration of decorating the quinoline core with alkylamino groups 
 at all possible positions . 
 these studies have led to the identification 
 of a novel tlr8 agonist that was 20-fold more potent than 
 the parent compound and displays prominent adjuvantic activity in 
 a rabbit model of immunization .