schizophrenia is a 
 complex mental illness characterized by positive 
 ( hallucinations , paranoia , disorganized behavior ) and negative symptoms 
 ( social withdrawal , anhedonia , flat affect ) as well as cognitive dysfunction 
 ( deficits in attention , learning , and memory ) . current treatments , including 
 typical and second - generation atypical antipsychotics , are based largely 
 upon the dopaminergic hypothesis of schizophrenia , which targets overactivation 
 of subcortical dopamine d2 receptors . both classes treat the positive symptoms ; however , neither 
 class of antipsychotics has made a substantial impact on the negative 
 and cognitive symptoms . after several decades of clinical use , a significant understanding 
 of the risks and side effect profiles that plague both classes of 
 antipsychotics has been gained . these include extrapyramidal side 
 effects , sexual dysfunction , weight gain ( metabolic syndrome ) , agranulocytosis , 
 increased cardiac risk , and poor patient compliance . the development of positive 
 allosteric modulators ( pams ) of metabotropic 
 glutamate receptor 5 ( mglu5 ) as a novel approach to test the glutamate or n - methyl - d - aspartate ( nmda ) receptor hypofunction hypothesis of schizophrenia has provided preclinical evidence for therapeutic potential in multiple 
 psychosis and cognitive animal models , and , for more than a decade , industry and academic drug discovery 
 groups have been in pursuit of brain penetrant small molecule mglu5 potentiators . a testament to the success of 
 the field has been the identification of over eight reported chemotypes with in vivo efficacy in preclinical models , beginning with 3-cyano - n-(1,3-diphenyl-1h - pyrazol-5-yl)benzamide 
 ( cdppb , 1 ) , piperidinyl 
 1,2,4-oxadiazoles from addex ( adx-47273 , 2 ) , and subsequently acetylenes n - methyl-5-(phenylethynyl)pyrimidin-2-amine 
 ( mppa , 3 ) , vu0360172 ( 4 ) , lsn2463359 ( 8) , piperazines vu0364289 ( 5 ) and 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone 
 ( cppz , 6 ) , ether vu0404251 
 ( 7 ) , and triazole lsn2814617 
 from lilly ( 9 ) . however , 
 recent findings from merck - addex and at the vanderbilt center for 
 neuroscience drug discovery ( vcndd ) have unveiled a target mediated 
 cns adverse - effect ( ae ) liability , driven by excessive glutamate fold 
 potentation or allosteric agonism , respectively ; suggesting that pams with lower functional cooperativity 
 with glutamate ( e.g. , glutamate fold - shift or potentiation ) and devoid 
 of allosteric agonism may be preferred for improved therapeutic index . more recently , we disclosed a phenoxy - based 
 ether dihydrothiazolopyridone series , with the representative in vivo tool compound vu0408899 ( 10 ) ( figure 1 ) . ether 10 represents , 
 in part , efforts to constrain amides of type 7 ; however , 
 due to inherent instability in acidic media ( 20% hp--cd in 
 water , ph = 4 ) leading to the corresponding 2-hydroxy - dihydrothiazolopyridone 
 fragment , full exploration of the analogous system containing a benzyloxy 
 ether linkage was not possible . activity 
 relationships ( sar ) suggested a preference for the phenoxy - based linker 
 within the dihydrothiazolopyridones ( two examples tested ) ; however , this trend was not found within monocyclic 
 nicotinamides of type 7 . thus , it was unknown if alteration of the linker moiety within the 
 context of a bicyclic system to more closely mimic 7 ( e.g. , 
 dihydronaphthyridinone core structures 1217 , figure 2 ) , would be productive 
 for mglu5 receptor potentiation . prior studies within a 
 dihydronaphthyridinone class utilizing an acetylene - based linker , 
 initially identified from an hts screen , led to pams with submicromolar 
 potency , including dihydronaphthyridinones 11 , suggested that linkers employing either ch2o or och2 might be successful 
 as an acetylene replacement . within acetylenic dihydronaphthyridinones 11 via simple amide congeners led to fundamental changes in 
 the mode of pharmacology ( e.g. , from pams to negative allosteric modulators 
 or nams ) . the pursuit of ethers 1220 were undertaken as part of a larger 
 comprehensive study aimed to target four key structure activity 
 relationships : ( 1 ) endocyclic versus exocyclic amide , ( 2 ) phenoxy 
 versus benzyloxy linker , ( 3 ) central ring pyridine isomers , and ( 4 ) 
 site of the pendant group attachment to central heterocycle . it was 
 unclear at the outset if ethers 1220 would exhibit subtle changes in the mode of pharmacology similar 
 to 11(32 ) and other chemotypes or would maintain a higher fidelity 
 for potentiation similar to cdppb ( 1),n-[5-chloro-2-[(1,3-dioxoisoindolin-2-yl)methyl]phenyl]-2-hydroxybenzamide 
 ( cppha ) , piperazines ( 56 ) , and glycine 
 sulfonamide ( ml332 ) based mglu5 pams . herein we describe the synthesis and pharmacological profile 
 of a series of tetrahydronaphthyridine and dihydronaphthyridinone 
 ethers ( 1220 ) as positive allosteric 
 modulators of mglu5 . low cooperativity pam 12c ( vu0405372 ) was ultimately identified and found to demonstrate a 
 suitable balance of in vitro and in vivo properties for proof - of - concept 
 studies in an amphetamine hyperlocomotor challenge model . in contrast 
 to previously reported and related monocyclic ether series , these bicyclic modulator scaffolds were overall found to exhibit 
 narrow sar , low efficacy , and have a higher propensity for pharmacological 
  mode switching  upon chemical 
 modification , demonstrating an sar profile reminiscent of related 
 acetylenic pams . mglu5 receptor 
 pams with reported efficacy in preclinical 
 models of schizophrenia and cognition . evolution of tetrahydronaphthyridine and dihydronaphthyridinone 
 ether based mglu5 allosteric modulators ( 1220 ) from nicotinamide 7(26 ) and acetylenic modulator scaffold 11 . to prepare bicyclic analogues 
 that most closely 
 mimic the structural features found in 11 , we initially 
 prepared a series of dihydro-1,6-naphthyridin-5(6h)-ones ( 12a12 t , scheme 1 ) . starting from known compound 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 21a , supporting 
 information ) , substitution under basic conditions in the presence 
 of various alcohols with heating afforded 12a12h , 12q , and 12s in low to moderate 
 yield . a subset was treated with alkylating agents under basic conditions , 
 or with various aryl and heteroaryl halides using a copper iodide diamine 
 ligand protocol to effect a modified ullman lactam cross - coupling 
 to give the respective alkyl ( 12i12n , 12s12 t ) and n - aryl ( 12o12p , 12r ) lactam products . reagents and conditions : ( a ) 21a , rch2oh , kot - bu , 
 dmf , 100 c , 416 h , 2363% ; ( b ) lactam , rch2br or rch2cl , kot - bu , dmf , 60 c , 24 h , 4075% ; ( c ) 
 lactam , cui ( 0.2 equiv ) , ar / hetbr , n , n - dimethylethylene diamine , k2co3 , toluene , 
 120 c , 18 h , 3559% . evaluation 
 of the alternate ether linkage based upon an aryloxy 
 methyl dihydronaphthyridinone , series 13 , began synthetically 
 from 21a as shown in scheme 2 . protection with para - methoxybenzyl chloride ( pmbcl ) 
 afforded 21b in good yield . miyaura coupling 
 under molander s conditions using vinyl potassium trifluoroborate 
 salt gave vinyl dihydronaphthyridinone intermediate 22 . mitsunobu alkylation in the presence of either phenol 
 or 3-fluorophenol provided 23a23b in 3540% yield over two steps from 22 . removal 
 of the pmb protecting group using cerium ammonium nitrate proceeded 
 smoothly to give 13a13b . remaining 
 analogues 13c13i were prepared similarly 
 as demonstrated for series 12 , using alkylating conditions 
 or copper - based arylation conditions to give alkyl and n - aryl lactam products 13c13e and 13f13i , respectively . reagents and conditions : ( a ) 21a , pmbcl , kot - bu , dmf , 60 c , 2 h , 
 66% ; ( b ) vinyl potassium trifluoroborate , cs2co3 , pd(pph3)4 , etoh , 80 c , 2 h , 74% ; ( c ) 
 o3 , ch2cl2:meoh , 78 c , 
 40 min , nabh4 , 0 c ; ( d ) roh , diad , pph3 , thf , 0 c , 16 h , 3540% ( over two steps ) ; ( e ) 
 can , ch3cn : h2o , rt , 50 min , 6065% ; ( f ) 13a  b , rch2br or 
 rch2cl , kot - bu , dmf , 60 c , 
 24 h , 4468% ; ( g ) 13a  b , cui ( 0.2 equiv ) , ar / hetbr , n , n - dimethylethylene diamine , k2co3 , toluene , 
 120 c , 18 h , 2589% . within a 
 third class of lactams 14 ( scheme 3 ) , a benzyloxy linkage was retained and an alternate 
 dihydro-1,7-naphthyridinone ring system was explored , placing the 
 pyridine nitrogen at the position ortho to the lactam amide . synthesis 
 of series 14 began with borane reduction of 5,6-dichloronicotinic 
 acid 24 and subsequent cyanation using pd(0 ) catalysis 
 to give 25 in moderate yield . treatment of 25 under mitsunobu conditions using phenol and di - tert - butyl azodicarboxylate ( dbab ) , followed by hydrolysis and ester 
 formation , afforded chloroester 27 . allylation of 27 using stille conditions with allyl tributyltin , followed 
 by osmium tetroxide sodium periodate mediated cleavage , provided 
 key aldehyde intermediate 28 in good yield for the two 
 steps . at this stage , installation of the lactam could be accomplished 
 using a variety of amines in a one - pot two - step reductive alkylation , 
 ring - cyclization reaction using sodium triacetoxyborohydride in thf reagents and conditions : 
 ( a ) 24 , bh3 thf , 0 c , 16 h , 68% ; ( b ) 
 zn(cn)2 , cs2co3 , pd(pph3)4 , dmf , 100 c , 3 h , 56% ; ( c ) dbab , phenol , pph3 , 
 thf , 0120 c , 40 min , 80% ; ( d ) naoh , 115 c , 3 h , 
 quant ; ( e ) ch3i , k2co3 , dmf , rt , 
 16 h , 73% ; ( f ) bu3sn(allyl ) , pd(pph3)4 , toluene , 125 c , 16 h , 56% ; ( g ) naio4 , oso4 , thf , 0 c  rt , 2 h , 74% ; ( h ) rnh2 , 
 thf - meoh , nabh(oac)3 , rt , 16 h , 1525% . synthesis of phenoxymethyl - dihydro-2,7-naphthyridinones 15 was accomplished using a seven - step sequence from methyl-6-methylnicotinate 
 ( 29 ) as shown in scheme 4 and 
 represented the fourth class of dihydronaphthyridinones investigated . chlorination followed by pyridine n - oxide formation 
 using catalytic methyltrioxorhenium(vii ) in the presence of hydrogen 
 peroxide gave intermediate 30 . installation of the vinyl moiety using stille conditions 
 provided key pyridine intermediate 33 in good yield . vinylpyridine 33 smoothly underwent one - pot michael addition 
 and lactam cyclization using ammonia or several alkyl and aryl amines 
 to give 15a15d in moderate to excellent 
 yield . reagents and conditions : ( a ) 29 , pocl3 , 16 h , 95 c , 68% ; ( b ) cat . ch3reo3 , h2o2 , ch2cl2 , 16 h , rt , quant ; ( c ) ac2o , 120 c , 
 45 min , 11% ; ( d ) k2co3 , meoh , rt , 15 min , 92% ; 
 ( e ) dbab , phenol , pph3 , thf , 60 c , 16 h , 73% ; ( f ) 
 bu3(vinyl)sn , pd(pph3)4 , toluene dioxane , 
 90 c , 16 h , 76% ; ( g ) amine / ammonia in thf or meoh , sealed tube , 
 100 c , 16 h , 3993% . alternative 
 tetrahydronaphthyridines wherein the ether linkage 
 is appended at the 3-position as a benzyloxy with an exocyclic amide 
 were achieved according to scheme 5 . starting 
 bromide 34 ullman - based 
 etherification using cui and 1,10-phenanthroline in toluene with heat 
 afforded targets 20d20 g . the related 
 aryloxymethyl targets retaining a similar substitution pattern and 
 acyl - tetrahydronaphthyridine were prepared according to scheme 6 . introduction of 4-fluoro - benzamide provided 
 intermediate 35c , followed by stille cross - coupling to 
 give vinyl 36 . subsequent ozonolysis and reductive work - up 
 gave alcohol 37 , which was elaborated via mitsunobu alkylation 
 to provide 20a20c in low to moderate 
 yields . reagents 
 and conditions : ( a ) 34 , hatu , dipea , dmf , rt , 16 h , 6090% ; 
 ( b ) 35 , arch2oh , cs2co3 , 1,10-phen , cui , 
 toluene , 110 c , 4 h , 2035% . reagents and conditions : 
 ( a ) 34 , 4-fluorobenzoic acid , hatu , dipea , dmf , rt , 16 
 h , 96% ; 
 ( b ) bu3(vinyl)sn , pd(pph3)4 , toluene , 
 120 c , 1 h , 82% ; ( c ) o3 , ch2cl2:meoh , 78 c , 30 min , nabh4 , 0 c ; ( d ) 
 roh , diad , polystyrene pph3 , thf , rt , 
 16 h , 2540% ( over two steps ) . as reported previously , compounds were profiled in a rat mglu5 low receptor expression 
 cell line using a triple add  
 protocol allowing for detection of agonism as well as positive and 
 negative allosteric modulation simultaneously . importantly , we have shown that detection of allosteric agonism 
 observed using the low receptor expressing cell line correlates with 
 functional response observed in native systems and modulators with 
 an allosteric agonist profile have been shown to 
 be associated with epileptiform activity and behavioral convulsions 
 in rodents . in contrast , an allosteric modulator devoid of agonism 
 in these low - expressing mglu5 cells was found not to display 
 a seizure liability in vivo . triple 
 add  protocol provided an important initial in vitro assessment 
 of both agonist and potentiation activity at mglu5 , which 
 could be utilized to prioritize analogues for further progression . sar are shown in tables 14 ( 1215 , 20 ) and figures 46 ( 1619 ) and summarize in 
 vitro potency optimization efforts from 
 10 distinct subseries . initial efforts within tables 12 were focused on further expanding 
 our previously utilized pharmacophore noted within several chemotypes , 
 including acetylene 4 , ethers 7 and 10 , which retain a pyridyl 
 nitrogen and an amide oxygen as potential hydrogen bond accepting 
 groups in an optimal spatial arrangement as illustrated in figure 3 . as can be seen in table 1 , in the case of 2-benzyloxy 
 derivatives , half of the analogues were active as mglu5 pams . among actives , the parent lactams ( r = h , 12a12h , 12q ) showed a range of potency 
 from 62 to 1171 nm with low to moderate maximal efficacy ( glutamate 
 max 4368% ) . notably , no evidence for allosteric agonism was 
 observed for actives in the series . sar on the r group 
 demonstrated toleration for meta - substituted fluorine and chlorine 
 congeners 12c and 12e , respectively , representing 
 two of the more interesting actives from this subseries on the basis 
 of overall potency and efficacy ( ec50 < 250 nm , glu 
 max > n - butyloxy derivatives 12q and 12r , reminiscent of a previously disclosed non - mpep 
 ether pam , were not successful . the 2 
 and 3-pyridyl congeners 12 g and 12h were 
 also not tolerated at r. additionally , eastern alkyl derivatives 12i12n containing linear , branched , cyclic , 
 phenyl , and heterocyclic systems were either inactive ( a < 10% increase 
 in the baseline ec20 glutamate fluorescence response in 
 calcium assays at the highest concentration tested of 30 m 
 is defined as  inactive  ) or had moderate potency and 
 low efficacy ( ec50 > 300 nm , pam glu max < 40% , 12l12n , 12s12 t ) . n - aryl congeners 12o and 12p demonstrated 
 comparable potency below 250 nm , with 12p slightly more 
 efficacious ( glu max 50% vs 38% ) . both the western fluoro and the 
 eastern amide n - aryl sar parallel the recently reported 
 thiazolyl core structure system ; however , 
 interestingly , n - alkyl derivatives were significantly 
 better tolerated in the thiazolyl ring system . calcium mobilization 
 assay using 
 hek293 cells stably expressing rat mglu5 receptors ; values 
 are the average of three or more independent determinations ; inactive , 
 less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response 
 using 30 m test compound ( percentage of maximal response versus 
 100 m glutamate ) . heterocycle , 
 amide , linker pharmacophore relationships , and relative 
 profile for  endocyclic  series 1215 dihydronaphthyridinones and hypothetical benzyloxy lactam 
 core regioisomers of 1415 . studies 
 by astrazeneca pharmaceuticals and within 
 the dihydrothiazolopyridone series 10(31 ) have demonstrated successful utilization of the both benzyloxy- 
 and phenoxymethyl - based spacers as replacements for the acetylene 
 moiety , with varied potency depending on the nature of the heterocycle 
 scaffold . interestingly , in contrast to the unsubstituted benzyloxy lactams 12a12b , alkoxymethyl- dihydronaphthyridinones 13a13b were weak pams ( a weak pam results 
 in a response that is greater than a 10% increase compared to the 
 submaximal glutamate addition ( ec20 ) but does not potentiate 
 the overall glutamate ec20 response by at least 2-fold ) . 
 similarly , n - alkyl derivatives were not productive 
 as pams , leading to compounds categorized as weak or inactive ( 13c13e , 13i ) . direct n - aryl derivatives were more beneficial , with the western 
 3-fluorophenyl congener 13 g bearing an n-4-fluoro - phenyl proving to be potent ( ec50 = 97 nm ) and 
 efficacious ( glu max = 72% ) ; the 2-pyridyl derivative 13h was 5-fold less active ( ec50 = 595 nm ) and less efficacious . fortunately , introduction of a 3-fluoro moiety on the distal phenyl 
 consistently boosted potency ( 23-fold ) and proved 
 transferrable among the subseries ( e.g. , 13f vs 13 g ) . the potency for 13g13h was overall similar to the benzyloxy direct comparators 12o12p ; however , phenoxymethyl - based pams 13g13h have a 2030% elevated 
 glu max response . despite modification of the linker , calcium mobilization 
 assay using 
 hek293 cells stably expressing rat mglu5 receptors ; values 
 are the average of three or more independent determinations ; inactive , 
 less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response 
 using 30 m test compound ( percentage of maximal response versus 
 100 m glutamate ) . in parallel , we evaluated isomeric dihydronaphthyridinones 1415 ( table 3 ) , which retain the linkage 
 site for the distal phenyl para to the lactam amide ; 
 however , the pyridine nitrogen is adjacent to the lactam amide in 
 a position reminiscent of a hydrogen bond accepting ( hba ) pharmacophore 
 proposed in a related ether series by varnes and co - workers . within both series 14 ( y = n ) and 
 series 15 ( x = n ) nh lactams ( 14a , 15a ) , n - methyl analogues ( 14b , 15b ) , and chiral ( r)-3,3-dimethylbutan-2-yl 
 substitute analogues , inspired by monocyclic ether 7 , 
 all proved to be inactive . the lack of activity in the calcium assay 
 for parent lactams 14a and 15a versus the 
 benzyloxy comparator 12a , in addition to western 3-fluoro 
 congeners 13b ( inactive ) versus 12c ( ec50 = 225 nm ) , suggests that for series 1215 wherein the distal phenyl is maintained para to the lactam amide , the heteroatom arrangement between the linker 
 oxygen and the pyridine nitrogen within series 12 is 
 more preferred . however , n - aryl analogues have a 
 unique sar , suggesting that in this context the central tetrahydronaphthyridine 
 scaffold is more critical for mglu5 potentiation than the 
 nature of the linker , as 12 and 13 are equally 
 tolerated in the context of an n - aryl substituent . n-4-fluorophenyl pams 14d and 15d , which differ in the location of the central pyridine nitrogen , 
 have comparable potency with dissimilar efficacy , with pyridine isomer 14d being one of the most efficacious pams reported across 
 the tetrahydronaphthyridine subseries described . the related congener 13f was 2.5-fold more potent ( ec50 = 198 
 nm ) with 68% glutamate max , and 13 g was 2-fold more potent 
 than 13f . relative to series 12 , 12o is equipotent to 13 g ; however , 13 g revealed 
 a 2-fold higher maximal glutamate response ( 72% vs 38% ) . although 
 a more comprehensive survey was undertaken for series 1213 relative to 1415 , on the basis of direct comparisons , general trends were 
 noted between the series . neither molecular 
 switches nor allosteric agonism proved to be issues for 1215 and sar was generally steep . active compounds 
 within 1213 have capacity for high 
 potency ( ec50  200 nm ) and moderate to high efficacy 
 ( % glu max 50% ) , whereas actives within series 1415 , although more limited , exhibited moderate 
 potency and variable efficacy ( % glu max 30% and 81% ) . subtle changes 
 in structure  efficacy / cooperativity relationships are best 
 addressed via glutamate fold - shift experiments ; however , glutamate 
 max trends were consistent within series 1215 , and series 12 shows variable efficacies that 
 encompass the full range reported for all subseries ( figure 3 ) . in general , phenoxy linkages show improved efficacy 
 over benzyloxy linkages for n - arylated analogues . the emerging pharmacophore for 1213 suggested these dihydronaphthyridinone scaffolds interact at the 
 allosteric site in a manner distinct than that proposed by varnes 
 and co - workers and likely more similar 
 to a monocyclic series of nicotinamides previously described . although formally benzyloxy variants of 14 and 15 ( figure 3 ) remained 
 as potential mglu5 pams , the template was not pursued . this decision was made in part on the basis of the overall efficacy 
 and potency observed within subseries 13 , thus phenoxy - based 
 systems remained higher priority for the remaining survey . calcium mobilization assay using 
 hek293 cells stably expressing rat mglu5 receptors ; values 
 are the average of three or more independent determinations ; inactive , 
 less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response 
 using 30 m test compound ( percentage of maximal response versus 
 100 m glutamate ) . we next pursued alternative sites for linker attachment of the 
 pendant aryl examining 3-substituted dihydronaphthyridinones based 
 on either a benzyloxy ( 16 ) or phenoxy - based ( 17 ) system ( figure 4 ) . brief surveys of both n - alkyl and n - aryl and heteroaryl substituents , including several congeners related 
 to those previously described ( table 13 ) , were unsuccessful in identifying favorable starting 
 points and led to modulators which were either inactive or weak pams 
 ( ec50 > 10 m ) . interestingly , a related more 
 rigid , 
 acetylene tetralone scaffold recently reported by merz pharmaceuticals 
 gmbh displays a similar array of hba 
 moieties and was reported to have excellent pam activity ( ec50 < 100 nm ) in a recombinant cho cell line expressing human mglu5 . inspired in part by the 
 addex piperidine chemotype 2 , we postulated that perhaps a more extended pharmacophore placing 
 the amide carbonyl moiety exocyclic of the tetrahydronaphthyridine 
 scaffold as found within the addex chemotype might prove viable ( figure 5 ) . pam 12c served as a precursor ( scheme 1 ) and allowed 
 for facile access to representative benzyloxy targets 18a  d in two steps via borane reduction and subsequent 
 amide coupling . intriguingly , all proved to be weak to moderate antagonists of mglu5 ( figure 5 ) . at this juncture , in light of the unexpected switch in 
 the mode of pharmacology for subseries 18 , as well as 
 the general challenges of multidimensional combinations of linker , 
 core , attachment site , and lactam versus exocyclic amide ( minimum 
 of 24 formal combinations / scaffolds ) , we employed a ligand - based computational 
 tool previously reported to prioritize 
 and potentially streamline medicinal chemistry efforts and ligand 
 design . using this approach , we assessed ligand - based conformational 
 ensembles of known highly efficacious pams , including addex ligand 2(52 ) and other lead molecules , with pam activity below 150 nm and robust glu max values above 70% . hypotheses  
 or reference pams for a mutual flexible low energy shape - based alignment 
 of proposed dihydronaphthyridinone and tetrahydronaphthyridines using 
 the surflex - sim algorithm as implemented in sybyl . the molecular similarity and alignment optimization algorithms used 
 in surflex - sim docking utilize a functional term to minimize the volume 
 of molecular superpositions to construct an objective function for 
 scoring superpositions of multiple molecules . the objective function can then be used as targets for virtual screening , or in this case , 
 for scaffold hopping prioritization . the results from these studies 
 using 2 are summarized in figure 6 with a depicted overlay 
 of the lowest energy structures for 2 versus 2-phenoxy 
 methyl model amide 19 and 3-phenoxy methyl model amide 20 . a surflex - sim score ( ss ) that approaches the reference 
 hypothesis , arbitrarily set to 10 , is indicative of a template that 
 more closely mimics the reference conformer ensemble . using 2 as a template , series 20 , containing a 3-phenoxymethyl , was predicted to have greater molecular similarity ( ss = 9.43 ) than 
 the 2-phenoxymethyl prototype 19 ( ss = 8.90 ) . unlike 
 2-benzyloxy derivatives within series 18 , compounds within 
 series 19 were not antagonists and proved inactive in 
 the primary calcium assay ( nine analogues were prepared and tested 
 from a 3-fluoro 1-(2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)ethanone scaffold , see supporting 
 information ) ; however , at this time , it is unclear if compounds 
 within series 19 have neutral cooperativity and thus 
 retained affinity for mglu5 ( a silent allosteric modulator 
 or sam ) . with this caveat in mind , it is also recognized 
 that the pam ec50 is better described as a composite of 
 four parameters encompassing affinity of the modulator for the receptor , 
 allosteric effects on binding and signaling of the orthosteric ligand 
 and potential intrinsic agonist efficacy of the modulator . thus , although 
 within closely related series , the surflex - sim approach appears to 
 facilitate scaffold prioritization based on pam ec50 activity 
 at of glu5 , the power of the surflex - sim methodology warrants 
 a broader more rigorous retrospective analysis with these additional 
 facets of modulator activity taken into consideration . relationship between 2 and 1820 and resulting 
 first - generation exocyclic amides 18 utilizing 2-benzyloxy 
 linkage . example of flexible alignment of model 2 and 
 3-phenoxymethyl - tetrahydronaphthyridines 19 and 20 ( green surface ) vs 2 ( purple 
 surface ) . with some insight that perhaps 
 3-substituted tetrahyrdonaphthyridines 20 were preferred 
 from ligand - based modeling , additional analogues 
 were prioritized . selected derivatives 20a20c , which retains a 3-phenoxy methyl linker , and 3-benzyloxy 
 variants 20d20 g , are shown in table 4 . within benzyloxy derivatives 20d20 g , compounds covered a spectrum 
 of profiles ranging from inactive ( 20d , 20f ) , to weak pams ( 20e ) , to weak partial antagonists ( 20 g ) ; the latter effect driven by subtle changes in fluorine 
 substitution patterns , a phenomenon not uncommon among certain mglu5 modulator chemotypes . within 
 the phenoxy subseries analogues 20a20c , success was found with 4-fluorobenzamide derivatives ; in particular , 
 the western phenyl and 3-fluoro phenyl derivatives 20a and 20b have statistically comparable potency and efficacy , 
 with ec50s of 640 and 1090 nm , respectively , and a glu 
 max of 69% . . three significant findings are evident 
 among these lactam and exocyclic amide templates examined : ( 1 ) parent 
 dihydronaphthyridinone 12c remains a confounding singleton 
 relative to regioisomeric and positional congeners 13b , 14a , 15a , 16 , and 17 , ( 2 ) relative to earlier acetylenic dihyronaphthyridinones 11 , no molecular switches were 
 noted within dihydronaphthyridinone systems , however , steep sar was 
 evident , and ( 3 ) in contrast to dihydronaphthyridinones , exocyclic 
 acyl - tetrahydronaphthyridines were highly sensitive to the linker 
 attachment site , as 2-substituted based ethers 18 and 19 , proved to be either weak antagonists or functionally inactive 
 in the calcium assay . in addition , within the exocyclic acyl - tetrahydronaphthyridines , 
 phenoxymethyl - based ethers were better tolerated and less susceptible 
 to changes in the mode of pharmacology ( e.g. , 20a versus 20 g ) ; however , overall potency was generally diminished relative 
 to series 1213 . calcium 
 mobilization assay using 
 hek293 cells stably expressing rat mglu5 receptors ; values 
 are the average of three or more independent determinations ; inactive , 
 less than 10% change in calcium fluorescence compared to the ec20 glutamate response . expressed as amplitude of response 
 using 30 m test compound ( percentage of maximal response versus 
 100 m glutamate ) . weak antagonist , data represents 
 plc50/ic50 from ec80 window and % glu 
 represents emin not emax . in light 
 of the overall mglu5 pam potency ( ec50 < 
 250 nm ) and efficacy ( glu max > 50% ) observed for compounds 12c , 12p , 13f , and 13 g , these pams were selected for further characterization ( table 5 ) . in addition , the moderately 
 potent pam 14d ( ec50 = 538 nm ) was considered 
 for further evaluation based upon its enhanced efficacy ( glu max = 
 81% ) . for clint , intrinsic clearance ; 
 clhep , predicted hepatic clearance , human and rat ( h , r ) , 
 ml / min / kg . further examined , 12p , 13f , 13 g , and 14d have similar clogp and molecular 
 weight ( mw ) by virtue of their common lactam n - aryl 
 substituent , while the simple nh lactam 12c stands out 
 as a low mw modulator ( mw = 272 ) with excellent calculated ligand 
 efficiency metrics ( lipe and lelp , see table 5 ) . pam 12c maintains the highest 
 calculated lipe and lowest lelp among the set , suggesting a highly 
 favorable enthalpy driven effect on cooperativity . despite the less 
 favorable physicochemical and le properties for 12p , 13f , 13 g , and 14d relative to 12c , intrinsic clearance in human and rat microsomes suggested 
 moderate predicted hepatic clearance and 110% fraction unbound . subsequent selectivity profiling against mglu14,68 using 12c , 13 g , and 14d revealed 
 that n - aryl congeners 13 g and 14d were also active as mglu3 negative allosteric 
 modulators ( nams ) with negative cooperativity fold - shift values between 
 0.2 and 0.3 , while 12c was fully selective for mglu5 ( mglu14,68 ec50 > 
 10 
 m , see supporting information ) . in light of the unexpected lack of selectivity for 13 g and 14d which maintain a similar n - aryl endocyclic amide pharmacophore found within 12p and 13f , we elected to continue to profile 12c in more depth based on its overall potency , selectivity , 
 and excellent physicochemical and ligand efficiency parameters ( see 
 figure 7 for overall 
 profile ) . positive allosteric modulator activity was investigated 
 by fold - shift experiments to determine the degree of cooperativity 
 with glutamate . as shown in figure 8 , progressive fold - shift experiments utilizing the 
 rat mglu5 receptor revealed a small leftward shift in the 
 glutamate concentration response curve ( crc ) with increasing concentration 
 of 12c ( 55 nm to 30 m ) , with no significant effect 
 on the maximal response . a 2.2-fold leftward shift in the glutamate 
 ec50 was observed in the presence of 30 m 12c , and a predicted allosteric modulator affinity of 2.17 
 m and an efficacy cooperativity factor ( log  ) between 
 glutamate and indicated allosteric modulator of 0.23 ( cooperativity 
 1.71 ) was defined as calculated using the operational model 
 of allosterism . these results support 
 a positive allosteric interaction between glutamate and 12c and were confirmed in a cell line expressing the human mglu5 receptor , where 10 m 12c produced a 3.3-fold leftward shift in the glutamate crc 
 and a pam crc potency of 370 nm ( 71% glu max ) . in comparison to recently 
 reported pams from our lab that have been profiled in glutamate progressive 
 fold - shift studies up to 30 m , including acetylenic picolinamides 
 ( e.g. , ml254 , maximum rat mglu5 fold - shift 3.0 ) and dihydrothiazolopyridone 10 ( maximum 
 rat mglu5 fold - shift 5.4),12c represents one of the weakest mglu5 allosteric 
 modulators reported in terms of measurable positive cooperativity . profile 
 summary of 12c . rat brain 
 homogenate binding to assess fraction 
 unbound in brain revealed a reasonable brain fu of 2.0% for 12c . inhibition of the major human 
 cytochrome p450 ( cyp ) enzymes ( 2c9 , 2d6 , 3a4 , 1a2 ) was measured in 
 a cocktail assay using human liver microsomes and known substrates . pam 12c was found to display weak cyp inhibitory activity 
 at 1a2 ( ic50 = 25.7 m ) , while no activity was observed 
 against the other cyps tested ( ic50 > 30 m ) . 
 in 
 kinetic aqueous solubility assays , 12c displayed moderate 
 to good solubility ( ph 2 , 4 , 7.4 buffer : 38 , 29 , and 28 m ) 
 and moderate solubility in fasted simulated intestinal fluid ( fassif , 
 ph = 7.2 ) of 31 g / ml ( 114 m ) . in a nontoxic vehicle 
 screen using 20% hp -cyclodextrin , 12c was a solution 
 at 1 mg / ml and a microsuspension at 35 mg / ml ( ph 4.0 ) . rat 
 pharmacokinetic studies ( 1 mg / kg iv , 10 mg / kg po ) revealed a high 
 plasma clearance ( clp ) of 75 ml / min / kg and a short half - life 
 ( t1/2 ) of 0.3 h. after oral administration 
 ( 10 mg / kg , 0.5% methyl cellulose , 1 mg / ml , noncrossover ) to male sprague dawley 
 rats , 12c reached an average maximal concentration ( cmax ) of 3.36 m with a corresponding time 
 to reach cmax ( tmax ) of 0.5 h and an auc024h of 6.2 m - h , 
 thus affording an estimated % f of 75 . we performed 
 a single dose in vivo screen to study the ability of 12c to reverse amphetamine - induced hyperlocomotion in rats ( po , 56.6 
 mg / kg 20% hp -cyclodextrin ) . robust reversal of the hyperlocomotion 
 response was noted at this dose ( > 30% , data not shown ) and high 
 brain 
 exposure was observed at 1.5 h ( cbrain = 16.6 m , 
 332 nm unbound ) , producing an average brain - to - plasma ( b : p ) ratio 
 of 1.67 and unbound brain level above the rat in vitro ec50 . furthermore , in vivo b : p and in vitro fu , plasma / fu , brain ( 1.7 ) measures were identical , with a calculated kp , uu of 1.1 , suggesting passive cns penetration for 12c in rat . thus , 12c continued to demonstrate promising properties to warrant further 
 evaluation in vivo . with respect to ancillary pharmacology , in a broad 
 panel selectivity screen against 68 gpcrs , ion channels and transporters 
 using 10 m 12c , no significant off - target activity 
 was noted ( eurofins inc . ) . encouraged 
 by its overall profile as a low fold - shift pam , 12c was 
 evaluated across a range of doses for its ability to reverse amphetamine - induced 
 hyperlocomotion ( ahl ) , an established model of antipsychotic activity 
 ( figure 8) . as seen in 
 figure 9 , 12c dosed orally showed robust dose - dependent effects in reversal 
 of hyperlocomotion , with a maximal effect ( 55% ) observed at the highest 
 dose tested of 100 mg / kg and an average terminal unbound brain concentration 
 of 1.5 m . the lowest active dose of 30 mg / kg afforded an estimated 
 average terminal unbound brain concentration of 712 nm , which 
 represents a 3-fold multiple of the rat in vitro potency and according 
 to the progressive fold - shift studies ( figure 8) , represents a left - ward shift in the glutamate crc in the 1.21.4 
 fold range . despite the overall low positive cooperativity , 12c retains efficacy in ahl at unbound brain exposures that 
 are a multiple of the in vitro ec50 and thus illustrates 
 that high cooperativity with glutamate is not fully required for an 
 mglu5 pam to maintain activity in the ahl model . this observation 
 appears to be in contrast to data reported recently for low fold - shift 
 pams 89 reported by lilly , wherein 
 little effect was noted in a similar amphetamine - induced hyperlocomotor 
 activity assay using male lister hooded rats . rotarod studies were performed using 12c at a doses 
 of 30 , 56.6 , and 100 mg / kg po ( 20% hp--cd in water ) to assess 
 balance and motor coordination ( see supporting 
 information ) . pam 12c showed no statistically 
 significant effect on general motor output ( 120 s cutoff ) . in a modified 
 irwin neurological test battery in rats , 12c , when dosed 
 at 100 mg / kg po ( 20% hp--cd in water ) , had no performance deficits 
 on any autonomic or somatomotor nervous system functions out to 4 
 h. collectively , these results highlight that pam 12c is free from overt neurological side effects and motor behavior 
 deficits at doses that produce maximal efficacy in reversal of amphetamine - induced 
 hyperlocomotion ( 100 mg / kg po ) . dose - dependent effect and calculated terminal 
 unbound brain of 12c on the reversal of amphetamine - induced 
 hyperlocomotion 
 in rats . diverse 
 mglu5 modulator pharmacological profiles were 
 attained within a series of tetrahydronaphthyridine and dihydronaphthyridinone 
 scaffolds through modifications of heteroatoms within the linker , 
 core structure , and location of the pendant linkage moiety . up to 10 diverse subseries were examined , and five of these series , 1215 and 20 , were described 
 in detail , leading to preferred endocyclic series 12 and 13 which , although free from allosteric agonism and molecular 
 switches , tended to have steep sar . n - aryl dihydronaphthyridinone 
 congeners were nonselective and displayed negative cooperativity at 
 mglu3 and thus were not further progressed . parent benzyloxy 
 lactam 12c emerged as a suitable tool compound with good 
 potency , excellent selectivity , and pharmacokinetic properties suitable 
 for acute studies . despite behaving in vitro as an ultralow cooperativity 
 pam in recombinant systems , 12c showed robust , dose - dependent 
 effects in the reversal of amphetamine - induced hyperlocomotion , with 
 a lowest active dose of 30 mg / kg po , that produced an estimated brain 
 unbound level 3-fold above its in vitro potency . maximal efficacy 
 of 12c was observed at a dose of 100 mg / kg with no significant 
 motor impairment or overt neurological side effects . 12c provides another tool compound to complement the available mglu5 pams with well characterized cooperativity profiles to better enable comparative studies with other chemotypes in native 
 systems and in preclinical animal models . all reagents purchased from commercial suppliers 
 were used without purification . unless noted , all solvents used were 
 anhydrous and all reactions were carried out under argon atmosphere . microwave assisted reactions were performed in a single - mode reactor : 
 emrys optimizer microwave reactor ( biotage ) . analytical thin layer 
 chromatography was performed on analtech silica gel gf 250 m 
 plates and on silica gel 60 f254 plates ( merck ) under standard techniques . 
 unless otherwise specified , preparative reverse - phase high performance 
 liquid chromatography ( rp - hplc ) purification was performed on a gilson 
 inc . preparative uv - based system using a phenomenex luna c18 column 
 ( 50 mm  30 mm i.d . , 5 m ) , with an acetonitrile ( unmodified)0.1% 
 trifluoroacetic acid in water gradient . normal - phase silica gel preparative 
 purification was performed using an automated combi - flash rf from 
 isco using either isco or merck ready - to - connect cartridges . analytical 
 lc / ms was performed using the following instruments : ( a ) agilent 1200 
 series with uv detection at 215 and 254 nm and elsd detection ( polymer 
 laboratories pl - els 2100 ) , utilizing an accucore c18 2.6  , 
 2.1 mm  30 mm column , a 1.1 min gradient , 7% [ ch3cn/0.1%tfa]95% [ ch3cn/0.1%tfa ] and a g6130 single 
 quadrupole mass spectrometer or ( b ) ultra performance liquid chromatography 
 ( uplc ) measurement performed using an acquity uplc ( waters ) system 
 comprising a sampler organizer , a binary pump with degasser , a four 
 column s oven , a diode array detector ( dad ) , and a beh - c18 
 column ( 1.7 m , 2.1 mm  50 mm ) from waters , with a flow 
 rate of 1.0 ml / min at 50 c without split to the ms detector . the gradient conditions consisted of 95% a ( 0.5 g / l ammonium acetate 
 solution + 5% acetonitrile ) , 5% b ( acetonitrile ) , to 40% a , 60% b 
 in 3.8 min , to 5% a , 95% b in 4.6 min , kept until 5.0 min without 
 split to the ms detector . the ms detector was configured with an esci 
 dual ionization source ( electrospray combined with atmospheric pressure 
 chemical ionization ) . low - resolution mass 
 spectra ( single quadrupole , sqd detector ) were acquired by scanning 
 from 100 to 1000 in 0.1 s using an interchannel delay of 0.08 s. the 
 capillary needle voltage was 3 kv . the cone voltage was 25 v for positive 
 ionization mode and 30 v for negative ionization mode . gc / ms measurement was 
 performed using a 6890 series gas chromatograph ( agilent technologies ) 
 system comprising a 7683 series injector and autosampler , coupled 
 to a 5973n msd mass selective detector ( single quadrupole , agilent 
 technologies ) . the ms detector was configured with an electronic impact 
 ionization source / chemical ionization source ( ei / ci ) . ei low - resolution 
 mass spectra were acquired by scanning from 50 to 550 at a rate of 
 14.29 scans / s . gc / ms was carried out on a 
 j&w hp-5ms column ( 20 m  0.18 mm , 0.18 m ) from agilent 
 technologies , with a flow rate of 0.7 ml / min . the temperature gradient 
 applied was : initial temperature 50 c , hold for 2.0 min , then 
 a 50 c / min ramp applied for 5.0 min until 300 c and hold 
 for 3.0 min in a 10 min run . split injection mode was used , 0.2 l injection volume , with 
 a 50/1 ratio into the gc / ms system . hrms were obtained using a micromass 
 ( waters ) q - tof api - us calibrated and verified with sodium iodide . the samples were diluted with a 50:50 0.1% formic acid ( in milli - q):acetonitrile 
 solution , directly infused using leucine  enkephalin ( m + h 
 = 556.2771 ) as a lockmass . scan range was from 100 to 1000 da , using 
 a scan time of one second . the [ m + h ] or [ m + na ] ion was observed . nmr spectra 
 were recorded on either a bruker dpx-400 or a bruker av-500 mhz spectrometer 
 with standard pulse sequences . h chemical shifts are reported 
 as  values in cdcl3 or dmso - d6 . data are reported as follows : chemical shift , integration , 
 multiplicity ( s = singlet , bs = broad singlet , d = doublet , t = triplet , 
 q = quartet , p = pentet , hex = hextet , sep = septet , dd = doublet 
 of doublets , dt = doublet of triplets , dq = doublet of quartets , m 
 = multiplet ) , coupling constant j reported in hz . c chemical shifts are reported in  values in cdcl3 as follows : chemical shift , c  f coupling constants 
 ( jc  f ) reported in hz . for a number 
 of compounds , melting points ( mp ) were determined in open capillary 
 tubes on a fp62 or on a fp81htfp90 apparatus ( mettler ) . melting points 
 were measured with a temperature gradient of 10 c / min ( maximum 
 temperature 300 c ) , and the melting point was read from a digital 
 display . melting point values are peak values and were obtained with 
 experimental uncertainties that are commonly associated with this 
 analytical method . experimental procedures , analytical data , hrms , and nmr 
 spectra can be found within supporting information . benzyl alcohol ( 85 l , 
 0.82 
 mmol ) was dissolved into dmf ( 1.6 ml ) and treated with kot - bu ( 184 mg , 1.64 mmol ) and stirred for 30 min . the mixture was treated 
 with 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one 
 ( 20 , 100 mg , 0.55 mmol , see supporting 
 information ) and heated to 100 c for 4 h. the mixture 
 was cooled to rt , acidified with 6 n hcl , and extracted with etoac 
 ( 3 ) . the combined organic layers were washed with brine , dried 
 over na2so4 , and evaporated to dryness . the 
 crude product mixture was purified by rp - hplc ( eluting with 4090% 
 mecn / h2o with 0.1% tfa modifier ) to afford the title compound 
 ( 73 mg , 53% ) . h nmr ( 400 mhz , cdcl3 )  
 8.20 ( 1h , d , j = 8.6 hz ) , 7.46 ( 2h , m ) , 7.36 ( 3h , 
 m ) , 6.75 ( 1h , d , j = 8.6 hz ) , 5.85 ( 1h , bs ) , 5.43 
 ( 2h , s ) , 3.62 ( 2h , t , j = 6.8 hz ) , 3.09 ( 2h , t , j = 6.8 hz ) . c nmr ( 100 mhz , cdcl3 )  166.4 , 165.2 , 158.0 , 138.3 , 136.7 , 128.4 , 128.0 , 127.9 , 
 118.3 , 109.8 , 68.1 , 39.2 , 30.7 . hrms ( es+ , m + h ) calcd for c15h15n2o2 , 255.1134 , found , 
 255.1130 . 3-fluorobenzyl alcohol 
 ( 1.66 g , 13.14 mmol ) was dissolved into dmf ( 25 ml ) and treated with 
 kot - bu ( 3.2 g , 26.29 mmol ) and stirred for 30 min . the mixture was treated with 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 20 , 1.49 g , 8.82 mmol , see supporting information ) and heated to 100 c 
 overnight . the mixture was adsorbed onto silica and purified ( 0 to 
 80% etoac / hexanes ) to give a semipure solid . this material was further 
 purified on rp - hplc ( eluting with 4090% mecn / h2o with 0.1% tfa modifier ) to give 840 mg of the title compound ( 35% ) . h nmr ( 400 mhz , cdcl3 )  8.44 ( 1h , d , j = 7.6 hz ) , 7.38 ( 1h , dd , j = 14.6 , 7.6 
 hz ) , 7.24 ( 1h , d , j = 7.5 hz ) , 7.22 ( 1h , d , j = 9.6 hz ) , 7.04 ( 1h , t , j = 8 hz ) , 6.79 
 ( 1h , d , j = 8.4 hz ) , 5.79 ( 1h , br s ) , 5.45 ( 2h , s ) , 
 3.64 ( 2h , t , j = 6.4 hz ) , 3.10 ( 2h , t , j = 6.4 hz ) . lcms tr = 0.736 min , > 98% at 215 and 254 
 nm , m / z = 273.1 [ m + h ] . hrms ( es+ , 
 m + h ) calcd for c15h14fn2o2 , 273.0961 ; found , 273.0963 . starting from 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 100 mg , 0.55 mmol ) and following the general procedure 
 outlined in 12a and 12c , rp - hplc afforded 
 the title compound as a powder ( 72 mg , 60% ) . h nmr ( 400 
 mhz , cdcl3 )  8.17 ( 1h , d , j = 8.6 
 hz ) , 6.67 ( 1h , d , j = 8.6 hz ) , 5.84 ( 1h , bs ) , 4.33 
 ( 2h , t , j = 6.6 hz ) , 3.61 ( 2h , t , j = 6.8 hz ) , 3.06 ( 2h , t , j = 6.8 hz ) , 1.77 ( 2h , 
 p , j = 7.9 hz ) , 1.48 ( 2h , hex , j = 7.5 hz ) , 0.98 ( 3h , t , j = 7.4 hz ) . c nmr ( 100 mhz , cdcl3 )  166.6 , 165.7 , 158.1 , 138.1 , 
 117.9 , 109.4 , 66.3 , 39.2 , 30.9 , 30.8 , 19.1 , 13.7 . hrms ( es+ , m + h ) 
 calcd for c12h17n2o2 , 
 221.1290 ; found , 221.1290 . 2-butoxy-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 12q , 50 mg , 0.23 mmol ) , 1-bromo-2,4-difluorobenzene 
 ( 52 l , 0.46 mmol ) , cui ( 9.1 mg , 0.047 mmol ) , n , n - dimethylethane-1,2-diamine 
 ( 14 l , 0.129 mmol ) , and potassium carbonate ( 64 mg , 0.46 mmol ) 
 were combined in a 2.0 ml microwave vial and placed under an argon 
 atmosphere . degassed toluene was added and the mixture heated at 110 
 c for 6 h. the mixture was cooled to rt , filtered over celite , 
 and the filtrate concentrated to dryness . rp - hplc purification afforded 
 the title compound as a powder ( 45 mg , 59% ) . h nmr ( 400 
 mhz , cdcl3 )  8.22 ( 1h , d , j = 8.6 
 hz ) , 7.32 ( 1h , m ) , 6.92 ( 2h , m ) , 6.68 ( 1h , d , j = 
 8.6 hz ) , 4.35 ( 2h , t , j = 6.6 hz ) , 3.91 ( 2h , t , j = 6.7 hz ) , 3.19 ( 2h , t , j = 6.6 hz ) , 
 1.77 ( 2h , p , j = 7.9 hz ) , 1.48 ( 2h , hex , j = 7.6 hz ) , 0.98 ( 3h , t , j = 7.4 hz ) . c nmr ( 100 mhz , cdcl3 )  165.9 , 163.9 , 161.4(dd , jc f = 247.6 , 11.3 hz ) , 157.8 ( dd , jc  f = 250.8 , 12.3 hz ) , 157.66 , 139.00 , 
 129.61 ( dd , jc f = 9.8 , 2.6 hz ) , 
 126.42 , ( dd , jc f = 12.8 , 3.9 hz ) , 
 118.0 , 111.55 ( dd , jc f = 22.2 , 
 3.6 hz ) , 109.7 , 104.9 ( dd , jc  f = 26.0 , 24.0 hz ) , 66.4 , 48.5 , 31.2 , 31.0 , 19.2 , 13.8 . hrms ( es+ , 
 m + h ) calcd for c18h19f2n2o2 , 333.1415 ; found , 333.1413 . ( 19 l , 0.158 mmol ) were dissolved into dmf ( 0.5 ml ) 
 and kot - bu added ( 16 mg , 0.143 mmol ) . the mixture 
 was stirred for 3 h at 60 c and then cooled to rt . water ( 1.0 
 ml ) was added to the mixture and extracted with etoac ( 3 ) . the 
 combined organic layers were evaporated to dryness and the crude product 
 mixture purified by rp - hplc to afford 16 mg of the desired product 
 ( 40% ) . h nmr ( 400 mhz , cdcl3 )  8.28 
 ( 1h , d , j = 8.6 hz ) , 7.45 ( 2h , m ) , 7.33 ( 8h , m ) , 
 6.76 ( 1h , d , j = 8.6 hz ) , 5.41 ( 2h , s ) , 4.77 ( 2h , 
 s ) , 3.53 ( 2h , t , j = 6.9 hz ) , 3.02 ( 2h , t , j = 6.9 hz ) . c nmr ( 100 mhz , cdcl3 )  165.1 , 164.1 , 156.9 , 139.0 , 137.3 , 136.8 , 128.7 , 128.5 , 
 128.1 , 128.0 , 127.9 , 127.5 , 118.7 , 109.9 , 68.1 , 50.2 , 44.5 , 30.7 . hrms ( es+ , m + h ) calcd for c22h21n2o2 , 345.1603 ; found , 345.1600 . ( 40 mg , 0.158 mmol ) were dissolved into dmf ( 0.5 ml ) and kot - bu added ( 36 mg , 1.35 mmol ) . the mixture was stirred for 
 3 h at 60 c and then cooled to rt . water ( 1.0 ml ) the combined organic 
 layers were evaporated to dryness and the crude product mixture purified 
 by rp - hplc to afford 16.2 mg of 12 t ( 40% ) . h nmr ( 400 mhz , cdcl3 )  0.8.55 ( 1h , d , j = 4.6 hz ) , 8.25 ( 1h , d , j = 8.6 hz ) , 
 7.66 ( 1h , dt , j = 7.6 , 1.7 hz ) , 7.45 ( 2h , m ) , 7.35 
 ( 4h , m ) , 7.2 ( 1h , m ) , 6.74 ( 1h , d , j = 8.6 hz ) , 5.41 
 ( 2h , s ) , 4.88 ( 2h , s ) , 3.70 ( 2h , t , j = 6.9 hz ) , 
 3.07 ( 2h , t , j = 6.9 hz ) . c nmr ( 100 
 mhz , cdcl3 )  165.1 , 164.2 , 157.3 , 157.2 , 149.2 , 
 140.0 , 136.9 , 136.8 , 128.5 , 128.1 , 128.0 , 122.4 , 122.3 , 118.6 , 109.8 , 
 68.1 , 52.4 , 45.6 , 30.7 . hrms ( es+ , m + h ) calcd for c21h20n3o2 , 346.1556 ; found , 346.1553 . [ 2-(phenoxy)methyl)-6-(4-methoxybenzyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 14 mg , 0.0.037 mmol ) was dissolved in ch3cn ( 0.25 ml ) and water ( 0.1 ml ) added . cerium ammonium nitrate ( 24.3 
 mg , 0.0.04 mmol ) was added , and after 15 min , the reaction was partitioned 
 between etoac ( 5 ml ) and brine ( 1 ml ) . the mixture was extracted with 
 etoac ( 2 ) , and the organic layers combined , concentrated , and 
 purified by rp - hplc to give title compound ( 2.6 mg , 28% ) . h nmr ( 400 mhz , cdcl3 )  8.32 ( 1h , d , j = 8.0 hz ) , 7.49 ( 1h , d , j = 8.0 hz ) , 7.25 ( 2h , 
 m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 6.49 ( 1h , bs ) , 5.10 ( 2h , s ) , 3.65 
 ( 2h , dt , j = 6.9 , 2.5 hz ) , 3.20 ( 2h , t , j = 6.7 hz ) . lcms tr = 0.732 min , > 
 98% 
 at 215 and 254 nm , m / z = 255.1 [ m 
 + h ] . hrms ( es+ , m + h ) calcd for c15h14n2o2 , 255.1055 ; found , 255.1057 . [ 2-((3-fluorophenoxy)methyl)-6-(4-methoxybenzyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 23b , 300 mg , 0.77 mmol ) was dissolved 
 in ch3cn ( 5 ml ) and water ( 2 ml ) added . cerium ammonium 
 nitrate ( 419 mg , 0.77 mmol ) was added , and after 15 min , the reaction 
 was partitioned between etoac ( 10 ml ) and brine ( 10 ml ) . the organic 
 layers were concentrated and purified by rp - hplc to give title compound 
 as a solid ( 74 mg , 76% ) . h nmr ( 400 mhz , cdcl3 )  8.36 ( 1h , d , j = 8.0 hz ) , 7.53 ( 1h , d , j = 8.0 hz ) , 7.25 ( 1h , m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 6.49 
 ( 1h , bs ) , 5.20 ( 2h , s ) , 3.68 ( 2h , dt , j = 6.7 , 2.5 
 hz ) , 3.21 ( 2h , t , j = 6.7 hz ) . c nmr 
 ( 100 mhz , cdcl3 )  165.3 , 163.6 ( d , jc f = 10.9 hz ) , 158.3 , 136.8 , 130.4 ( d , jc  f = 9.8 hz ) , 123.9 , 119.9 , 110.5 ( d , jc f = 2.9 hz ) , 108.3 ( d , jc  f = 21.1 hz ) , 102.7 ( d , jc f = 24.9 hz ) , 70.5 , 39.4 , 30.9 . hrms ( es+ , m + h ) 
 calcd for c15h14fn2o2 , 
 273.1039 ; found , 273.1037 . 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 10.0 mg , 0.04 mmol ) was dissolved 
 in dmf ( 0.5 ml ) . nah ( 2.0 mg , 0.08 mmol ) was added into the solution 
 and stirred at rt . after 30 min , mei ( 5.0 l ) was added into 
 the mixture and allowed to stir for 1 h. the mixture was then quenched 
 with h2o ( 2.0 ml ) and extracted with etoac ( 3 ) . the 
 organic layers were combined and concentrated to afford product as 
 a white solid ( 9.2 mg , 87% ) . lcms tr = 
 0.81 min , > 98% at 215 and 254 nm , m / z = 287.2 [ m + h ] . h nmr ( 400 mhz , cdcl3 )  
 8.30 ( 1h , d , j = 8.0 hz ) , 7.45 ( 1h , d , j = 8.0 hz ) , 7.25 ( 2h , m ) , 6.76 ( 1h , m ) , 6.62 ( 2h , m ) , 5.10 ( 2h , s ) , 
 3.65 ( 2h , dt , j = 6.9 , 2.5 hz ) , 3.20 ( 2h , t , j = 6.7 hz ) , 3.16 ( s , 3 h ) . 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) was dissolved 
 in dmf ( 2.0 ml ) . kot - bu ( 55 mg , 0.5 mmol ) was added 
 followed by 1-bromo-2-methylpropane ( 70 l , 0.5 mmol ) . the mixture 
 was heated to 60 c for 4 h. the mixture was then quenched with 
 h2o ( 2.0 ml ) and extracted with etoac ( 3 ) . the resulting 
 crude mixture was purified by rp - hplc to give title compound as a 
 powder ( 43 mg , 44% ) . h nmr ( 400 mhz , cdcl3 ) 
  8.37 ( 1h , d , j = 8.0 hz ) , 7.51 , ( 1h , d , j = 8.0 hz ) , 7.22 ( 1h , m ) , 6.76 ( 1h , m ) , 6.69 ( 2h , m ) , 5.19 
 ( 2h , s ) , 3.65 ( 2h , t , j = 6.8 hz ) , 3.40 ( 2h , d , j = 7.5 hz ) , 3.19 ( 2h , t , j = 6.7 hz ) , 
 2.05 ( 1h , sep , j = 6.8 hz ) , 0.97 ( 6h , d , j = 6.7 hz ) . f = 10.7 
 hz ) , 159.1 , 157.4 , 137.1 , 130.4 ( d , jc f = 10.0 hz ) , 124.5 , 119.9 , 110.5 ( d , jc  f = 2.9 hz ) , 108.2 ( d , jc f = 21.2 
 hz ) , 102.7 ( d , jc  f = 24.9 hz ) , 
 70.5 , 54.7 , 45.8 , 30.9 , 27.2 , 20.1 . hrms ( es+ , m + h ) calcd for c19h22fn2o2 , 329.1665 ; found , 
 329.1662 . starting from 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) and ( bromomethyl)cyclopropane 
 ( 50 l , 0.50 mmol ) , the general procedure outlined in 13d was performed . rp - hplc afforded the title compound as 
 a powder ( 64 mg , 66% ) . h nmr ( 400 mhz , cdcl3 )  8.36 ( 1h , d , j = 8.0 hz ) , 7.50 ( 1h , d , j = 8.0 hz ) , 7.23 ( 1h , m ) , 6.75 ( 1h , m ) , 6.68 ( 2h , m ) , 5.18 
 ( 2h , s ) , 3.75 ( 2h , t , j = 6.7 hz ) , 3.47 ( 2h , d , j = 7.0 hz ) , 3.21 ( 2h , t , j = 6.7 hz ) , 
 1.07 ( 1h , m ) , 0.55 ( 2h , m ) , 0.31 ( 2h , m ) . c nmr ( 100 
 mhz , cdcl3 )  163.6 ( d , jc  f = 244.2 . f = 10.8 hz ) , 159.1 , 157.5 , 137.0 , 130.3 ( d , jc f = 9.9 hz ) , 124,4 , 119.8 , 110.5 ( d , jc f = 2.8 hz ) , 108.2 ( d , jc  f = 21.2 hz ) , 102.7 ( d , jc f = 24.8 hz ) , 70.5 , 51.4 , 45.2 , 30.8 , 9.5 , 3.5 . hrms ( es+ , m + h ) 
 calcd for c19h20fn2o2 , 
 327.1509 ; found , 327.1508 . 2-(phenoxymethyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13a , 50 mg , 0.19 mmol ) , 1-bromo-2,4-difluorobenzene 
 ( 42 l , 0.38 mmol ) , cui ( 7.2 mg , 0.038 mmol ) , and potassium 
 carbonate ( 52 mg , 0.38 mmol ) were combined in a 2.0 ml microwave vial 
 and placed under an argon atmosphere . degassed toluene was added followed 
 by n , n - dimethylethane-1,2-diamine 
 ( 12 l , 0.110 mmol ) and the mixture heated at 120 c for 
 16 h. the mixture was cooled to rt , filtered over celite , and the 
 filtrate concentrated to dryness . rp - hplc purification afforded the 
 title compound as a powder ( 16 mg , 31% ) . h nmr ( 400 mhz , 
 cdcl3 )  8.43 ( 1h , d , j = 8.0 hz ) , 
 7.60 ( 1h , d , j = 8.0 hz ) , 7.33 ( 4h , m ) , 7.12 ( 2h , 
 m ) , 6.99 ( 3h , m ) , 5.25 ( 2h , m ) , 4.06 ( 2h , t , j = 
 6.7 hz ) , 3.36 ( 2h , t , j = 6.6 hz ) . c 
 nmr ( 100 mhz , cdcl3 )  163.3 , 160.9 ( d , jc  f = 245.0 hz ) , 160.7 , 158.1 , 157.5 , 138.4 ( d , jc f = 3.2 hz ) , 137.5 , 129.6 , 127.0 ( d , jc f = 8.5 hz ) , 124.3 , 121.4 , 120.1 , 115.9 
 ( d , jc f = 22.5 hz ) , 114.8 , 70.1 , 
 48.7 , 31.2 . hrms ( es+ , m + h ) calcd for c21h18fn2o2 , 349.1352 ; found , 349.1351 . 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b , 26 mg , 0.095 mmol ) , 2-bromo-5-fluoropyridine 
 ( 33 mg , 0.19 mmol ) , cui ( 3.8 mg , 0.02 mmol ) , and potassium carbonate 
 ( 26 mg , 0.19 mmol ) were combined in a 2.0 ml microwave vial and placed 
 under an argon atmosphere . degassed toluene ( 1.0 ml ) was added followed 
 by n , n - dimethylethane-1,2-diamine 
 ( 5.8 l , 0.053 mmol ) and the mixture heated at 120 c for 
 16 h. the mixture was cooled to rt and filtered over celite . the filtrate 
 was diluted with h2o and extracted with etoac ( 3 ) . rp - hplc 
 purification afforded the title compound as a powder ( 31 mg , 89% ) . h nmr ( 400 mhz , cdcl3 )  8.46 ( 1h , d , j = 8.0 hz ) , 8.29 ( 1h , d , j = 3.0 hz ) , 
 8.01 ( 1h , dd , j = 9.1 , 4.0 hz ) , 7.57 ( 1h , d , j = 8.1 hz ) , 7.47 ( 1h , m ) , 7.23 ( 1h , m ) , 6.77 ( 1h , m ) , 6.71 
 ( 2h , m ) , 5.23 ( 2h , s ) , 4.38 ( 2h , t , j = 6.7 hz ) , 
 3.32 ( 2h , t , j = 6.6 hz ) . c nmr ( 100 
 mhz , cdcl3 )  163.6 ( d , jc f = 244.4 hz ) , 163.6 , 160.4 , 159.4 ( d , jc f = 10.8 hz ) , 158.4 , 156.8 ( d , jc  f = 252.0 hz ) , 149.5 ( d , jc f = 
 25.2 hz ) , 130.4 ( d , jc  f = 9.9 
 hz ) , 124.4 , 124.3 ( d , jc f = 2.9 hz ) , 
 108.3 ( d , jc  f = 21.1 hz ) , 102.7 
 ( d , jc f = 24.9 hz ) , 70.5 , 45.0 , 
 31.2 . hrms ( es+ , m + h ) calcd for c20h16f2n3o2 , 368.1211 ; found , 368.1210 . starting from 2-((3-fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6h)-one ( 13b ) ( 100 mg , 0.41 mmol ) and ( bromomethyl)benzene 
 ( 67 l , 0.55 mmol ) , the general procedure outlined in 13d was performed . rp - hplc afforded the title compound as 
 a powder ( 74 mg , 66% ) . h nmr ( 400 mhz , cdcl3 )  8.44 ( 1h , d , j = 8.0 hz ) , 7.53 ( 1h , d , j = 8.0 hz ) , 7.32 ( 5h , m ) , 7.24 ( 1h , m ) , 6.76 ( 1h , m ) , 6.69 
 ( 2h , m ) , 5.19 ( 2h , s ) , 4.80 ( 2h , s ) , 3.59 ( 2h , t , j = 6.8 hz ) , 3.15 ( 2h , t , j = 6.8 hz ) . f = 244.3 hz ) , 163.4 , 159.4 ( d , jc  f = 10.6 hz ) , 159.3 , 157.5 , 137.2 , 136.9 , 130.4 
 ( d , jc  f = 10.0 hz ) , 128.8 , 128.1 , 
 127.7 , 124.2 , 119.9 , 110.5 ( d , jc f = 2.9 hz ) , 108.2 ( d , jc  f = 21.4 
 hz ) , 102.7 ( d , jc  f = 24.9 hz ) , 
 70.5 , 50.4 , 44.5 , 30.7 . hrms ( es+ , m + h ) calcd for c22h20fn2o2 , 363.1509 ; found , 363.1507 . ring - closure step : starting from 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic 
 acid methyl ester ( 128 mg , 0.45 mmol ) and benzylamine ( 0.059 ml , 0.54 
 mmol ) in ch2cl2 ( 8 ml ) , the mixture was stirred 
 at rt for 1 h and sodium triacetoxyborohydride ( 95 mg , 0.45 mmol ) 
 was added and the mixture was stirred at rt for 16 h. the mixture 
 was diluted with ch2cl2 and washed with a saturated 
 solution of nahco3 and brine . the organic layer was separated , 
 dried ( na2so4 ) , filtered , and the solvents evaporated 
 in vacuo . the crude product was purified by flash column chromatography 
 ( silica ; 7n solution of ammonia in methanol in ch2cl2 0/100 to 4/96 ) . the desired fractions were collected and 
 concentrated to yield , 7-benzyl-3-phenoxymethyl-6,7-dihydro-5h - naphthyridin-8-one as an oil ( 41 mg , 23% ) . lcms tr = 2.83 min , > 90% at 215 and 254 nm , m / z = 255.1 [ m + h ] . debenzylation step : 
 trifluoromethanesulfonic acid 0.020 ml , 0.23 mmol ) was added to a 
 solution of 7-benzyl-3-phenoxymethyl-6,7-dihydro-5h - naphthyridin-8-one ( 20 mg , 0.058 mmol ) in toluene ( 0.5 ml ) 
 in a sealed tube . dowex 1  2100 ( strongly 
 basic anion exchanger ) ( 300 mg ) was added , followed by meoh ( 1 ml ) . the mixture was shaken 4 h at rt , and the resin was filtered and washed 
 with ch2cl2 ( 1 ml ) , meoh ( 1 ml ) , ch2cl2 ( 1 ml ) , and meoh ( 1 ml ) . the crude product was purified by flash column chromatography 
 ( silica , 7 m ammonia in methanol in ch2cl2 0/100 
 to 5/95 ) . the desired fractions were collected and the solvents evaporated 
 in vacuo to yield an impure fraction which was purified by rp - hplc 
 on ( c18 xbridge 19 mm  100 mm 5 m ) . mobile phase ( gradient 
 from 80% 0.1% nh4co3h / nh4oh ph 9 
 solution in water , 20% ch3cn to 0% 0.1% nh4co3h / nh4oh ph 9 solution in water , 100% ch3cn ) to yield 14a as a white solid ( 6.26 mg , 42% yield ) . lcms tr = 1.36 min , > 98% at 215 and 
 254 
 nm , m / z = 255.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 )  8.74 ( d , j = 1.4 hz , 1 h ) , 7.73 ( br . s , 1 h ) , 7.397.29 ( m , 2 h ) , 7.24 
 ( br . s , 1 h ) , 7.056.94 ( m , 3 h ) , 5.14 ( s , 2 h ) , 3.64 ( td , j = 6.6 , 2.9 hz , 2 h ) , 3.09 ( t , j = 6.6 
 hz , 2 h ) . methylamine ( 2.0 m in 
 thf ) ( 2 ml ) was added to a stirred solution of 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic 
 acid methyl ester ( 40 mg , 0.14 mmol ) in ch2cl2 ( 2 ml ) . the mixture was stirred at rt for 1 h. then , sodium triacetoxyborohydride 
 ( 45 mg , 0.21 mmol ) was added and the mixture was stirred at rt for 
 16 h. the mixture was diluted with ch2cl2 and 
 washed with a saturated solution of nahco3 and brine . the 
 organic layer was separated , dried ( na2so4 ) , 
 filtered , and the solvents evaporated in vacuo . the crude product 
 was purified by flash column chromatography ( silica ; 7n solution of 
 ammonia in methanol in ch2cl2 0/100 to 4/96 ) . the desired fractions were collected and the solvents evaporated in 
 vacuo to yield an impure fraction which was repurified by rp - hplc 
 on ( c18 xbridge 19 mm  mm 100 5 m ) . mobile phase ( gradient 
 from 80% 0.1% nh4co3h / nh4oh ph 9 
 solution in water , 20% ch3cn to 0% 0.1% nh4co3h / nh4oh ph 9 solution in water , 100% ch3cn ) to yield 14b as a white solid ( 5.8 mg , 15% ) . lcms tr = 1.59 min , > 98% at 215 and 254 nm , m / z = 269.1 [ m + h ] . h nmr 
 ( 500 mhz , cdcl3 )  8.73 ( d , j = 
 1.7 hz , 1 h ) , 7.68 ( s , 1 h ) , 7.367.28 ( m , 2 h ) , 7.086.90 
 ( m , 3 h ) , 5.13 ( s , 2 h ) , 3.62 ( t , j = 6.6 hz , 2 h ) , 
 3.22 ( s , 3 h ) , 3.08 ( t , j = 6.8 hz , 2 h ) . . starting 
 from 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic acid methyl 
 ester ( 40 mg , 0.14 mmol ) and ( r)-(+)-3,3-dimethyl 
 2-aminobutane 0.039 ml , 0.34 mmol ) following the procedure described 
 for compound 14a  b , compound 14c was obtained as a beige solid ( 24 mg , 25% ) . lcms tr = 2.82 min , > 98% at 215 and 254 nm , m / z = 339.1 [ m + h ] . h nmr 
 ( 500 mhz , cdcl3 )  8.73 ( d , j = 
 1.7 hz , 1 h ) , 7.68 ( s , 1 h ) , 7.287.37 ( m , 2 h ) , 6.927.04 
 ( m , 3 h ) , 5.13 ( s , 2 h ) , 4.92 ( q , j = 7.2 hz , 1 h ) , 
 3.623.46 ( m , 2 h ) , 3.102.98 ( m , 1 h ) , 2.982.84 
 ( m , 1 h ) , 1.22 ( d , j = 7.2 hz , 3 h ) , 1.00 ( s , 9 h ) ; 
 d = 15.7 ( c = 0.55 w / v% , meoh ) . 4-fluoroaniline 
 ( 0.032 ml , 0.34 mmol ) was added to a stirred solution of 3-(2-oxo - ethyl)-5-phenoxymethyl - pyridine-2-carboxylic 
 acid methyl ester ( 80 mg , 0.28 mmol ) in ch2cl2 ( 4 ml ) . the mixture was stirred at rt for 1 h. then , sodium triacetoxyborohydride 
 ( 89 mg , 0.42 mmol ) was added and the mixture was stirred at rt for 
 16 h. then , acetic acid ( 0.040 ml ) was added and the mixture was stirred 
 at rt for additional 20 h. the mixture was diluted with ch2cl2 and washed with a saturated solution of nahco3 and brine . the organic layer was separated , dried ( na2so4 ) , filtered , and the solvents evaporated in 
 vacuo . the crude product was purified by flash column chromatography 
 ( silica ; 7n solution of ammonia in methanol in ch2cl2 0/100 to 4/96 ) . the desired fractions were collected and 
 the solvents evaporated in vacuo to yield an impure fraction which 
 was repurified by rp - hplc on ( c18 xbridge 19 mm  100 mm , 5 m ) . 
 mobile phase ( gradient from 80% 0.1% nh4co3h / nh4oh ph 9 solution in water , 20% ch3cn to 0% 0.1% 
 nh4co3h / nh4oh ph 9 solution in water , 
 100% ch3cn ) , to yield 14d as a white solid 
 ( 15 mg , 15% ) . lcms tr = 2.00 min , > 
 98% 
 at 215 and 254 nm , m / z = 349.1 [ m 
 + h ] . h nmr ( 500 mhz , cdcl3 )  8.78 ( d , j = 1.7 hz , 1 h ) , 7.76 ( br . s , 1 h ) , 7.427.36 ( m , 
 2 h ) , 7.357.29 ( m , 2 h ) , 7.167.07 ( m , 2 h ) , 7.01 ( t , j = 7.5 hz , 1 h ) , 6.98 ( d , j = 7.8 hz , 
 2 h ) , 5.17 ( s , 2 h ) , 4.01 ( t , j = 6.4 hz , 2 h ) , 3.22 
 ( t , j = 6.4 hz , 2 h ) . a 7n solution of ammonia in methanol ( 5.5 
 ml ) was added to 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester 
 ( 33 , 55 mg , 0.2 mmol ) . the mixture was stirred at 100 
 c for 16 h. the solvents were evaporated in vacuo . the crude 
 product was purified by flash chromatography ( silica ; acoet in ch2cl2 0/100 to 100/0 ) . the desired fractions were 
 collected and the solvents evaporated in vacuo to yield an impure 
 fraction which was triturated with et2o and repurified 
 by rp - hplc on ( c18 xbridge 30 mm  100 mm , 5 m ) . mobile 
 phase ( gradient from 80% 0.1% nh4co3h / nh4oh ph 9 solution in water , 20% ch3cn to 0% 0.1% 
 nh4co3h / nh4oh ph 9 solution in water , 
 100% ch3cn ) to yield 15a as a white solid 
 ( 20 mg , 39% ) ; mp 193 c . lcms tr = 
 1.47 min , > 98% at 215 and 254 nm , m / z = 255.1 [ m + h ] . h nmr ( 400 mhz , cdcl3 )  
 9.18 ( s , 1 h ) , 7.43 ( d , j = 0.5 hz , 1 h ) , 7.367.28 
 ( m , 2 h ) , 7.066.94 ( m , 3 h ) , 6.26 ( br s , 1 h ) , 5.24 ( s , 2 
 h ) , 3.61 ( td , j = 6.6 , 2.9 hz , 2 h ) , 3.03 ( t , j = 6.7 hz , 2 h ) .. starting from 6-phenoxymethyl-4-vinyl - nicotinic 
 acid methyl ester ( 33 , 55 mg , 0.2 mmol ) and 2 m methylamine 
 in thf ( 5.5 ml ) following the procedure described for compound 15a , compound 15b was obtained as a white solid 
 ( 51 mg , 93% ) ; mp 134 c . lcms tr = 
 1.72 min , > 98% at 215 and 254 nm , m / z = 269.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 )  
 9.18 ( s , 1 h ) , 7.38 ( s , 1 h ) , 7.31 ( s , 2 h ) , 7.046.95 ( m , 
 3 h ) , 5.23 ( s , 2 h ) , 3.59 ( t , j = 6.6 hz , 2 h ) , 3.16 
 ( s , 3 h ) , 3.03 ( t , j = 6.6 hz , 2 h ) .. acetic 
 acid ( 0.006 ml , 0.11 mmol ) was added to a solution of 6-phenoxymethyl-4-vinyl - nicotinic 
 acid methyl ester ( 33 , 29 mg , 0.11 mmol ) and ( r)-(+)-3,3-dimethyl 2-aminobutane ( 0.030 ml , 0.225 mmol ) 
 in meoh ( 1 ml ) . the mixture was stirred at 100 c for 16 h. then 
 ( r)-(+)-3,3-dimethyl 2-aminobutane ( 0.030 ml , 0.225 
 mmol ) and acetic acid ( 0.006 ml , 0.11 mmol ) were added , and the mixture 
 was heated at 100 c for 2 days . the solvents were evaporated 
 in vacuo and the residue diluted with ch2cl2 and extracted with a saturated solution of nahco3 . the 
 organic layer was separated , dried ( na2so4 ) , 
 filtered , and the solvents evaporated in vacuo . the crude product 
 was purified by flash chromatography ( silica ; acoet in ch2cl2 0/100 to 30/70 ) . the desired fractions were collected 
 and the solvents evaporated in vacuo to yield compound 15c as a colorless oil which solidified upon standing at room temperature 
 ( 29 mg , 79% ) . lcms tr = 3.10 min , > 
 98% 
 at 215 and 254 nm , m / z = 339.1 [ m 
 + h ] . h nmr ( 500 mhz , cdcl3 )  9.19 ( s , 
 1 h ) , 7.38 ( s , 1 h ) , 7.31 ( t , j = 8.1 hz , 2 h ) , 7.036.92 
 ( m , 3 h ) , 5.24 ( s , 2 h ) , 4.83 ( q , j = 7.2 hz , 1 h ) , 
 3.593.43 ( m , 2 h ) , 3.062.93 ( m , 1 h ) , 2.932.81 
 ( m , 1 h ) , 1.21 ( d , j = 6.9 hz , 3 h ) , 0.99 ( s , 9 h ) . starting from 
 6-phenoxymethyl-4-vinyl - nicotinic acid methyl ester ( 33 , 30 mg , 0.11 mmol ) and 4-fluoroaniline ( 0.044 ml , 0.47 mmol ) following 
 the procedure described for compound 15c , title compound 15d was obtained as a white solid ( 24 mg , 62% ) ; mp 198.8 c . lcms tr = 2.66 min , > 98% at 215 and 
 254 
 nm , m / z = 349.1 [ m + h ] . h nmr ( 500 mhz , cdcl3 )  9.24 ( s , 1 h ) , 7.46 ( s , 
 1 h ) , 7.397.29 ( m , 4 h ) , 7.177.08 ( m , 2 h ) , 7.046.96 
 ( m , 3 h ) , 5.27 ( s , 2 h ) , 3.99 ( t , j = 6.5 hz , 2 h ) , 
 3.17 ( t , j = 6.4 hz , 2 h ) .. 3-(3-bromo-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)(cyclopropyl)methanone ( 35a , 35 mg , 0.125 
 mmol ) , 3-fluorobenzyl alcohol ( 15 mg , 0.125 mmol ) , cesium carbonate 
 ( 59 mg , 0.18 mmol ) , cui ( 3 mg , 0.01 mmol ) , and 1,10-phenanthroline 
 ( 3.9 mg , 0.02 mmol ) were combined in a 2.0 ml microwave vial and placed 
 under an argon atmosphere . degassed toluene was added and the mixture 
 heated at 110 c for 4 h. the mixture was cooled to rt , filtered 
 over celite , and the filtrate concentrated to dryness . rp - hplc purification 
 afforded 20d as an off - white powder ( 15 mg , 33% ) . lcms tr = 0.79 min , > 98% at 215 and 254 nm , m / z = 328.1 [ m + h ] . hrms ( es+ , m + h ) 
 calcd for c22h20fn2o2 , 
 327.1509 ; found , 327.1512 . 3-bromo-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)(4-fluorophenyl)methanone 
 ( 35c , 28 mg , 
 0.09 mmol ) , benzyl alcohol ( 9.6 mg , 0.09 mmol ) , cesium carbonate ( 43 
 mg , 0.13 mmol ) , cui ( 2 mg , 0.009 mmol ) , and 1,10-phenanthroline ( 3.2 
 mg , 0.018 mmol ) were combined in a 2.0 ml microwave vial and placed 
 under an argon atmosphere . degassed toluene was added and the mixture 
 heated at 110 c for 4 h. the mixture was cooled to rt , filtered 
 over celite , and the filtrate concentrated to dryness . rp - hplc purification 
 afforded the 20 g as an off - white powder ( 6.9 mg , 22% ) . lcms tr = 0.85 min , > 98% at 215 and 
 254 
 nm , m / z = 363.1 [ m + h ] . hrms ( es+ , 
 m + h ) calcd for c22h22fn2o2 , 363.1509 ; found , 363.1503 . ( 4-fluorophenyl)(3-(hydroxymethyl)-7,8-dihydro-1,6-naphthyridin-6(5h)-yl)methanone ( 39 , 40 mg , 0.14 mmol ) , phenol 
 ( 40 mg , 0.43 mmol ) , and polystyrene supported triphenylphosphine ( 150 
 mg , 0.45 mmol ) were combined in thf ( 6 ml ) and diisopropyl azodicarboxylate 
 ( 42 mg , 0.21 mmol ) was added , and the reaction was allowed to stir 
 overnight at rt . the residue was purified via rp - hplc ( 1090% 
 gradient of ch3cn in water with 0.1% tfa modifier ) to afford 
 title compound as an off - white powder ( 35 mg , 69% ) . lcms tr = 0.75 min , > 98% at 215 and 254 nm , m / z = 363.1 [ m + h ] . h nmr ( 400 mhz , 
 cdcl3 )  8.77 ( s , 1h ) , 8.06 ( s , 1h ) , 7.537.49 
 ( m , 3h ) , 7.36 ( t , j = 8.0 , 2h ) , 7.18 ( t , j = 8.0 , 2h ) , 7.06 ( t , j = 8.2 , 1h ) , 6.87 
 ( d , j = 8.0 , 1h ) , 5.18 ( s , 2h ) , 4.98 ( s , 2h ) , 3.92 
 ( s , 2h ) , 3.36 ( t , j = 6.0 , 2h ) . hrms ( es+ , m + h ) 
 calcd for c22h19fn2o2 , 
 362.1431 ; found , 362.1434 . for 
 measurement of compound - evoked increases in intracellular calcium , hek293 cells stably expressing rat mglu5 were plated in 
 384-well , poly - d - lysine coated , 
 black - walled , clear - bottomed plates in 20 l of assay medium 
 ( dmem supplemented with 10% dialyzed fetal bovine serum , 20 mm hepes , 
 and 1 mm sodium pyruvate ) at a density of 15000 cells / well . the next day , 
 medium was removed and cells were incubated with 20 l / well 
 of 1 m fluo-4am ( invitrogen , carlsbad , california ) prepared 
 as a 2.3 mm stock in dimethyl sulfoxide ( dmso ) and mixed in a 1:1 
 ratio with 10% ( w / v ) pluronic acid f-127 and diluted in calcium assay 
 buffer ( hank s balanced salt solution ( hbss ; invitrogen , carlsbad , 
 ca ) supplemented with 20 mm hepes and 2.5 mm probenecid , ph 7.4 ) for 
 50 min at 37 c . dye loading solution was then removed and replaced 
 with 20 l / well of assay buffer . for pam potency curves , mglu5 compounds were diluted in calcium assay buffer and added 
 to the cells , followed by the addition of an ec20 concentration 
 of glutamate 140 s later and then an ec80 concentration 
 of glutamate 90120 s later . for fold - shift experiments , either 
 a single concentration ( 10 m ) or multiple fixed concentrations 
 ( 55 nm to 30 m ) of mglu5 compound or vehicle was 
 added , followed by the addition of a concentration calcium flux was measured over 
 time as an increase in fluorescence using a functional drug screening 
 system 6000 ( fdss 6000 , hamamatsu , japan ) . the change in relative 
 fluorescence over basal was calculated before normalization to the 
 maximal response to glutamate . a 2-fold enhancement over basal response 
 at the maximum concentration was sufficient criteria for weak pam 
 activity ( e.g. , 40% glu max at an ec20 add of glutamate ; 
 the basal ec20 response range allowed was 1030% ) . to assess the effect 
 of test compounds 
 at mglu1 , ca mobilization assays were performed 
 as described previously . briefly , hek293 cells 
 stably expressing rat mglu1 were plated in black - walled , 
 clear - bottomed , poly - d - lysine coated 384-well plates ( greiner 
 bio - one , monroe , nc ) in assay medium at a density of 20000 cells / well . calcium flux was measured over time as an increase in fluorescence 
 of the ca indicator dye , fluo-4am using a fdss 6000 . either vehicle or a fixed concentration of test compound ( 10 m , 
 final concentration ) was added , followed 140 s later by a crc of glutamate . the functional activity 
 of the compounds of interest was assessed at the rat group ii and 
 iii mglu receptors by measuring thallium flux through girk channels 
 as previously described . briefly , hek293-girk 
 cells expressing mglu subtypes 2 , 3 , 4 , 6 , 7 , or 8 were plated into 
 384-well , black - walled , clear - bottom poly - d - lysine coated 
 plates at a density of 15000 cells / well in assay medium . a single 
 concentration of test compound ( 10 m ) or vehicle was added , 
 followed 140 s later by a crc of glutamate ( or l - ap4 for mglu7 ) diluted in thallium buffer ( 125 mm nahco3 , 1 
 mm mgso4 , 1.8 mm caso4 , 5 mm glucose , 12 mm 
 thallium sulfate , 10 mm hepes ) , and fluorescence was measured using 
 a fdss 6000 . animals were housed in the animal 
 care facility certified by the american association for the accreditation 
 of laboratory animal care ( aaalac ) under a 12 h light / dark cycle ( lights 
 on , 7 a.m. ; lights off , 7 p.m. ) and had free access to food and water . the animals used in these experiments were food - deprived the evening 
 before experimentation for oral administration of test compound . the 
 experimental protocols performed during the light cycle were approved 
 by the institutional animals care and use committee of vanderbilt 
 university and conformed to the guidelines established by the national 
 research council guide for the care and use of laboratory animals . 12c was formulated 
 in volumes specific to the number of animals dosed each day . the solutions 
 were formulated so that animals were injected with a maximal dosing 
 volume of 10 ml / kg . the appropriate amount according to the dosage was mixed into a 20% 2-(hydroxypropyl)--cyclodextrin in sterile 
 water ( hp--cd ; sigma , catalogue no . c0926 - 10 g ) solution . each 
 mixture was ultrahomogenized on ice for 23 min using a hand - held 
 tissue homogenizer . next , the ph of all solutions was checked using 
 014 emd ph strips and adjusted to a ph of 67 if necessary 
 with 1n naoh . the mixtures were then vortexed and stored in a sonication 
 bath at 40 c until time of injection . d - amphetamine 
 hemisulfate ( amp ) was obtained from sigma ( catalogue no . a5880 - 1 g ; 
 st . salt - correction was used to determine the correct 
 amount of the d - amphetamine hemisulfate form in mg 
 to add to sterile water in order to yield a 1 mg / ml solution , injected 
 with a maximal dosing volume of 10 ml / kg . studies 
 were conducted using smart open field activity chambers ( 27 cm  
 27 cm  20 cm ) ( kinder scientific , poway , ca ) equipped with 16 
 horizontal ( x- and y - axes ) infrared 
 photobeams . changes in locomotor activity were measured as the number 
 of photobeam breaks over time and were recorded with a pentium i computer 
 equipped with rat activity monitoring system software ( motor monitor , 
 kinder scientific , poway , ca ) . male harlan sprague  dawley rats 
 ( harlan laboratories , indianapolis , in ) weighing 250375 g 
 were used . animals were next pretreated for an additional 
 30 min with either vehicle or a dose of 12c po , followed 
 by a subcutaneous injection of 1 mg / kg amphetamine or vehicle and 
 monitored for an additional 60 min . dose group 1 , vamp = 20% -cd 
 ( 12c vehicle ) , po + 1 mg / kg amp , sc ( n = 8) . dose group 2 , 3amp = 3 mg / kg 12c , po + 1 mg / kg 
 amp , sc ( n = 6 ) . dose group 3 , 10amp = 10 mg / kg 12c , po + 1 mg / kg amp , sc ( n = 7 ) . dose group 
 4 , 30amp = 30 mg / kg 12c , po + 1 mg / kg amp , sc ( n = 8) . mg / kg 12c , po + 1 mg / kg amp , sc ( n = 8) . mg / kg 12c , po + 1 mg / kg amp , sc ( n = 8) . dose group 7 , 100v = 100 mg / kg 12c , po + sterile 
 water ( amp vehicle ) , sc ( n = 8) . data were expressed 
 as changes in ambulation defined as the total number of photobeam 
 breaks per 5 min interval . at the end of this behavioral study , each 
 animal was euthanized , then decapitated , and the plasma and brain 
 tissues were collected for the evaluation of exposure levels of 12c . behavioral data were analyzed using a 
 one - way anova with main effects of treatment and time . post hoc analyses 
 were performed using a dunnett s t - test with 
 all treatment groups compared to the vamp group using jmp 8.0 ( sas 
 institute , cary , nc ) statistical software . data were graphed using 
 sigmaplot for windows version 11.0 ( saugua , ma ) . a probability of p  0.05 was taken as the level of statistical significance . percent effect and reversal calculations for the 3amp , 10amp , 30amp , 
 56.6amp , and 100amp treatment groups were performed with the following 
 formula relative to the vamp treatment group : ( 1 ) total number of 
 photobeam breaks in the time interval from t = 60 
 to t = 120 was calculated for each rat in each treatment 
 group , ( 2 ) mean total number of photobeam breaks in the time interval 
 from t = 60 to t = 120 was calculated 
 for the vamp group , ( 3 ) percent effect = ratio of the total number 
 of photobeam breaks for each rat in each treatment group divided by 
 the mean total number of photobeam breaks in the time interval from t = 60 to t = 120 of the vamp group multiplied 
 by 100 , ( 4 ) percent reversal = 100  the percent effect for 
 each rat in each treatment group , ( 5 ) finally , the mean  se 
 percent reversal for each treatment group was calculated from the 
 individual percent reversal values . percent effect and change calculations 
 for the vamp and 100 v treatment groups relative to the vv treatment 
 group were also performed with the following formula : ( 1 ) total number 
 of photobeam breaks in the time interval from t = 
 60 to t = 120 was calculated for each rat in each 
 treatment group , ( 2 ) mean total number of photobeam breaks in the 
 time interval from t = 60 to t = 
 120 was calculated for the vv group ; ( 3 ) percent effect = ratio of 
 the total number of photobeam breaks for each rat in each treatment 
 group divided by the mean total number of photobeam breaks in the 
 time interval from t = 60 to t = 
 120 of the vv group multiplied by 100 , ( 4 ) percent change = the percent 
 effect for each rat in each treatment group  100 , ( 5 ) finally , 
 the mean  se percent change for each treatment group was calculated 
 from the individual percent change values . brain samples were homogenized in 3 ml of 70:30 2-propanol : water 
 and then centrifuged at 3500 rpm for 5 min . resulting brain sample 
 supernatant and plasma samples were transferred into a 96-well plate 
 containing plasma blanks , plasma double blank , a standard curve of 
 the test article , and quality control samples for subsequent lc - ms / ms 
 analysis . each sample was diluted with acetonitrile containing 50 
 nm carbamazepine ( internal standard ) , centrifuged at 3500 rpm , and 
 the resulting supernatant was transferred to a new 96-well plate . after an equal volume of water was added to each sample ( to provide 
 50:50 acetonitrile : water solution ) , the plate was analyzed via esi 
 on a triple - quadrupole mass spectrometer ( ab sciex api-4000 ) coupled 
 with an autosampling liquid chromatography system ( shimadzu lc-10ad 
 pumps , leap technologies ctc pal autosampler ) . analyte ( test article ) 
 was separated by gradient elution using a c18 3.0 mm  50 mm , 
 3 m column ( fortis technologies ) thermostated at 40 c . hplc mobile phase a was 0.1% formic acid in water ( ph unadjusted ) , 
 mobile phase b was 0.1% formic acid in acetonitrile ( ph unadjusted ) , 
 and the gradient used was 3090% mobile phase b with 0.2 min 
 hold at 30% b , linear increase to 90% b over 0.8 min , hold at 90% 
 b for 0.6 min , and a return to 30% b over 0.1 min followed by a re - equilibration 
 ( 0.5 min ) to provide a 2.0 min total run time per sample . hplc flow 
 rate was 0.5 ml / min , source temperature was 500 c , and mass 
 spectral analyses were performed using the following mrm transitions : 
 353.5  259.2 and 353.5  123.2 m / z . esi was achieved by a turbo - ionspray source in positive 
 ionization mode ( 5.0 kv spray voltage ) . the raw plasma and brain concentration 
 data obtained by lc - ms / ms were analyzed by analyst software ( ab sciex , 
 version 1.5.1 ) , which used the ratio of peak area responses of drug 
 relative to internal standard to construct a standard curve with a 
 dynamic range covering the concentrations found in the samples . free 
 plasma and brain concentrations were calculated by multiplication 
 of the total concentration values by fu , plasma and fu , brain , respectively , based on 
 data from in vitro rat plasma protein and rat brain homogenate binding 
 experiments . the effects of 12c on motor performance 
 were evaluated using an automated rotarod setup with a rotarod ( 7.0 
 cm in diameter ) rotating at a constant speed of 20 rotations / min ( medassociates , 
 inc . , male harlan sprague  dawley rats ( harlan 
 laboratories , indianapolis , in ) weighing 250300 g were used . animals were given two training trials of 120 s on the rotarod with 
 a 10 min interval between trials , followed by baseline assessment 
 of performance with a 120 s trial , and any animals that did not reach 
 a performance criteria of 85 s were excluded from the study . animals 
 were next pretreated for 30 min with vehicle ( 20% hp--cd ) or 
 a dose of 12c ( n = 6 each group ) and 
 then tested on the rotarod using a 120 s trial . the amount of time 
 in seconds that each animal remained on the rotarod was recorded ; 
 animals not falling off of the rotarod were given a maximal score 
 of 120 s. data were expressed as the mean latency to fall off the 
 rotarod in seconds for each treatment group . behavioral data were analyzed using a 
 one - way anova with main effects of treatment . post hoc analyses were 
 performed using a dunnett s t test with all 
 treatment groups compared to the vehicle group using jmp 8.0 ( sas 
 institute , cary , nc ) statistical software . data were graphed using 
 sigmaplot for windows version 11.0 ( saugua , ma ) . a probability of p  0.05 was taken as the level of statistical significance . male harlan 
 sprague  dawley rats ( harlan laboratories , indianapolis , in ) 
 weighing 250300 g were used . animals were evaluated in the 
 modified irwin neurological test battery at t = 0 
 to provide a baseline measurement across each functional end point . animals were next administered either vehicle ( 20% hp--cd ) 
 or a 100 mg / kg po dose of 12c ( n = 6 
 each group ) and then assessed after a 30 min , 1 h , and 4 h pretreatment 
 interval in the modified irwin neurological test battery . changes 
 in the different functional end points of the irwin test battery were 
 given a score of 0 , 1 or 2 , with 0 = no effect , 1 = moderate effect , 
 and 2 = robust full effect . autonomic nervous system functions : ptosis , exophthalmos , miosis , 
 mydriasis , corneal reflex , pinna reflex , piloerection , respiratory 
 rate , writhing , tail erection , lacrimation , salivation , vasodilation , 
 skin color , irritability , and rectal temperature . somatomotor nervous 
 system functions : motor activity , ataxia , arch / roll , tremors , leg 
 weakness , rigid stance , spraddle , placing loss , grasping loss , righting 
 loss , catalepsy , tail pinch reaction , escape loss , and physical appearance . mean change values 
 for each functional 
 end point in the irwin test battery of each treatment group were calculated 
 using microsoft excel . behavioral data were then analyzed using a 
 one - way anova with main effect of treatment . post hoc analyses were 
 performed using a dunnett s t test with all 
 treatment groups compared to the vehicle group using jmp 8.0 ( sas 
 institute , cary , nc ) statistical software . data were graphed using 
 sigmaplot for windows version 11.0 ( saugua , ma ) . a probability of p  0.05 was taken as the level of statistical significance .
positive 
 allosteric modulators ( pams ) of metabotropic glutamate 
 receptor 5 ( mglu5 ) represent a promising therapeutic strategy 
 for the treatment of schizophrenia . starting from an acetylene - based 
 lead from high throughput screening , 
 an evolved bicyclic dihydronaphthyridinone 
 was identified . 
 we describe further refinements leading to both dihydronaphthyridinone 
 and tetrahydronaphthyridine mglu5 pams containing an alkoxy - based 
 linkage as an acetylene replacement . 
 exploration of several structural 
 features including western pyridine ring isomers , positional amides , 
 linker connectivity / position , and combinations thereof , reveal that 
 these bicyclic modulators generally exhibit steep sar and within specific 
 subseries display a propensity for pharmacological mode switching 
 at mglu5 as well as antagonist activity at mglu3 . structure 
 activity relationships within a dihydronaphthyridinone 
 subseries uncovered 12c ( vu0405372 ) , a selective mglu5 pam with good in vitro potency , low glutamate fold - shift , 
 acceptable dmpk properties , and in vivo efficacy in an amphetamine - based 
 model of psychosis .